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Sample records for monitoring single-cell activities

  1. Bacterial Activity in the Rhizosphere Analyzed at the Single-Cell Level by Monitoring Ribosome Contents and Synthesis Rates

    PubMed Central

    Ramos, Cayo; Mølbak, Lars; Molin, Søren

    2000-01-01

    The growth activity of Pseudomonas putida cells colonizing the rhizosphere of barley seedlings was estimated at the single-cell level by monitoring ribosomal contents and synthesis rates. Ribosomal synthesis was monitored by using a system comprising a fusion of the ribosomal Escherichia coli rrnBP1 promoter to a gene encoding an unstable variant of the green fluorescent protein (Gfp). Gfp expression in a P. putida strain carrying this system inserted into the chromosome was strongly dependent on the growth phase and growth rate of the strain, and cells growing exponentially at rates of ≥0.17 h−1 emitted growth rate-dependent green fluorescence detectable at the single-cell level. The single-cell ribosomal contents were very heterogeneous, as determined by quantitative hybridization with fluorescently labeled rRNA probes in P. putida cells extracted from the rhizosphere of 1-day-old barley seedlings grown under sterile conditions. After this, cells extracted from the root system had ribosomal contents similar to those found in starved cells. There was a significant decrease in the ribosomal content of P. putida cells when bacteria were introduced into nonsterile bulk or rhizosphere soil, and the Gfp monitoring system was not induced in cells extracted from either of the two soil systems. The monitoring system used permitted nondestructive in situ detection of fast-growing bacterial microcolonies on the sloughing root sheath cells of 1- and 2-day-old barley seedlings grown under sterile conditions, which demonstrated that it may be possible to use the unstable Gfp marker for studies of transient gene expression in plant-microbe systems. PMID:10653754

  2. A single-cell correlative nanoelectromechanosensing approach to detect cancerous transformation: monitoring the function of F-actin microfilaments in the modulation of the ion channel activity

    NASA Astrophysics Data System (ADS)

    AbdolahadThe Authors With Same Contributions., Mohammad; Saeidi, Ali; Janmaleki, Mohsen; Mashinchian, Omid; Taghinejad, Mohammad; Taghinejad, Hossein; Azimi, Soheil; Mahmoudi, Morteza; Mohajerzadeh, Shams

    2015-01-01

    Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such responses were extremely weak for malignant cells. Confocal microscopy results showed the impact of actin microfilament remodelling on the reduction of the electrical response for aspirated cancer cells due to the significant role of actin in modulating the ion channel activity in the cell membrane.Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such

  3. Microwave-induced thermogenetic activation of single cells

    SciTech Connect

    Safronov, N. A.; Fedotov, I. V.; Ermakova, Yu. G.; Matlashov, M. E.; Belousov, V. V.; Sidorov-Biryukov, D. A.; Fedotov, A. B.; Zheltikov, A. M.

    2015-04-20

    Exposure to a microwave field is shown to enable thermogenetic activation of individual cells in a culture of cell expressing thermosensitive ion channels. Integration of a microwave transmission line with an optical fiber and a diamond quantum thermometer has been shown to allow thermogenetic single-cell activation to be combined with accurate local online temperature measurements based on an optical detection of electron spin resonance in nitrogen–vacancy centers in diamond.

  4. Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

    PubMed

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Chen, Chia-Hung

    2016-07-15

    Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. PMID:26995287

  5. Monitoring single-cell bioenergetics via the coarsening of emulsion droplets

    PubMed Central

    Boitard, L.; Cottinet, D.; Kleinschmitt, C.; Bremond, N.; Baudry, J.; Yvert, G.; Bibette, J.

    2012-01-01

    Microorganisms are widely used to generate valuable products, and their efficiency is a major industrial focus. Bioreactors are typically composed of billions of cells, and available measurements only reflect the overall performance of the population. However, cells do not equally contribute, and process optimization would therefore benefit from monitoring this intrapopulation diversity. Such monitoring has so far remained difficult because of the inability to probe concentration changes at the single-cell level. Here, we unlock this limitation by taking advantage of the osmotically driven water flux between a droplet containing a living cell toward surrounding empty droplets, within a concentrated inverse emulsion. With proper formulation, excreted products are far more soluble within the continuous hydrophobic phase compared to initial nutrients (carbohydrates and salts). Fast diffusion of products induces an osmotic mismatch, which further relaxes due to slower diffusion of water through hydrophobic interfaces. By measuring droplet volume variations, we can deduce the metabolic activity down to isolated single cells. As a proof of concept, we present the first direct measurement of the maintenance energy of individual yeast cells. This method does not require any added probes and can in principle apply to any osmotically sensitive bioactivity, opening new routes for screening, and sorting large libraries of microorganisms and biomolecules. PMID:22538813

  6. Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution

    PubMed Central

    Ludlow, Andrew T.; Robin, Jerome D.; Sayed, Mohammed; Litterst, Claudia M.; Shelton, Dawne N.; Shay, Jerry W.; Wright, Woodring E.

    2014-01-01

    The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase. PMID:24861623

  7. Fast serial analysis of active cholesterol at the plasma membrane in single cells.

    PubMed

    Tian, Chunxiu; Zhou, Junyu; Wu, Zeng-Qiang; Fang, Danjun; Jiang, Dechen

    2014-01-01

    Previously, our group has utilized the luminol electrochemiluminescence to analyze the active cholesterol at the plasma membrane in single cells by the exposure of one cell to a photomultiplier tube (PMT) through a pinhole. In this paper, fast analysis of active cholesterol at the plasma membrane in single cells was achieved by a multimicroelectrode array without the pinhole. Single cells were directly located on the microelectrodes using cell-sized microwell traps. A cycle of voltage was applied on the microelectrodes sequentially to induce a peak of luminescence from each microelectrode for the serial measurement of active membrane cholesterol. A minimal time of 1.60 s was determined for the analysis of one cell. The simulation and the experimental data exhibited a semisteady-state distribution of hydrogen peroxide on the microelectrode after the reaction of cholesterol oxidase with the membrane cholesterol, which supported the relative accuracy of the serial analysis. An eight-microelectrode array was demonstrated to analyze eight single cells in 22 s serially, including the channel switching time. The results from 64 single cells either activated by low ion strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT) revealed that most of the cells analyzed had the similar active membrane cholesterol, while few cells had more active cholesterol resulting in the cellular heterogeneity. The fast single-cell analysis platform developed will be potentially useful for the analysis of more molecules in single cells using proper oxidases. PMID:24328095

  8. Modulation of Neural Network Activity through Single Cell Ablation: An in Vitro Model of Minimally Invasive Neurosurgery.

    PubMed

    Soloperto, Alessandro; Bisio, Marta; Palazzolo, Gemma; Chiappalone, Michela; Bonifazi, Paolo; Difato, Francesco

    2016-01-01

    The technological advancement of optical approaches, and the growth of their applications in neuroscience, has allowed investigations of the physio-pathology of neural networks at a single cell level. Therefore, better understanding the role of single neurons in the onset and progression of neurodegenerative conditions has resulted in a strong demand for surgical tools operating with single cell resolution. Optical systems already provide subcellular resolution to monitor and manipulate living tissues, and thus allow understanding the potentiality of surgery actuated at single cell level. In the present work, we report an in vitro experimental model of minimally invasive surgery applied on neuronal cultures expressing a genetically encoded calcium sensor. The experimental protocol entails the continuous monitoring of the network activity before and after the ablation of a single neuron, to provide a robust evaluation of the induced changes in the network activity. We report that in subpopulations of about 1000 neurons, even the ablation of a single unit produces a reduction of the overall network activity. The reported protocol represents a simple and cost effective model to study the efficacy of single-cell surgery, and it could represent a test-bed to study surgical procedures circumventing the abrupt and complete tissue removal in pathological conditions. PMID:27527143

  9. Single-cell bacteria growth monitoring by automated DEP-facilitated image analysis.

    PubMed

    Peitz, Ingmar; van Leeuwen, Rien

    2010-11-01

    Growth monitoring is the method of choice in many assays measuring the presence or properties of pathogens, e.g. in diagnostics and food quality. Established methods, relying on culturing large numbers of bacteria, are rather time-consuming, while in healthcare time often is crucial. Several new approaches have been published, mostly aiming at assaying growth or other properties of a small number of bacteria. However, no method so far readily achieves single-cell resolution with a convenient and easy to handle setup that offers the possibility for automation and high throughput. We demonstrate these benefits in this study by employing dielectrophoretic capturing of bacteria in microfluidic electrode structures, optical detection and automated bacteria identification and counting with image analysis algorithms. For a proof-of-principle experiment we chose an antibiotic susceptibility test with Escherichia coli and polymyxin B. Growth monitoring is demonstrated on single cells and the impact of the antibiotic on the growth rate is shown. The minimum inhibitory concentration as a standard diagnostic parameter is derived from a dose-response plot. This report is the basis for further integration of image analysis code into device control. Ultimately, an automated and parallelized setup may be created, using an optical microscanner and many of the electrode structures simultaneously. Sufficient data for a sound statistical evaluation and a confirmation of the initial findings can then be generated in a single experiment. PMID:20842296

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

    PubMed

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

    2015-10-22

    Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer. PMID:26375009

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

    Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer.

  12. Monitoring metabolic responses to chemotherapy in single cells and tumors using nanostructure-initiator mass spectrometry (NIMS) imaging

    PubMed Central

    2013-01-01

    Background Tissue imaging of treatment-induced metabolic changes is useful for optimizing cancer therapies, but commonly used methods require trade-offs between assay sensitivity and spatial resolution. Nanostructure-Initiator Mass Spectrometry imaging (NIMS) permits quantitative co-localization of drugs and treatment response biomarkers in cells and tissues with relatively high resolution. The present feasibility studies use NIMS to monitor phosphorylation of 3′-deoxy-3′-fluorothymidine (FLT) to FLT-MP in lymphoma cells and solid tumors as an indicator of drug exposure and pharmacodynamic responses. Methods NIMS analytical sensitivity and spatial resolution were examined in cultured Burkitt’s lymphoma cells treated briefly with Rapamycin or FLT. Sample aliquots were dispersed on NIMS surfaces for single cell imaging and metabolic profiling, or extracted in parallel for LC-MS/MS analysis. Docetaxel-induced changes in FLT metabolism were also monitored in tissues and tissue extracts from mice bearing drug-sensitive tumor xenografts. To correct for variations in FLT disposition, the ratio of FLT-MP to FLT was used as a measure of TK1 thymidine kinase activity in NIMS images. TK1 and tumor-specific luciferase were measured in adjacent tissue sections using immuno-fluorescence microscopy. Results NIMS and LC-MS/MS yielded consistent results. FLT, FLT-MP, and Rapamycin were readily detected at the single cell level using NIMS. Rapid changes in endogenous metabolism were detected in drug-treated cells, and rapid accumulation of FLT-MP was seen in most, but not all imaged cells. FLT-MP accumulation in xenograft tumors was shown to be sensitive to Docetaxel treatment, and TK1 immunoreactivity co-localized with tumor-specific antigens in xenograft tumors, supporting a role for xenograft-derived TK1 activity in tumor FLT metabolism. Conclusions NIMS is suitable for monitoring drug exposure and metabolite biotransformation with essentially single cell resolution, and

  13. Application of laser tweezers Raman spectroscopy techniques to the monitoring of single cell response to stimuli

    NASA Astrophysics Data System (ADS)

    Chan, James W.; Liu, Rui; Matthews, Dennis L.

    2012-06-01

    Laser tweezers Raman spectroscopy (LTRS) combines optical trapping with micro-Raman spectroscopy to enable label-free biochemical analysis of individual cells and small biological particles in suspension. The integration of the two technologies greatly simplifies the sample preparation and handling of suspension cells for spectroscopic analysis in physiologically meaningful conditions. In our group, LTRS has been used to study the effects of external perturbations, both chemical and mechanical, on the biochemistry of the cell. Single cell dynamics can be studied by performing longitudinal studies to continuously monitor the response of the cell as it interacts with its environment. The ability to carry out these measurements in-vitro makes LTRS an attractive tool for many biomedical applications. Here, we discuss the use of LTRS to study the response of cancer cells to chemotherapeutics and bacteria cells to antibiotics and show that the life cycle and apoptosis of the cells can be detected. These results show the promise of LTRS for drug discovery/screening, antibiotic susceptibility testing, and chemotherapy response monitoring applications. In separate experiments, we study the response of red blood cells to the mechanical forces imposed on the cell by the optical tweezers. A laser power dependent deoxygenation of the red blood cell in the single beam trap is reported. Normal, sickle cell, and fetal red blood cells have a different behavior that enables the discrimination of the cell types based on this mechanochemical response. These results show the potential utility of LTRS for diagnosing and studying red blood cell diseases.

  14. Nystatin-induced changes in yeast monitored by time-resolved automated single cell electrorotation.

    PubMed

    Hölzel, R

    1998-10-23

    A widespread use of electrorotation for the determination of cellular and subcellular properties has been hindered so far by the need for manual recording of cell movements. Therefore a system has been developed that allows the automatic collection of electrorotation spectra of single cells in real time. It employs a hardware based registration of image moments from which object orientation is calculated. Since the camera's video signal is processed without intermediate image storage a high data throughput of about two recordings per second could be achieved independently of image resolution. This made it possible to monitor changes in cell membrane and cytoplasm of the yeast Saccharomyces cerevisiae under the influence of the antibiotic nystatin with a temporal resolution of 3 min. Up to 20 electrorotation spectra of an individual cell could be collected in the frequency range between 1 kHz and 1 GHz. Two distinct events 7 and 75 min after addition of nystatin were observed with a fast increase in membrane permeability accompanied by a nearly simultaneous drop in cytoplasmic conductivity. PMID:9795246

  15. Raman-activated cell sorting based on dielectrophoretic single-cell trap and release.

    PubMed

    Zhang, Peiran; Ren, Lihui; Zhang, Xu; Shan, Yufei; Wang, Yun; Ji, Yuetong; Yin, Huabing; Huang, Wei E; Xu, Jian; Ma, Bo

    2015-02-17

    Raman-activated cell sorting (RACS) is a promising single-cell technology that holds several significant advantages, as RACS is label-free, information-rich, and potentially in situ. To date, the ability of the technique to identify single cells in a high-speed flow has been limited by inherent weakness of the spontaneous Raman signal. Here we present an alternative pause-and-sort RACS microfluidic system that combines positive dielectrophoresis (pDEP) for single-cell trap and release with a solenoid-valve-suction-based switch for cell separation. This has allowed the integration of trapping, Raman identification, and automatic separation of individual cells in a high-speed flow. By exerting a periodical pDEP field, single cells were trapped, ordered, and positioned individually to the detection point for Raman measurement. As a proof-of-concept demonstration, a mixture of two cell strains containing carotenoid-producing yeast (9%) and non-carotenoid-producing Saccharomyces cerevisiae (91%) was sorted, which enriched the former to 73% on average and showed a fast Raman-activated cell sorting at the subsecond level. PMID:25607599

  16. Dynamic monitoring of single cell lysis in an impedance-based microfluidic device.

    PubMed

    Zhou, Ying; Basu, Srinjan; Laue, Ernest D; Seshia, Ashwin A

    2016-08-01

    A microfluidic device that is capable of trapping and sensing dynamic variations in the electrical properties of individual cells is demonstrated. The device is applied to the real-time recording of impedance measurements of mouse embryonic stem cells (mESCs) during the process of membrane lysis, with the resulting changes in the electrical properties of cells during this process being quantitatively tracked over time. It is observed that the impedance magnitude decreases dramatically after cell membrane lysis. A significant shift in the phase spectrum is also observed during the time course of this process. By fitting experimental data to physical models, the electrical parameters of cells can be extracted and parameter variations quantified during the process. In the cell lysis experiments, the equivalent conductivity of the cell membrane is found to increase significantly due to pore formation in the membrane during lysis. An increase in the specific capacitance of the membrane is also observed. On the other hand, the conductivity of the cytoplasm is observed to decrease, which may be explained the fact that excess water enters the cell through the gradual permeabilization of the membrane during lysis. Cells can be trapped in the device for periods up to several days, and their electrical response can be monitored by real-time impedance measurements in a label-free and non-invasive manner. Furthermore, due to the highly efficient single cell trapping capacity of the device, a number of cells can be trapped and held in separate wells for concurrent parallel experiments, allowing for the possibility of stepped parametric experiments and studying cell heterogeneity by combining measurements across the array. PMID:27299468

  17. Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of Calcein Acetoxymethyl Ester

    PubMed Central

    Krämer, Christina E. M.; Singh, Abhijeet; Helfrich, Stefan; Grünberger, Alexander; Wiechert, Wolfgang; Nöh, Katharina; Kohlheyer, Dietrich

    2015-01-01

    Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single cell cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing cells with metabolic activity as well as non-growing and non-fluorescent cells with no detectable esterase activity. Microfluidic single cell cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non

  18. Automated single cell microbioreactor for monitoring intracellular dynamics and cell growth in free solution†

    PubMed Central

    Johnson-Chavarria, Eric M.; Agrawal, Utsav; Tanyeri, Melikhan; Kuhlman, Thomas E.

    2014-01-01

    We report an automated microfluidic-based platform for single cell analysis that allows for cell culture in free solution with the ability to control the cell growth environment. Using this approach, cells are confined by the sole action of gentle fluid flow, thereby enabling non-perturbative analysis of cell growth away from solid boundaries. In addition, the single cell microbioreactor allows for precise and time-dependent control over cell culture media, with the combined ability to observe the dynamics of non-adherent cells over long time scales. As a proof-of-principle demonstration, we used the platform to observe dynamic cell growth, gene expression, and intracellular diffusion of repressor proteins while precisely tuning the cell growth environment. Overall, this microfluidic approach enables the direct observation of cellular dynamics with exquisite control over environmental conditions, which will be useful for quantifying the behaviour of single cells in well-defined media. PMID:24836754

  19. A monolithic glass chip for active single-cell sorting based on mechanical phenotyping.

    PubMed

    Faigle, Christoph; Lautenschläger, Franziska; Whyte, Graeme; Homewood, Philip; Martín-Badosa, Estela; Guck, Jochen

    2015-03-01

    The mechanical properties of biological cells have long been considered as inherent markers of biological function and disease. However, the screening and active sorting of heterogeneous populations based on serial single-cell mechanical measurements has not been demonstrated. Here we present a novel monolithic glass chip for combined fluorescence detection and mechanical phenotyping using an optical stretcher. A new design and manufacturing process, involving the bonding of two asymmetrically etched glass plates, combines exact optical fiber alignment, low laser damage threshold and high imaging quality with the possibility of several microfluidic inlet and outlet channels. We show the utility of such a custom-built optical stretcher glass chip by measuring and sorting single cells in a heterogeneous population based on their different mechanical properties and verify sorting accuracy by simultaneous fluorescence detection. This offers new possibilities of exact characterization and sorting of small populations based on rheological properties for biological and biomedical applications. PMID:25537986

  20. Research Resource: Monitoring Endoplasmic Reticulum Membrane Integrity in β-Cells at the Single-Cell Level

    PubMed Central

    Kanekura, Kohsuke; Ou, Jianhong; Hara, Takashi; Zhu, Lihua J.

    2015-01-01

    Endoplasmic reticulum (ER) membrane integrity is an emerging target for human chronic diseases associated with ER stress. Despite the underlying importance of compromised ER membrane integrity in disease states, the entire process leading to ER membrane permeabilization and cell death is still not clear due to technical limitations. Here we describe a novel method for monitoring ER membrane integrity at the single-cell level in real time. Using a β-cell line expressing ER-targeted redox sensitive green fluorescent protein, we could identify a β-cell population undergoing ER membrane permeabilization induced by palmitate and could monitor cell fate and ER stress of these cells at the single-cell level. Our method could be used to develop a novel therapeutic modality targeting the ER membrane for ER-associated disorders, including β-cell death in diabetes, neurodegeneration, and Wolfram syndrome. PMID:25584413

  1. Femtosecond laser fabricated microfluorescence-activated cell sorter for single cell recovery

    NASA Astrophysics Data System (ADS)

    Bragheri, F.; Paiè, P.; Nava, G.; Yang, T.; Minzioni, P.; Martinez Vazquez, R.; Bellini, N.; Ramponi, R.; Cristiani, I.; Osellame, R.

    2014-03-01

    Manipulation, sorting and recovering of specific live cells from samples containing less than a few thousand cells is becoming a major hurdle in rare cell exploration such as stem cell research or cell based diagnostics. Moreover the possibility of recovering single specific cells for culturing and further analysis would be of great impact in many biological fields ranging from regenerative medicine to cancer therapy. In recent years considerable effort has been devoted to the development of integrated and low-cost optofluidic devices able to handle single cells, which usually rely on microfluidic circuits that guarantee a controlled flow of the cells. Among the different microfabrication technologies, femtosecond laser micromachining (FLM) is ideally suited for this purpose as it provides the integration of both microfluidic and optical functions on the same glass chip leading to monolithic, robust and portable devices. Here a new optofluidic device is presented, which is capable of sorting and recovering of single cells, through optical forces, on the basis of their fluorescence and. Both fluorescence detection and single cell sorting functions are integrated in the microfluidic chip by FLM. The device, which is specifically designed to operate with a limited amount of cells but with a very high selectivity, is fabricated by a two-step process that includes femtosecond laser irradiation followed by chemical etching. The capability of the device to act as a micro fluorescence-activated cell sorter has been tested on polystyrene beads and on tumor cells and the results on the single live cell recovery are reported.

  2. Single-cell bioelectrical impedance platform for monitoring cellular response to drug treatment.

    PubMed

    Asphahani, Fareid; Wang, Kui; Thein, Myo; Veiseh, Omid; Yung, Sandy; Xu, Jian; Zhang, Miqin

    2011-02-01

    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. PMID:21301069

  3. Living at the border: A community and single-cell assessment of lake bacterioneuston activity

    PubMed Central

    Hörtnagl, Paul; Pérez, María Teresa; Sommaruga, Ruben

    2010-01-01

    We assessed the physicochemical properties of the surface microlayer (SML: first 900 μm) and its underlying water (ULW: 0.2–0.5-m depth) and compared the composition and activity of their bacterial communities in six lakes located across an altitude gradient. Activity was assessed at both the community level, by measuring leucine bulk incorporation, and at the single-cell level, by using microautoradiography. Catalyzed reporter deposition fluorescence in situ hybridization was used to quantitatively assess the structure of the bacterial assemblage. Dissolved organic matter at the SML was significantly enriched in small-size molecules as compared to the ULW. Bacterial abundance in the SML ranged from 3.2 × 105 cells mL−1 to 3.2 × 106 cells mL−1 and was enriched in four out of six lakes when compared to the ULW. The SML and ULW showed lake-specific differences in bacterial community composition, although in most cases, both layers were dominated by Betaproteobacteria. This group also contributed the most to total activity in both layers in all lakes, followed by Actinobacteria. Despite large differences in environmental conditions among lakes, the fraction of active neustonic bacteria was very similar in most of them. Both bulk and single-cell activities are not necessarily lower in the SML than in the ULW, and well-adapted bacteria exist in the extreme conditions found in this habitat. PMID:20401318

  4. Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes.

    PubMed

    van Unen, Jakobus; Rashidfarrokhi, Ali; Hoogendoorn, Eelco; Postma, Marten; Gadella, Theodorus W J; Goedhart, Joachim

    2016-09-01

    Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide sufficient contrast and specificity to measure activity of G-protein-coupled receptors. The four histamine receptor subtypes (H1R, H2R, H3R, and H4R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein-mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H1R activates Gαq. We also observed that H1R activates Gαi, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gαs, we found that the H2R induces Gαq-mediated calcium release. The H3R and H4R activated Gαi with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves. PMID:27358232

  5. Measurement of Protein Tyrosine Phosphatase Activity in Single Cells by Capillary Electrophoresis

    PubMed Central

    Phillips, Ryan M.; Bair, Eric; Lawrence, David S.; Sims, Christopher E.; Allbritton, Nancy L.

    2013-01-01

    A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates, and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 nM and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn2+ and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min−1 mg−1) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min−1 mg−1). Zn2+, 1,2-naphthoquinone and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 μM, and 100 nM, respectively. Dephosphorylation of the reporter following loading into living single cells occurred at rates of at least 2 pmol min−1 mg−1. When single cells were exposed to 1,2-naphthoquinone (50 μM), Zn2+ (100 μM), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens. PMID:23682679

  6. Metagenomics, metatranscriptomics and single cell genomics reveal functional response of active Oceanospirillales to Gulf oil spill

    SciTech Connect

    Mason, Olivia U.; Hazen, Terry C.; Borglin, Sharon; Chain, Patrick S. G.; Dubinsky, Eric A.; Fortney, Julian L.; Han, James; Holman, Hoi-Ying N.; Hultman, Jenni; Lamendella, Regina; Mackelprang, Rachel; Malfatti, Stephanie; Tom, Lauren M.; Tringe, Susannah G.; Woyke, Tanja; Zhou, Jizhong; Rubin, Edward M.; Jansson, Janet K.

    2012-06-12

    The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

  7. Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring

    PubMed Central

    Kvastad, L.; Werne Solnestam, B.; Johansson, E.; Nygren, A. O.; Laddach, N.; Sahlén, P.; Vickovic, S.; Bendigtsen, Schirmer C.; Aaserud, M.; Floer, L.; Borgen, E.; Schwind, C.; Himmelreich, R.; Latta, D.; Lundeberg, J.

    2015-01-01

    Single cell analysis techniques have great potential in the cancer genomics field. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics. PMID:26558529

  8. Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring.

    PubMed

    Kvastad, L; Werne Solnestam, B; Johansson, E; Nygren, A O; Laddach, N; Sahlén, P; Vickovic, S; Bendigtsen, Schirmer C; Aaserud, M; Floer, L; Borgen, E; Schwind, C; Himmelreich, R; Latta, D; Lundeberg, J

    2015-01-01

    Single cell analysis techniques have great potential in the cancer genomics field. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics. PMID:26558529

  9. Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells.

    PubMed

    Luo, Yuping; Coskun, Volkan; Liang, Aibing; Yu, Juehua; Cheng, Liming; Ge, Weihong; Shi, Zhanping; Zhang, Kunshan; Li, Chun; Cui, Yaru; Lin, Haijun; Luo, Dandan; Wang, Junbang; Lin, Connie; Dai, Zachary; Zhu, Hongwen; Zhang, Jun; Liu, Jie; Liu, Hailiang; deVellis, Jean; Horvath, Steve; Sun, Yi Eve; Li, Siguang

    2015-05-21

    The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation. PMID:26000486

  10. Single-cell vs. bulk activity properties of coastal bacterioplankton over an annual cycle in a temperate ecosystem.

    PubMed

    Morán, Xosé Anxelu G; Calvo-Díaz, Alejandra

    2009-01-01

    The connections between single-cell activity properties of heterotrophic planktonic bacteria and whole community metabolism are still poorly understood. Here, we show flow cytometry single-cell analysis of membrane-intact (live), high nucleic acid (HNA) content and actively respiring (CTC+) bacteria with samples collected monthly during 2006 in northern Spain coastal waters. Bulk activity was assessed by measuring 3H-Leucine incorporation and specific growth rates. Consistently, different single-cell relative abundances were found, with 60-100% for live, 30-84% for HNA and 0.2-12% for CTC+ cells. Leucine incorporation rates (2-153 pmol L(-1) h(-1)), specific growth rates (0.01-0.29 day(-1)) and the total and relative abundances of the three single-cell groups showed marked seasonal patterns. Distinct depth distributions during summer stratification and different relations with temperature, chlorophyll and bacterial biovolume suggest the existence of different controlling factors on each single-cell property. Pooled leucine incorporation rates were similarly correlated with the abundance of all physiological groups, while specific growth rates were only substantially explained by the percentage of CTC+ cells. However, the ability to reduce CTC proved notably better than the other two single-cell properties at predicting bacterial bulk rates within seasons, suggesting a tight linkage between bacterial individual respiration and biomass production at the community level. PMID:19120458

  11. Single-cell activity of freshwater aerobic anoxygenic phototrophic bacteria and their contribution to biomass production.

    PubMed

    Garcia-Chaves, Maria C; Cottrell, Matthew T; Kirchman, David L; Ruiz-González, Clara; Del Giorgio, Paul A

    2016-07-01

    Aerobic anoxygenic phototrophic (AAP) bacteria are photoheterotrophs that despite their low abundances have been hypothesized to play an ecologically and biogeochemically important role in aquatic systems. Characterizing this role requires a better understanding of the in situ dynamics and activity of AAP bacteria. Here we provide the first assessment of the single-cell activity of freshwater AAP bacteria and their contribution to total bacterial production across lakes spanning a wide trophic gradient, and explore the role of light in regulating AAP activity. The proportion of cells that were active in leucine incorporation and the level of activity per cell were consistently higher for AAP than for bulk bacteria across lakes. As a result, AAP bacteria contributed disproportionately more to total bacterial production than to total bacterial abundance. Interestingly, although environmentally driven patterns in activity did not seem to differ largely between AAP and bulk bacteria, their response to light did, and exposure to light resulted in increases in the proportion of active AAP bacteria with no clear effect on their cell-specific activity. This suggests that light may play a role in the activation of AAP bacteria, enabling these photoheterotrophs to contribute more to the carbon cycle than suggested by their abundance. PMID:26771928

  12. Single-cell western blotting

    PubMed Central

    Hughes, Alex J.; Spelke, Dawn P.; Xu, Zhuchen; Kang, Chi-Chih; Schaffer, David V.; Herr, Amy E.

    2014-01-01

    To measure cell-to-cell variation in protein-mediated functions — a hallmark of biological processes — we developed an approach to conduct ~103 concurrent single-cell western blots (scWesterns) in ~4 hours. A microscope slide supporting a 30 µm-thick photoactive polyacrylamide gel enables western blotting comprised of: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins, and antibody probing. We apply this scWestern to monitor single rat neural stem cell differentiation and responses to mitogen stimulation. The scWestern quantifies target proteins even with off-target antibody binding, multiplexes to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supports analyses of low starting cell numbers (~200) when integrated with fluorescence activated cell sorting. The scWestern thus overcomes limitations in single-cell protein analysis (i.e., antibody fidelity, sensitivity, and starting cell number) and constitutes a versatile tool for the study of complex cell populations at single-cell resolution. PMID:24880876

  13. Nanocoating of single cells: from maintenance of cell viability to manipulation of cellular activities.

    PubMed

    Park, Ji Hun; Yang, Sung Ho; Lee, Juno; Ko, Eun Hyea; Hong, Daewha; Choi, Insung S

    2014-04-01

    The chronological progresses in single-cell nanocoating are described. The historical developments in the field are divided into biotemplating, cytocompatible nanocoating, and cells in nano-nutshells, depending on the main research focuses. Each subfield is discussed in conjunction with the others, regarding how and why to manipulate living cells by nanocoating at the single-cell level. PMID:24452932

  14. Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging.

    PubMed

    Pliss, Artem; Peng, Xiao; Liu, Lixin; Kuzmin, Andrey; Wang, Yan; Qu, Junle; Li, Yuee; Prasad, Paras N

    2015-01-01

    Molecular organization of a cell is dynamically transformed along the course of cellular physiological processes, pathologic developments or derived from interactions with drugs. The capability to measure and monitor concentrations of macromolecules in a single cell would greatly enhance studies of cellular processes in heterogeneous populations. In this communication, we introduce and experimentally validate a bio-analytical single-cell assay, wherein the overall concentration of macromolecules is estimated in specific subcellular domains, such as structure-function compartments of the cell nucleus as well as in nucleoplasm. We describe quantitative mapping of local biomolecular concentrations, either intrinsic relating to the functional and physiological state of a cell, or altered by a therapeutic drug action, using two-photon excited fluorescence lifetime imaging (FLIM). The proposed assay utilizes a correlation between the fluorescence lifetime of fluorophore and the refractive index of its microenvironment varying due to changes in the concentrations of macromolecules, mainly proteins. Two-photon excitation in Near-Infra Red biological transparency window reduced the photo-toxicity in live cells, as compared with a conventional single-photon approach. Using this new assay, we estimated average concentrations of proteins in the compartments of nuclear speckles and in the nucleoplasm at ~150 mg/ml, and in the nucleolus at ~284 mg/ml. Furthermore, we show a profound influence of pharmaceutical inhibitors of RNA synthesis on intracellular protein density. The approach proposed here will significantly advance theranostics, and studies of drug-cell interactions at the single-cell level, aiding development of personal molecular medicine. PMID:26155309

  15. Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging

    PubMed Central

    Pliss, Artem; Peng, Xiao; Liu, Lixin; Kuzmin, Andrey; Wang, Yan; Qu, Junle; Li, Yuee; Prasad, Paras N

    2015-01-01

    Molecular organization of a cell is dynamically transformed along the course of cellular physiological processes, pathologic developments or derived from interactions with drugs. The capability to measure and monitor concentrations of macromolecules in a single cell would greatly enhance studies of cellular processes in heterogeneous populations. In this communication, we introduce and experimentally validate a bio-analytical single-cell assay, wherein the overall concentration of macromolecules is estimated in specific subcellular domains, such as structure-function compartments of the cell nucleus as well as in nucleoplasm. We describe quantitative mapping of local biomolecular concentrations, either intrinsic relating to the functional and physiological state of a cell, or altered by a therapeutic drug action, using two-photon excited fluorescence lifetime imaging (FLIM). The proposed assay utilizes a correlation between the fluorescence lifetime of fluorophore and the refractive index of its microenvironment varying due to changes in the concentrations of macromolecules, mainly proteins. Two-photon excitation in Near-Infra Red biological transparency window reduced the photo-toxicity in live cells, as compared with a conventional single-photon approach. Using this new assay, we estimated average concentrations of proteins in the compartments of nuclear speckles and in the nucleoplasm at ~150 mg/ml, and in the nucleolus at ~284 mg/ml. Furthermore, we show a profound influence of pharmaceutical inhibitors of RNA synthesis on intracellular protein density. The approach proposed here will significantly advance theranostics, and studies of drug-cell interactions at the single-cell level, aiding development of personal molecular medicine. PMID:26155309

  16. Measuring activity in the ubiquitin-proteasome system: From large scale discoveries to single cells analysis

    PubMed Central

    Melvin, Adam T.; Woss, Gregery S.; Park, Jessica H.; Waters, Marcey L.; Allbritton, Nancy L.

    2013-01-01

    The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS have provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington’s disease. These reporters, usually consisting of a recognition sequences fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes (DUBs). This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, recent work is presented highlighting the development of novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples. PMID:23686610

  17. Capturing Single Cell Genomes of Active Polysaccharide Degraders: An Unexpected Contribution of Verrucomicrobia

    PubMed Central

    Martinez-Garcia, Manuel; Brazel, David M.; Swan, Brandon K.; Arnosti, Carol; Chain, Patrick S. G.; Reitenga, Krista G.; Xie, Gary; Poulton, Nicole J.; Gomez, Monica Lluesma; Masland, Dashiell E. D.; Thompson, Brian; Bellows, Wendy K.; Ziervogel, Kai; Lo, Chien-Chi; Ahmed, Sanaa; Gleasner, Cheryl D.; Detter, Chris J.; Stepanauskas, Ramunas

    2012-01-01

    Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Genomic sequencing of five cells, representing the most predominant, polysaccharide-active Verrucomicrobia phylotype, revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases and esterases, confirming that these organisms were well equipped for the hydrolysis of diverse polysaccharides. Remarkably, this enrichment was on average higher than in the sequenced representatives of Bacteroidetes, which are frequently regarded as highly efficient biopolymer degraders. These findings shed light on the ecological roles of uncultured Verrucomicrobia and suggest specific taxa as promising bioprospecting targets. The employed method offers a powerful tool to rapidly identify and recover discrete genomes of active players in polysaccharide degradation, without the need for cultivation. PMID:22536372

  18. Automated platform for multiparameter stimulus response studies of metabolic activity at the single-cell level

    NASA Astrophysics Data System (ADS)

    Ashili, Shashanka P.; Kelbauskas, Laimonas; Houkal, Jeff; Smith, Dean; Tian, Yanqing; Youngbull, Cody; Zhu, Haixin; Anis, Yasser H.; Hupp, Michael; Lee, Kristen B.; Kumar, Ashok V.; Vela, Juan; Shabilla, Andrew; Johnson, Roger H.; Holl, Mark R.; Meldrum, Deirdre R.

    2011-02-01

    We have developed a fully automated platform for multiparameter characterization of physiological response of individual and small numbers of interacting cells. The platform allows for minimally invasive monitoring of cell phenotypes while administering a variety of physiological insults and stimuli by means of precisely controlled microfluidic subsystems. It features the capability to integrate a variety of sensitive intra- and extra-cellular fluorescent probes for monitoring minute intra- and extra-cellular physiological changes. The platform allows for performance of other, post- measurement analyses of individual cells such as transcriptomics. Our method is based on the measurement of extracellular metabolite concentrations in hermetically sealed ~200-pL microchambers, each containing a single cell or a small number of cells. The major components of the system are a) a confocal laser scan head to excite and detect with single photon sensitivity the emitted photons from sensors; b) a microfluidic cassette to confine and incubate individual cells, providing for dynamic application of external stimuli, and c) an integration module consisting of software and hardware for automated cassette manipulation, environmental control and data collection. The custom-built confocal scan head allows for fluorescence intensity detection with high sensitivity and spatial confinement of the excitation light to individual pixels of the sensor area, thus minimizing any phototoxic effects. The platform is designed to permit incorporation of multiple optical sensors for simultaneous detection of various metabolites of interest. The modular detector structure allows for several imaging modalities, including high resolution intracellular probe imaging and extracellular sensor readout. The integrated system allows for simulation of physiologically relevant microenvironmental stimuli and simultaneous measurement of the elicited phenotypes. We present details of system design, system

  19. Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

    PubMed Central

    Ranganathan, Gayathri Nattar; Koester, Helmut J.

    2012-01-01

    Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (>97% spike detection efficiency), has a low rate of false positive spike detection (< 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4. PMID:22972033

  20. Complement activation and cytokine response by BioProtein, a bacterial single cell protein.

    PubMed

    Sikkeland, L I B; Thorgersen, E B; Haug, T; Mollnes, T E

    2007-04-01

    The bacterial single cell protein (BSCP), BioProtein, is dried bacterial mass derived from fermentation of the gram negative bacteria Methylococcus capsulatus, used for animal and fish feed. Workers in this industry suffer frequently from pulmonary and systemic symptoms which may be induced by an inflammatory reaction. The aim of the present study was to examine the effect of BSCP on inflammation in vitro as evaluated by complement activation and cytokine production. Human serum was incubated with BSCP and complement activation products specific for all pathways were detected by enzyme-linked immunosorbent assay (ELISA). Human whole blood anti-coagulated with lepirudin was incubated with BSCP and a panel of 27 biological mediators was measured using multiplex technology. BSCP induced a dose-dependent complement activation as revealed by a pronounced increase in alternative and terminal pathway activation (fivefold and 20-fold, respectively) at doses from 1 microg BSCP/ml serum and a similar, but less extensive (two- to fourfold) increase in activation of the lectin and classical pathways at doses from 100 and 1000 microg BSCP/ml serum, respectively. Similarly, BSCP induced a dose-dependent production of a number of cytokines, chemokines and growth factors in human whole blood. At doses as low as 0 x 05-0 x 5 microg BSCP/ml blood a substantial increase was seen for tumour necrosis factor (TNF)-alpha, interleukin (IL)-1-beta, IL-6, interferon (IFN)-gamma, IL-8, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, IL-4, IL-9, IL-17, IL-1Ra, granulocyte-colony-stimulating factor (G-CSF) and vascular endothelial growth factor (VEGF). Thus, BSCP induced a substantial activation of all three initial complement pathways as well as a pronounced cytokine response in vitro, indicating a potent inflammatory property of this agent. PMID:17302729

  1. Single-cell protein production from spent sulfite liquor utilizing cell-recycle and computer monitoring

    SciTech Connect

    Gold, D.; Mohagheghi, A.; Cooney, C.L.; Wang, D.I.C.

    1981-01-01

    To reduce the BOD of spent sulfite liquor before disposal, torula yeast (Candida utilis) is produced by a continuous culture process, the productivity of which is limited by sugar concentration and cell growth rate. To increase productivity, a recycle system has been designed and tested. Cells were sedimented continuously with a flocculating agent (bentonite) before being recycled to the fermentor. A bentonite concentration of 0.02 g/g cell was required. A computer monitoring system based on material balancing techniques was developed to monitor and control the recycle system. With this computer system, productivity was raised to 6.1 g/L-h, with cell concentrations of less than or equal to 65 g/L in the recycle stream and 24 g/L in the fermentor. This represents a productivity increase of 150% over continuous culture with no recycle.

  2. Single-cell telomere-length quantification couples telomere length to meristem activity and stem cell development in Arabidopsis.

    PubMed

    González-García, Mary-Paz; Pavelescu, Irina; Canela, Andrés; Sevillano, Xavier; Leehy, Katherine A; Nelson, Andrew D L; Ibañes, Marta; Shippen, Dorothy E; Blasco, Maria A; Caño-Delgado, Ana I

    2015-05-12

    Telomeres are specialized nucleoprotein caps that protect chromosome ends assuring cell division. Single-cell telomere quantification in animals established a critical role for telomerase in stem cells, yet, in plants, telomere-length quantification has been reported only at the organ level. Here, a quantitative analysis of telomere length of single cells in Arabidopsis root apex uncovered a heterogeneous telomere-length distribution of different cell lineages showing the longest telomeres at the stem cells. The defects in meristem and stem cell renewal observed in tert mutants demonstrate that telomere lengthening by TERT sets a replicative limit in the root meristem. Conversely, the long telomeres of the columella cells and the premature stem cell differentiation plt1,2 mutants suggest that differentiation can prevent telomere erosion. Overall, our results indicate that telomere dynamics are coupled to meristem activity and continuous growth, disclosing a critical association between telomere length, stem cell function, and the extended lifespan of plants. PMID:25937286

  3. Single-cell codetection of metabolic activity, intracellular functional proteins, and genetic mutations from rare circulating tumor cells.

    PubMed

    Zhang, Yu; Tang, Yin; Sun, Shuai; Wang, Zhihua; Wu, Wenjun; Zhao, Xiaodong; Czajkowsky, Daniel M; Li, Yan; Tian, Jianhui; Xu, Ling; Wei, Wei; Deng, Yuliang; Shi, Qihui

    2015-10-01

    The high glucose uptake and activation of oncogenic signaling pathways in cancer cells has long made these features, together with the mutational spectrum, prime diagnostic targets of circulating tumor cells (CTCs). Further, an ability to characterize these properties at a single cell resolution is widely believed to be essential, as the known extensive heterogeneity in CTCs can obscure important correlations in data obtained from cell population-based methods. However, to date, it has not been possible to quantitatively measure metabolic, proteomic, and genetic data from a single CTC. Here we report a microchip-based approach that allows for the codetection of glucose uptake, intracellular functional proteins, and genetic mutations at the single-cell level from rare tumor cells. The microchip contains thousands of nanoliter grooves (nanowells) that isolate individual CTCs and allow for the assessment of their glucose uptake via imaging of a fluorescent glucose analog, quantification of a panel of intracellular signaling proteins using a miniaturized antibody barcode microarray, and retrieval of the individual cell nuclei for subsequent off-chip genome amplification and sequencing. This approach integrates molecular-scale information on the metabolic, proteomic, and genetic status of single cells and permits the inference of associations between genetic signatures, energy consumption, and phosphoproteins oncogenic signaling activities in CTCs isolated from blood samples of patients. Importantly, this microchip chip-based approach achieves this multidimensional molecular analysis with minimal cell loss (<20%), which is the bottleneck of the rare cell analysis. PMID:26378744

  4. Single-Cell Analysis of Phosphoinositide 3-Kinase (PI3K) and Phosphatase and Tensin Homolog (PTEN) Activation

    PubMed Central

    Jiang, Dechen; Sims, Christopher Eldridge; Allbritton, Nancy Lynn

    2010-01-01

    Summary A single-cell assay was developed to measure the activation of phosphoinositide 3-kinase (PI3K) using microanalytical chemical separations and a fluorescently labeled lipid substrate. Phosphatidyl-inositol 4,5 bisphosphate labeled on its acyl chain with Bodipy fluorescein (Bodipy Fl PIP2) was utilized as a substrate for both in vitro and cell-based assays. Detection limits for the substrate and product of the PI3K reaction were 10 to 20 zeptomoles. In vitro assays with PI3K with and without pharmacologic inhibitors demonstrated that Bodipy Fl PIP2 was converted to phosphatidyl-inositol 3,4,5 trisphosphate (Bodipy Fl PIP3 ). Bodipy Fl PIP3 could be back converted to Bodipy Fl PIP2 by the phosphatase PTEN. When Bodipy Fl PIP2 was added to a cell lysate, 1.4 fmoles of the Bodipy Fl PIP3 were produced per ng of protein in the cytoplasmic extract in 10 min. Addition of Bodipy Fl PIP3 to a cell lysate yielded 3 fmoles of Bodipy Fl PIP2 per ng of protein in 8 min. Both Bodipy Fl PIP2 and Bodipy Fl PIP3 were measureable in single cells and the two species could be inter-converted. Under the appropriate conditions, a fluorescent diacylglycerol was also detected in single cells. When the FcεR1 receptor on the cells loaded with the fluorescent lipid was cross-linked, the amount of Bodipy Fl PIP3 generated per cell increased 4-fold over that of unstimulated cells. This production of Bodipy Fl PIP3 was blocked by wortmannin. Chemical cytometry utilizing the fluorescent lipids will be of value in understanding lipid metabolism at the single-cell level. PMID:21221426

  5. Monitoring the permeabilization of a single cell in a microfluidic device, through the estimation of its dielectric properties based on combined dielectrophoresis and electrorotation in situ experiments.

    PubMed

    Trainito, Claudia Irene; Français, Olivier; Le Pioufle, Bruno

    2015-05-01

    The electric field is commonly used in microdevices to handle, treat, or monitor living cells for various biological or biomedical applications (cells electrofusion, gene electrotransfer, drugs injection, cell sorting, …). Dielectrophoresis (DEP) forces, using stationary waves (conventional DEP) or traveling waves, are widely used for the cell handling or sorting. Electrorotation, which is induced by a rotating electrical field, is used for the determination of cell dielectric parameters. The application of pulsed electric field (PEF) results in the cell membrane permeabilization that might allow the transfer of various molecules in the cytoplasm. In this paper, we propose a method to monitor in situ the level of electropermeabilization induced by PEF application on a single cell, by combining the dielectrophoresis force and the electrorotation torque within a microfluidic device. The method was experimented on two different cell lines (human leukemic T-cell lymphoblast and murine melanoma cell): a single cell is captured by dielectrophoresis while its dielectric properties (both permittivity and conductivity of cytoplasm and membrane) are estimated thanks to a rotating electric field, which is applied simultaneously. The permeabilization effect of PEF, applied to the single cell trapped in such conditions in the biodevice, could be monitored by the estimation of its dielectric properties before and after pulse application. PMID:25641658

  6. Single-cell microinjection assay indicates that 7-hydroxycoumarin induces rapid activation of caspase-3 in A549 cancer cells

    PubMed Central

    SOTO-NUÑEZ, MARIBEL; DÍAZ-MORALES, KAREN AZUCENA; CUAUTLE-RODRÍGUEZ, PATRICIA; TORRES-FLORES, VÍCTOR; LÓPEZ-GONZÁLEZ, JOSÉ SULLIVAN; MANDOKI-WEITZNER, JUAN JOSÉ; MOLINA-GUARNEROS, JUAN ARCADIO

    2015-01-01

    Coumarins have attracted intense interest in recent years due to their apoptogenic effects. The aim of the present study was to determine whether 7-hydroxycoumarin (7-HC) induces changes in caspase-3 (C-3) activity in A549 human lung carcinoma cells. A range of analytical techniques, including colorimetric and fluorometric assays, western blotting, single-cell microinjection, fluorescence microscopy and image analysis were conducted to elucidate the effects of 7-HC. A 24-h exposure to 1.85 mM 7-HC induced a 65% increase in C-3 activity, and a notable conversion of procaspase-3 to C-3, in addition to poly(ADP-ribose)polymerase cleavage. Furthermore, morphological changes associated with apoptosis were observed. Exposure of the cells to 7-HC for 3 or 6 h increased calcium conductance by 27%. By performing the single-cell microinjection of a specific fluorescent substrate of C-3 into previously 7-HC-exposed cells, a typical enzymatic kinetic profile of C-3 activation was identified a number of hours prior to the morphological and biochemical changes associated with apoptosis being observed. These results suggest that the rapid in vivo activation of C-3 is induced by 7-HC, the most relevant biotransformation product of coumarin in humans. PMID:26640551

  7. An automated programmable platform enabling multiplex dynamic stimuli delivery and cellular response monitoring for high-throughput suspension single-cell signaling studies.

    PubMed

    He, Luye; Kniss, Ariel; San-Miguel, Adriana; Rouse, Tel; Kemp, Melissa L; Lu, Hang

    2015-03-21

    Cell signaling events are orchestrated by dynamic external biochemical cues. By rapidly perturbing cells with dynamic inputs and examining the output from these systems, one could study the structure and dynamic properties of a cellular signaling network. Conventional experimental techniques limit the implementation of these systematic approaches due to the lack of sophistication in manipulating individual cells and the fluid microenvironment around them; existing microfluidic technologies thus far are mainly targeting adherent cells. In this paper we present an automated platform to interrogate suspension cells with dynamic stimuli while simultaneously monitoring cellular responses in a high-throughput manner at single-cell resolution. We demonstrate the use of this platform in an experiment to measure Jurkat T cells in response to distinct dynamic patterns of stimuli; we find cells exhibit highly heterogeneous responses under each stimulation condition. More interestingly, these cells act as low-pass filters, only entrained to the low frequency stimulus signals. We also demonstrate that this platform can be easily programmed to actively generate arbitrary dynamic signals. We envision our platform to be useful in other contexts to study cellular signaling dynamics, which may be difficult using conventional experimental methods. PMID:25609410

  8. Measurement of enzyme activity in single cells by voltammetry using a microcell with a positionable dual electrode.

    PubMed

    Gao, Ning; Zhao, Minghui; Zhang, Xiaoli; Jin, Wenrui

    2006-01-01

    The electrochemical single-cell analysis for enzyme activity was developed using microcells on a microcell array coupled with a positionable dual microelectrode. The microcell array with the nanoliter-scale microcells was constructed using simple chemical etching without photolithographic techniques. The positionable dual microelectrodes consisted of the nanometer-to-micrometer-radius Au disk working electrode and a approximately 80-microm-radius Ag/AgCl reference electrode. Peroxidase was chosen as the model enzyme. Factors that concern electrochemical single-cell analysis in microcells such as solution evaporation, interference of soluble oxygen, electrode size, solution volume, and electrode fouling were investigated and discussed. The 20 or 100 nL of detection volume was found to be suitable for peroxidase determination in single neutrophils or single acute promyelocytic leukemia cells without interference from intracellular macromolecules and electrode fouling, when the dual electrode with a 10-microm-radius Au disk working electrode was used. Cells were perforated with digitonin before transferring them into the microcells, to lyse cells easily. The perforated cells were transferred into the microcells by pushing a microscope slide on a drop of the cell suspension on the microcell array. After a single cell in the microcell was lysed using a freeze-thawing technique and allowed to dry, physiological buffer saline containing 2.0 x 10(-3) mol/L hydroquinone and 2.0 x 10(-3) mol/L H2O2 as the substrates of the enzyme-catalyzed reaction was added. The microcell array was positioned in a constant-humidity chamber to prevent evaporation. Then the dual electrode was inserted into the microcell by means of a scanning electrochemical microscope and the product benzoquinone of the enzyme-catalyzed reaction was voltammetrically detected. Peroxidase activity could be quantified using the steady-state current on the voltammogram after subtracting the blank and using the

  9. A critical review of NanoSIMS in analysis of microbial metabolic activities at single-cell level.

    PubMed

    Gao, Dawen; Huang, Xiaoli; Tao, Yu

    2016-10-01

    Over 3.8 billion years of evolution has enabled many microbial species a versatile metabolism. However, limited by experimental methods, some unique metabolism remains unknown or unclear. A major obstacle is to attribute the incorporation of certain nutrients into a noncultivable species out of a complex microbial community. Such difficulty could be solved if we are able to directly observe substrate uptake at the single-cell level. Nanoscale secondary ion mass spectrometry (NanoSIMS) is a powerful tool for revealing element distribution in nanometer-scale resolution in the fields such as material sciences, geosciences and astronomy. In this review, we focus on another applicability of NanoSIMS in microbiology. In such fields, physiological properties and metabolic activities of microorganisms can be revealed with a single-cell scale resolution by NanoSIMS solely or in combination with other techniques. This review will highlight the features of NanoSIMS in analyzing the metabolic activities of carbon, nitrogen, metal irons by mixed-culture assemblies. Some values of NanoSIMS in environmental microbiology are expected to be discussed via this review. PMID:26177334

  10. Combined autoradiography and immunofluorescence for estimation of single cell activity by ammonium-oxidizing bacteria

    SciTech Connect

    Ward, B.B.

    1984-03-01

    Immunofluorescence and /sup 14/CO/sub 2/ autoradiography were used for simultaneously enumerating and assaying the autotrophic activity of ammonium-oxidizing bacteria in seawater. Relative activity (/sup 14/CO/sub 2/ assimilation as measured by autoradiography) and abundance were measured in simulated in situ incubations at seven stations in the primary NO/sub 2//sup -/ maximum region of the Northeast Pacific Ocean. More than 10/sup 4/ cells-liter/sup -1/ were present; relative activity often showed a peak near the surface and an increase in the NO/sub 2//sup -/ max region below the photic zone. The method permits assessment of individual cell activity; most cells at all depths were active in CO/sub 2/ assimilation, usually at low and quite variable levels. Relative activity was positively correlated with the abundance of ammonium-oxidizing bacteria, temperature, total dark CO/sub 2/ assimilation and phenopigment concentration.

  11. Spontaneous Neuronal Activity in Developing Neocortical Networks: From Single Cells to Large-Scale Interactions

    PubMed Central

    Luhmann, Heiko J.; Sinning, Anne; Yang, Jenq-Wei; Reyes-Puerta, Vicente; Stüttgen, Maik C.; Kirischuk, Sergei; Kilb, Werner

    2016-01-01

    Neuronal activity has been shown to be essential for the proper formation of neuronal circuits, affecting developmental processes like neurogenesis, migration, programmed cell death, cellular differentiation, formation of local and long-range axonal connections, synaptic plasticity or myelination. Accordingly, neocortical areas reveal distinct spontaneous and sensory-driven neuronal activity patterns already at early phases of development. At embryonic stages, when immature neurons start to develop voltage-dependent channels, spontaneous activity is highly synchronized within small neuronal networks and governed by electrical synaptic transmission. Subsequently, spontaneous activity patterns become more complex, involve larger networks and propagate over several neocortical areas. The developmental shift from local to large-scale network activity is accompanied by a gradual shift from electrical to chemical synaptic transmission with an initial excitatory action of chloride-gated channels activated by GABA, glycine and taurine. Transient neuronal populations in the subplate (SP) support temporary circuits that play an important role in tuning early neocortical activity and the formation of mature neuronal networks. Thus, early spontaneous activity patterns control the formation of developing networks in sensory cortices, and disturbances of these activity patterns may lead to long-lasting neuronal deficits. PMID:27252626

  12. Spontaneous Neuronal Activity in Developing Neocortical Networks: From Single Cells to Large-Scale Interactions.

    PubMed

    Luhmann, Heiko J; Sinning, Anne; Yang, Jenq-Wei; Reyes-Puerta, Vicente; Stüttgen, Maik C; Kirischuk, Sergei; Kilb, Werner

    2016-01-01

    Neuronal activity has been shown to be essential for the proper formation of neuronal circuits, affecting developmental processes like neurogenesis, migration, programmed cell death, cellular differentiation, formation of local and long-range axonal connections, synaptic plasticity or myelination. Accordingly, neocortical areas reveal distinct spontaneous and sensory-driven neuronal activity patterns already at early phases of development. At embryonic stages, when immature neurons start to develop voltage-dependent channels, spontaneous activity is highly synchronized within small neuronal networks and governed by electrical synaptic transmission. Subsequently, spontaneous activity patterns become more complex, involve larger networks and propagate over several neocortical areas. The developmental shift from local to large-scale network activity is accompanied by a gradual shift from electrical to chemical synaptic transmission with an initial excitatory action of chloride-gated channels activated by GABA, glycine and taurine. Transient neuronal populations in the subplate (SP) support temporary circuits that play an important role in tuning early neocortical activity and the formation of mature neuronal networks. Thus, early spontaneous activity patterns control the formation of developing networks in sensory cortices, and disturbances of these activity patterns may lead to long-lasting neuronal deficits. PMID:27252626

  13. Combined autoradiography and immunofluorescence for estimation of single cell activity by ammonium-oxidizing bacteria

    SciTech Connect

    Ward, B.B.

    1984-03-01

    Immunofluorescence and /sup 14/CO/sub 2/ autoradiography were used for simultaneously enumerating and assaying the autotrophic activity of ammonium-oxidizing bacteria in seawater. Relative activity (/sup 14/CO/sub 2/ assimilation as measured by autoradiography) and abundance were measured in simulated in situ incubations at seven stations in the primary NO/sub 2//sup -/ maximum region of the Northeast Pacific Ocean. More than 10/sup 4/ cells liter/sup -1/ were present; relative activity often showed a peak near the surface and an increase in the NO/sub 2//sup -/ max region below the photic zone. The method permits assessment of individual cell activity; most cells at all depths were active in CO/sub 2/ assimilation, usually at low and quite variable levels. There were no differences in relative activity between samples incubated under simulated in situ conditions and in the dark. Relative activity was positively correlated with the abundance of ammonium-oxidizing bacteria, temperature, total dark CO/sub 2/ assimilation (as measured by liquid scintillation counting of replicate samples), and pheopigment concentration, and negatively correlated with oxygen concentration.

  14. Spatiotemporal activity patterns detected from single cell measurements from behaving animals

    NASA Astrophysics Data System (ADS)

    Villa, Alessandro E. P.; Tetko, Igor V.

    1999-03-01

    Precise temporal patterning of activity within and between neurons has been predicted on theoretical grounds, and found in the spike trains of neurons recorded from anesthetized and conscious animals, in association with sensor stimuli and particular phases of task performance. However, the functional significance of such patterning in the generation of behavior has not been confirmed. We recorded from multiple single neurons in regions of rat auditory cortex during the waiting period of a Go/NoGo task. During this time the animal waited for an auditory signal with high cognitive load. Of note is the fact that neural activity during the period analyzed was essentially stationary, with no event related variability in firing. Detected patterns therefore provide a measure of brain state that could not be addressed by standard methods relying on analysis of changes in mean discharge rate. The possibility is discussed that some patterns might reflect a preset bias to a particular response, formed in the waiting period. Others patterns might reflect a state of prior preparation of appropriate neural assemblies for analyzing a signal that is expected but of unknown behavioral valence.

  15. Investigating Microbial Activity in Diazotrophic Methane Seep Sediment via Transcript Analysis and Single-Cell FISH-NanoSIMS

    NASA Astrophysics Data System (ADS)

    Dekas, A. E.; Connon, S. A.; Chadwick, G.; Orphan, V. J.

    2012-12-01

    Methane seep microbial ecosystems are phylogenetically diverse and physiologically complex, and require culture-independent techniques to accurately investigate metabolic activity. In the present study we combine an RNA analysis of four key microbial genes with FISH-NanoSIMS analysis of single cells to determine the diversity of nitrogen fixing microorganisms (diazotrophs) present at a deep-sea methane-seeping site, as well as investigate the methane-dependency of a variety of community members. Recently, methane-dependent nitrogen fixation was observed in Mound 12 Costa Rica sediments, and was spatially correlated with the abundance of aggregates of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacterial symbionts (SRB). Combined with the detection of 15N uptake from 15N2 in these aggregates, this suggested that the ANME-SRB aggregates are the primary diazotrophs in seep sediment. However, the diversity of dinitrogenase reductase (nifH) sequences recovered from several deep-sea locales, including Mound 12, suggests a greater diversity of diazotrophs in marine sediment. To investigate the activity of these potential diazotrophs in Mound 12 sediment, we investigated a suite of RNA transcripts in 15N2 incubations in both the presence and absence of methane: nifH, bacterial 16S rRNA, methyl coenzyme M reductase A (mcrA), and adenosine-5'-phosposulfate reductase alpha subunit (aprA). No nifH transcripts were recovered in incubations without methane, consistent with previous measurements lacking 15N2 uptake in the same sediments. The activity of the bacterial community in general, assessed by variable transcription, was also greatly affected by the presence or absence of methane. Single-cell fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS) was employed to confirm diazotrophic activity (15N2 uptake) and protein synthesis (15NH4+ uptake) of particular species implicated as ecologically important by the

  16. Monitoring human leukocyte antigen class I molecules by micro-Raman spectroscopy at single-cell level

    NASA Astrophysics Data System (ADS)

    Das, Gobind; La Rocca, Rosanna; Lakshmikanth, Tadepally; Gentile, Francesco; Tallerico, Rossana; Zambetti, Lia P.; Devitt, J.; Candeloro, Patrizio; de Angelis, Francesco; Carbone, Ennio; di Fabrizio, Enzo

    2010-03-01

    Human leukocyte antigen (HLA) class I molecules are formed by three immunoglobulin-like domains (α1, α2, and α3) once folded by peptide and β2-microglobulin show the presence of two α-helix streams and one β-sheet limiting the pocket for the antigenic peptide. The loss of HLA class I expression in tumors and virus-infected cells, on one hand, prevents T cell recognition, while on the other hand, it leads to natural killer (NK) cell mediated cytotoxicity. We propose the possibility of using Raman spectroscopy to measure the relative expression of HLA class I molecules at the single-cell level. Raman spectra are recorded for three cell lines (K562, T2, and T3) and monomers (HLA class I folded, unfolded and peptide+β2-microlobulin refolded) using 830 nm laser line. Our data are consistent with the hypothesis that in the Raman spectra, ranging from 1600 to 1800 cm-1, the intensity variation of cells associated with HLA class I molecules could be measured.

  17. Single cell wound repair

    PubMed Central

    Abreu-Blanco, Maria Teresa; Verboon, Jeffrey M

    2011-01-01

    Cell wounding is a common event in the life of many cell types, and the capacity of the cell to repair day-to-day wear-and-tear injuries, as well as traumatic ones, is fundamental for maintaining tissue integrity. Cell wounding is most frequent in tissues exposed to high levels of stress. Survival of such plasma membrane disruptions requires rapid resealing to prevent the loss of cytosolic components, to block Ca2+ influx and to avoid cell death. In addition to patching the torn membrane, plasma membrane and cortical cytoskeleton remodeling are required to restore cell function. Although a general understanding of the cell wound repair process is in place, the underlying mechanisms of each step of this response are not yet known. We have developed a model to study single cell wound repair using the early Drosophila embryo. Our system combines genetics and live imaging tools, allowing us to dissect in vivo the dynamics of the single cell wound response. We have shown that cell wound repair in Drosophila requires the coordinated activities of plasma membrane and cytoskeleton components. Furthermore, we identified an unexpected role for E-cadherin as a link between the contractile actomyosin ring and the newly formed plasma membrane plug. PMID:21922041

  18. Direct methods for dynamic monitoring of secretions from single cells by capillary electrophoresis and microscopy with laser-induced native fluorescence detection

    SciTech Connect

    Tong, W.

    1997-10-08

    Microscale separation and detection methods for real-time monitoring of dynamic cellular processes (e.g., secretion) by capillary electrophoresis (CE) and microscopic imaging were developed. Ultraviolet laser-induced native fluorescence (LINF) provides simple, sensitive and direct detection of neurotransmitters and proteins without any derivatization. An on-column CE-LINF protocol for quantification of the release from single cell was demonstrated. Quantitative measurements of both the amount of insulin released from and the amount remaining in the cell ({beta}TC3) were achieved simultaneously. Secretion of catecholamines (norepinephrine (NE) and epinephrine (E)) from individual bovine adrenal chromaffin cells was determined using the on-column CE-LINF. Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved by LINF imaging microscopy with high temporal and spatial resolution. The secretion of serotonin from individual leech Retzius neurons was directly characterized by LINF microscopy with high spatial resolution.

  19. Single Cell Physiology

    NASA Astrophysics Data System (ADS)

    Neveu, Pierre; Sinha, Deepak Kumar; Kettunen, Petronella; Vriz, Sophie; Jullien, Ludovic; Bensimon, David

    The possibility to control at specific times and specific places the activity of biomolecules (enzymes, transcription factors, RNA, hormones, etc.) is opening up new opportunities in the study of physiological processes at the single cell level in a live organism. Most existing gene expression systems allow for tissue specific induction upon feeding the organism with exogenous inducers (e.g., tetracycline). Local genetic control has earlier been achieved by micro-injection of the relevant inducer/repressor molecule, but this is an invasive and possibly traumatic technique. In this chapter, we present the requirements for a noninvasive optical control of the activity of biomolecules and review the recent advances in this new field of research.

  20. MCO Monitoring activity description

    SciTech Connect

    SEXTON, R.A.

    1998-11-09

    Spent Nuclear Fuel remaining from Hanford's N-Reactor operations in the 1970s has been stored under water in the K-Reactor Basins. This fuel will be repackaged, dried and stored in a new facility in the 200E Area. The safety basis for this process of retrieval, drying, and interim storage of the spent fuel has been established. The monitoring of MCOS in dry storage is a currently identified issue in the SNF Project. This plan outlines the key elements of the proposed monitoring activity. Other fuel stored in the K-Reactor Basins, including SPR fuel, will have other monitoring considerations and is not addressed by this activity description.

  1. Monitoring F1651 P-Like Fimbria Expression at the Single-Cell Level Reveals a Highly Heterogeneous Phenotype

    PubMed Central

    Graveline, Richard; Lavoie, Rémi; Garneau, Philippe; Daigle, France; Sénéchal, Serge; Martin, Christine

    2015-01-01

    F1651 and the pyelonephritis-associated pili (Pap) are two members of the type P family of adhesive factors. They play a key role in establishing disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) strains in animals and humans. Both F1651 and Pap are under the control of an epigenetic and reversible switch that defines the number of fimbriated (ON) and afimbriated (OFF) cells within a clonal population. Using the Gfp reporter system, we monitored in vitro the level of fluorescence intensity corresponding to the F1651 and Pap fimbrial synthesis. Monitoring individual Escherichia coli cells by flow cytometry and by real-time fluorescence microscopy, we identified cells associated with a low or high level of fluorescence intensity and a large amount of cells with partial levels of fluorescence, mostly present in the F1651 system. This mixed population identified through fluorescence intensity could be attributed to the high switching rate previously observed in F1651-positive bacteria. The fimbrial heterogeneous phenotype for these ExPEC could represent increased fitness in unpredictable environments. Our study illustrates that within the large repertoire of fimbrial variants such as the well-characterized Pap, F1651 is an exquisite example of regulatory expression that arms the bacterium with strategies for surviving in more than one particular environment. PMID:25712930

  2. Single Cell Electrical Characterization Techniques

    PubMed Central

    Mansor, Muhammad Asraf; Ahmad, Mohd Ridzuan

    2015-01-01

    Electrical properties of living cells have been proven to play significant roles in understanding of various biological activities including disease progression both at the cellular and molecular levels. Since two decades ago, many researchers have developed tools to analyze the cell’s electrical states especially in single cell analysis (SCA). In depth analysis and more fully described activities of cell differentiation and cancer can only be accomplished with single cell analysis. This growing interest was supported by the emergence of various microfluidic techniques to fulfill high precisions screening, reduced equipment cost and low analysis time for characterization of the single cell’s electrical properties, as compared to classical bulky technique. This paper presents a historical review of single cell electrical properties analysis development from classical techniques to recent advances in microfluidic techniques. Technical details of the different microfluidic techniques are highlighted, and the advantages and limitations of various microfluidic devices are discussed. PMID:26053399

  3. Metabolic activation of herbicide products by Vicia faba detected in human peripheral lymphocytes using alkaline single cell gel electrophoresis.

    PubMed

    Calderón-Segura, María Elena; Gómez-Arroyo, Sandra; Molina-Alvarez, Bertha; Villalobos-Pietrini, Rafael; Calderón-Ezquerro, Carmen; Cortés-Eslava, Josefina; Valencia-Quintana, Pedro Rafael; López-González, Lucina; Zúñiga-Reyes, Rubén; Sánchez-Rincón, José

    2007-09-01

    Ametryn and metribuzin S-triazines derivatives and EPTC thiocarbamate are herbicides used extensively in Mexican agriculture, for example in crops such as corn, sugar cane, tomato, wheat, and beans. The present study evaluated the DNA damage and cytotoxic effects of three herbicides after metabolism by Vicia faba roots in human peripheral lymphocytes using akaline single cell gel electrophoresis. Three parameters were scored as indicators of DNA damage: tail length, percentage of cells with DNA damage (with comet), and level DNA damage. The lymphocytes were treated for 2 h with 0.5-5.0 microg/ml ametryn or metribuzin and 1.5-10 microg/ml EPTC. Lymphocytes also were coincubated for 2 h with 20 microl V. faba roots extracts that had been treated for 4 h with 50-500 mg/l of the two triazines or with the thiocarbamate herbicide or with ethanol (3600 mg/l), as positive control. The lymphocytes treated with three pesticides without in vivo metabolic activation by V. faba root did not show significant differences in the mean values between genotoxic parameters compared with negative control. But when human cells were exposed to three herbicides after they had been metabolized the frequency of cell comet, tail length and level DNA damage all increased. At highest concentrations of the three herbicides produced severe DNA damage compared with S10 fraction and negative control. The linear regression analysis of the tail length values of three herbicides indicated that there was genotoxic effect concentration-response relationship with ametryn and ametribuzin but no EPTC. The ethanol induced major increase DNA damage compared with S10 fraction and the three pesticides. There were not effects in cell viability with treatment EPTC and metribuzin whether or not it had been metabolized. High concentrations of ametryn alone and after it had been metabolized decreased cell viability compared with the negative control. The results demonstrated that the three herbicides needed to be

  4. Fast methods for analysis of neurotransmitters from single cell and monitoring their releases in central nervous system by capillary electrophoresis, fluorescence microscopy and luminescence imaging

    SciTech Connect

    Wang, Ziqiang

    1999-12-10

    Fast methods for separation and detection of important neurotransmitters and the releases in central nervous system (CNS) were developed. Enzyme based immunoassay combined with capillary electrophoresis was used to analyze the contents of amino acid neurotransmitters from single neuron cells. The release of amino acid neurotransmitters from neuron cultures was monitored by laser induced fluorescence imaging method. The release and signal transduction of adenosine triphosphate (ATP) in CNS was studied with sensitive luminescence imaging method. A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Detection was based on monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. The detection limit of glutamate is down to 10{sup {minus}8} M level, which is 1 order of magnitude lower than the previously reported detection limit based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most of amino acids. The glutamate content in single human erythrocyte and baby rat brain neurons were determined with this method and results agreed well with literature values.

  5. Visualization of RelB expression and activation at the single-cell level during dendritic cell maturation in Relb-Venus knock-in mice.

    PubMed

    Seki, Takao; Yamamoto, Mami; Taguchi, Yuu; Miyauchi, Maki; Akiyama, Nobuko; Yamaguchi, Noritaka; Gohda, Jin; Akiyama, Taishin; Inoue, Jun-ichiro

    2015-12-01

    RelB is activated by the non-canonical NF-κB pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The Relb(Venus/Venus) mice developed without any abnormalities observed in the Relb(-/-) mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. Relb(Venus/Venus) DC analyses revealed that DCs consist of RelB(-), RelB(low) and RelB(high) populations. The RelB(high) population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelB(low) population was in the cytoplasm. Although both the RelB(low) and RelB(-) populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelB(low) than in the RelB(-) splenic conventional DCs. Taken together, our results identify the RelB(low) population as a possible novel intermediate maturation stage of cDCs and the Relb(Venus/Venus) mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-κB pathway. PMID:26115685

  6. Rare Earth Ion Mediated Fluorescence Accumulation on a Single Microbead: An Ultrasensitive Strategy for the Detection of Protein Kinase Activity at the Single-Cell Level.

    PubMed

    Zhang, Xiaobo; Liu, Chenghui; Wang, Honghong; Wang, Hui; Li, Zhengping

    2015-12-01

    A single microbead-based fluorescence imaging (SBFI) strategy that enables detection of protein kinase activity from single cell lysates is reported. We systematically investigated the ability of various rare earth (RE) ions, immobilized on the microbead, for specific capturing of kinase-induced phosphopeptides, and Dy(3+) was found to be the most prominent one. Through the efficient concentration of kinase-induced fluorescent phosphopeptides on a Dy(3+) -functionalized single microbead, kinase activity can be detected and quantified by reading the fluorescence on the microbead with a confocal fluorescence microscope. Owing to the extremely specific recognition of Dy(3+) towards phosphopeptides and the highly-concentrated fluorescence accumulation on only one microbead, ultrahigh sensitivity has been achieved for the SBFI strategy which allows direct kinase analysis at the single-cell level. PMID:26482714

  7. Small Active Radiation Monitor

    NASA Technical Reports Server (NTRS)

    Badhwar, Gautam D.

    2004-01-01

    A device, named small active radiation monitor, allows on-orbit evaluations during periods of increased radiation, after extravehicular activities, or at predesignated times for crews on such long-duration space missions as on the International Space Station. It also permits direct evaluation of biological doses, a task now performed using a combination of measurements and potentially inaccurate simulations. Indeed the new monitor can measure a full array of radiation levels, from soft x-rays to hard galactic cosmic-ray particles. With refinement, it will benefit commercial (nuclear power-plant workers, airline pilots, medical technicians, physicians/dentists, and others) and military personnel as well as the astronauts for whom thermoluminescent dosimeters are inadequate. Civilian and military personnel have long since graduated from film badges to thermoluminescent dosimeters. Once used, most dosimeters must be returned to a central facility for processing, a step that can take days or even weeks. While this suffices for radiation workers for whom exposure levels are typically very low and of brief duration, it does not work for astronauts. Even in emergencies and using express mail, the results can often be delayed by as much as 24 hours. Electronic dosimeters, which are the size of electronic oral thermometers, and tattlers, small electronic dosimeters that sound an alarm when the dose/dose rate exceeds preset values, are also used but suffer disadvantages similar to those of thermoluminescent dosimeters. None of these devices fully answers the need of rapid monitoring during the space missions. Instead, radiation is monitored by passive detectors, which are read out after the missions. Unfortunately, these detectors measure only the absorbed dose and not the biologically relevant dose equivalent. The new monitor provides a real-time readout, a time history of radiation exposures (both absorbed dose and biologically relevant dose equivalent), and a count of the

  8. SINGLE CELL GENOME SEQUENCING

    PubMed Central

    Yilmaz, Suzan; Singh, Anup K.

    2011-01-01

    Whole genome amplification and next-generation sequencing of single cells has become a powerful approach for studying uncultivated microorganisms that represent 90–99 % of all environmental microbes. Single cell sequencing enables not only the identification of microbes but also linking of functions to species, a feat not achievable by metagenomic techniques. Moreover, it allows the analysis of low abundance species that may be missed in community-based analyses. It has also proved very useful in complementing metagenomics in the assembly and binning of single genomes. With the advent of drastically cheaper and higher throughput sequencing technologies, it is expected that single cell sequencing will become a standard tool in studying the genome and transcriptome of microbial communities. PMID:22154471

  9. Single Cell Oncogenesis

    NASA Astrophysics Data System (ADS)

    Lu, Xin

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

  10. Magnetic levitation of single cells.

    PubMed

    Durmus, Naside Gozde; Tekin, H Cumhur; Guven, Sinan; Sridhar, Kaushik; Arslan Yildiz, Ahu; Calibasi, Gizem; Ghiran, Ionita; Davis, Ronald W; Steinmetz, Lars M; Demirci, Utkan

    2015-07-14

    Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10(-4) g ⋅ mL(-1). We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine. PMID:26124131

  11. Magnetic levitation of single cells

    PubMed Central

    Durmus, Naside Gozde; Tekin, H. Cumhur; Guven, Sinan; Sridhar, Kaushik; Arslan Yildiz, Ahu; Calibasi, Gizem; Davis, Ronald W.; Steinmetz, Lars M.; Demirci, Utkan

    2015-01-01

    Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10−4 g⋅mL−1. We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine. PMID:26124131

  12. Monitoring international nuclear activity

    SciTech Connect

    Firestone, R.B.

    2006-05-19

    The LBNL Table of Isotopes website provides primary nuclearinformation to>150,000 different users annually. We have developedthe covert technology to identify users by IP address and country todetermine the kinds of nuclear information they are retrieving. Wepropose to develop pattern recognition software to provide an earlywarning system to identify Unusual nuclear activity by country or regionSpecific nuclear/radioactive material interests We have monitored nuclearinformation for over two years and provide this information to the FBIand LLNL. Intelligence is gleaned from the website log files. Thisproposal would expand our reporting capabilities.

  13. Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform

    PubMed Central

    Nakamura, Kazuki; Iizuka, Ryo; Nishi, Shinro; Yoshida, Takao; Hatada, Yuji; Takaki, Yoshihiro; Iguchi, Ayaka; Yoon, Dong Hyun; Sekiguchi, Tetsushi; Shoji, Shuichi; Funatsu, Takashi

    2016-01-01

    Environmental microbes are a great source of industrially valuable enzymes with potent and unique catalytic activities. Unfortunately, the majority of microbes remain unculturable and thus are not accessible by culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied, opening access to untapped genetic resources. Here we present a methodological approach for the identification of genes that encode metabolically active enzymes in environmental microbes in a culture-independent manner. Our method is based on activity-based single-cell sequencing, which focuses on microbial cells showing specific enzymatic activities. First, at the single-cell level, environmental microbes were encapsulated in water-in-oil microdroplets with a fluorogenic substrate for the target enzyme to screen for microdroplets that contain microbially active cells. Second, the microbial cells were recovered and subjected to whole genome amplification. Finally, the amplified genomes were sequenced to identify the genes encoding target enzymes. Employing this method, we successfully identified 14 novel β-glucosidase genes from uncultured bacterial cells in marine samples. Our method contributes to the screening and identification of genes encoding industrially valuable enzymes. PMID:26915788

  14. Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform.

    PubMed

    Nakamura, Kazuki; Iizuka, Ryo; Nishi, Shinro; Yoshida, Takao; Hatada, Yuji; Takaki, Yoshihiro; Iguchi, Ayaka; Yoon, Dong Hyun; Sekiguchi, Tetsushi; Shoji, Shuichi; Funatsu, Takashi

    2016-01-01

    Environmental microbes are a great source of industrially valuable enzymes with potent and unique catalytic activities. Unfortunately, the majority of microbes remain unculturable and thus are not accessible by culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied, opening access to untapped genetic resources. Here we present a methodological approach for the identification of genes that encode metabolically active enzymes in environmental microbes in a culture-independent manner. Our method is based on activity-based single-cell sequencing, which focuses on microbial cells showing specific enzymatic activities. First, at the single-cell level, environmental microbes were encapsulated in water-in-oil microdroplets with a fluorogenic substrate for the target enzyme to screen for microdroplets that contain microbially active cells. Second, the microbial cells were recovered and subjected to whole genome amplification. Finally, the amplified genomes were sequenced to identify the genes encoding target enzymes. Employing this method, we successfully identified 14 novel β-glucosidase genes from uncultured bacterial cells in marine samples. Our method contributes to the screening and identification of genes encoding industrially valuable enzymes. PMID:26915788

  15. Mesopelagic prokaryotic bulk and single-cell heterotrophic activity and community composition in the NW Africa-Canary Islands coastal-transition zone

    NASA Astrophysics Data System (ADS)

    Gasol, Josep M.; Alonso-Sáez, Laura; Vaqué, Dolors; Baltar, Federico; Calleja, Maria Ll.; Duarte, Carlos M.; Arístegui, Javier

    2009-12-01

    Mesopelagic prokaryotic communities have often been assumed to be relatively inactive in comparison to those from epipelagic waters, and therefore unresponsive to the presence of nearby upwelled waters. We have studied the zonal (shelf-ocean), latitudinal, and depth (epipelagic-mesopelagic) variability of microbial assemblages in the NW Africa-Canary Islands coastal-transition zone (CTZ). Vertical profiles of bacterial bulk and single-cell activity through the epi- and mesopelagic waters were combined with point measurements of bacterial respiration, leucine-to-carbon conversion factors and leucine-to-thymidine incorporation ratios. The overall picture that emerges from our study is that prokaryotes in the mesopelagic zone of this area are less abundant than in the epipelagic but have comparable levels of activity. The relationship between prokaryotes and heterotrophic nanoflagellates, their main predators, remains constant throughout the water column, further contradicting the assumption that deep ocean bacterial communities are mostly inactive. Both bulk and single-cell activity showed clear differences between stations, with higher mesopelagic activities closer to the shelf or affected by upwelling features. We also tested whether differences in microbial function between stations could be related to differences in bacterial community structure, and conclude that bacterial communities are very similar at similar depths in the deep ocean, even if the stations present order-of-magnitude differences in bacterial function.

  16. Interplay between type IV pili activity and exopolysaccharides secretion controls motility patterns in single cells of Myxococcus xanthus

    PubMed Central

    Hu, Wei; Gibiansky, Maxsim L.; Wang, Jing; Wang, Chuandong; Lux, Renate; Li, Yuezhong; Wong, Gerard C. L.; Shi, Wenyuan

    2016-01-01

    Myxococcus xanthus performs coordinated social motility of cell groups through the extension and retraction of type IV pili (TFP) on solid surfaces, which requires both TFP and exopolysaccharides (EPS). By submerging cells in a liquid medium containing 1% methylcellulose, M. xanthus TFP-driven motility was induced in isolated cells and independently of EPS. We measured and analyzed the movements of cells using community tracking algorithms, which combine single-cell resolution with statistics from large sample populations. Cells without significant multi-cellular social interactions have surprisingly complex behaviors: EPS− cells exhibited a pronounced increase in the tendency to stand vertically and moved with qualitatively different characteristics than other cells. A decrease in the EPS secretion of cells correlates with a higher instantaneous velocity, but with lower directional persistence in trajectories. Moreover, EPS− cells do not adhere to the surface as strongly as wild-type and EPS overproducing cells, and display a greater tendency to have large deviations between the direction of movement and the cell axis, with cell velocity showing only minimal dependence on the direction of movement. The emerging picture is that EPS does not simply provide rheological resistance to a single mechanism but rather that the availability of EPS impacts motility pattern. PMID:26821939

  17. The 40-year history of modeling active dendrites in cerebellar Purkinje cells: emergence of the first single cell “community model”

    PubMed Central

    Bower, James M.

    2015-01-01

    The subject of the effects of the active properties of the Purkinje cell dendrite on neuronal function has been an active subject of study for more than 40 years. Somewhat unusually, some of these investigations, from the outset have involved an interacting combination of experimental and model-based techniques. This article recounts that 40-year history, and the view of the functional significance of the active properties of the Purkinje cell dendrite that has emerged. It specifically considers the emergence from these efforts of what is arguably the first single cell “community” model in neuroscience. The article also considers the implications of the development of this model for future studies of the complex properties of neuronal dendrites. PMID:26539104

  18. Single-cell proteins

    SciTech Connect

    Litchfield, J.H.

    1983-02-11

    Both photosynthetic and nonphotosynthetic microorganisms, grown on various carbon and energy sources, are used in fermentation processes for the production of single-cell proteins. Commercial-scale production has been limited to two algal processes, one bacterial process, and several yeast and fungal processes. High capital and operating costs and the need for extensive nutritional and toxicological assessments have limited the development and commercialization of new processes. Any increase in commercial-scale production appears to be limited to those regions of the world where low-cost carbon and energy sources are available and conventional animal feedstuff proteins, such as soybean meal or fish meal, are in short supply. (Refs. 59).

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

    PubMed

    Schmidt, Felix; Efferth, Thomas

    2016-01-01

    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. PMID:27322289

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

    PubMed Central

    Schmidt, Felix; Efferth, Thomas

    2016-01-01

    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. PMID:27322289

  1. Single cell profiling of surface carbohydrates on Bacillus cereus.

    PubMed

    Wang, Congzhou; Ehrhardt, Christopher J; Yadavalli, Vamsi K

    2015-02-01

    Cell surface carbohydrates are important to various bacterial activities and functions. It is well known that different types of Bacillus display heterogeneity of surface carbohydrate compositions, but detection of their presence, quantitation and estimation of variation at the single cell level have not been previously solved. Here, using atomic force microscopy (AFM)-based recognition force mapping coupled with lectin probes, the specific carbohydrate distributions of N-acetylglucosamine and mannose/glucose were detected, mapped and quantified on single B. cereus surfaces at the nanoscale across the entire cell. Further, the changes of the surface carbohydrate compositions from the vegetative cell to spore were shown. These results demonstrate AFM-based 'recognition force mapping' as a versatile platform to quantitatively detect and spatially map key bacterial surface biomarkers (such as carbohydrate compositions), and monitor in situ changes in surface biochemical properties during intracellular activities at the single cell level. PMID:25505137

  2. Single cell profiling of surface carbohydrates on Bacillus cereus

    PubMed Central

    Wang, Congzhou; Ehrhardt, Christopher J.; Yadavalli, Vamsi K.

    2015-01-01

    Cell surface carbohydrates are important to various bacterial activities and functions. It is well known that different types of Bacillus display heterogeneity of surface carbohydrate compositions, but detection of their presence, quantitation and estimation of variation at the single cell level have not been previously solved. Here, using atomic force microscopy (AFM)-based recognition force mapping coupled with lectin probes, the specific carbohydrate distributions of N-acetylglucosamine and mannose/glucose were detected, mapped and quantified on single B. cereus surfaces at the nanoscale across the entire cell. Further, the changes of the surface carbohydrate compositions from the vegetative cell to spore were shown. These results demonstrate AFM-based ‘recognition force mapping’ as a versatile platform to quantitatively detect and spatially map key bacterial surface biomarkers (such as carbohydrate compositions), and monitor in situ changes in surface biochemical properties during intracellular activities at the single cell level. PMID:25505137

  3. Specific Single-Cell Isolation of Escherichia coli O157 from Environmental Water Samples by Using Flow Cytometry and Fluorescence-Activated Cell Sorting.

    PubMed

    Ozawa, Shuji; Okabe, Satoshi; Ishii, Satoshi

    2016-08-01

    Contamination of food and water with pathogenic bacteria is of concern. Although culture-independent detection and quantification of pathogens is useful, isolation of pathogenic bacteria is still important when identifying the sources of pathogens. Here, we report the use of flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) to specifically detect and isolate individual Escherichia coli O157:H7 cells from water samples. When present at >10 cells/mL water, target pathogen was specifically detected and isolated. The FACS-sorted E. coli O157:H7 population reflected the original population diversity, in contrast to the populations obtained by immunomagnetic separation. Relative abundance of multiple pathogenic strains is important when performing source-tracking studies; therefore, single-cell isolation with FCM-FACS can be a useful tool to obtain pathogenic bacteria for source tracking purpose. PMID:27182755

  4. Single Cell Chemical Cytometry of Akt Activity in Rheumatoid Arthritis and Normal Fibroblast-like Synoviocytes in Response to Tumor Necrosis Factor α.

    PubMed

    Mainz, Emilie R; Serafin, D Stephen; Nguyen, Tuong T; Tarrant, Teresa K; Sims, Christopher E; Allbritton, Nancy L

    2016-08-01

    The etiology of rheumatoid arthritis (RA) is poorly understood, and 30% of patients are unresponsive to established treatments targeting tumor necrosis factor α (TNFα). Akt kinase is implicated in TNFα signaling and may act as a barometer of patient responses to biologic therapies. Fluorescent peptide sensors and chemical cytometry were employed to directly measure Akt activity as well as proteolytic activity in individual fibroblast-like synoviocytes (FLS) from RA and normal subjects. The specificity of the peptide reporter was evaluated and shown to be a valid measure of Akt activity in single cells. The effect of TNFα treatment on Akt activity was highly heterogeneous between normal and RA subjects, which was not observable in bulk analyses. In 2 RA subjects, a bimodal distribution of Akt activity was observed, primarily due to a subpopulation (21.7%: RA Subject 5; 23.8%: RA Subject 6) of cells in which >60% of the reporter was phosphorylated. These subjects also possessed statistically elevated proteolytic cleavage of the reporter relative to normal subjects, suggesting heterogeneity in Akt and protease activity that may play a role in the RA-affected joint. We expect that chemical cytometry studies pairing peptide reporters with capillary electrophoresis will provide valuable data regarding aberrant kinase activity from small samples of clinical interest. PMID:27391352

  5. Quantitative determination of enzyme activity in single cells by scanning microelectrode coupled with a nitrocellulose film-covered microreactor by means of a scanning electrochemical microscope.

    PubMed

    Zhang, Xiaoli; Sun, Fuchan; Peng, Xuewei; Jin, Wenrui

    2007-02-01

    An electrochemical method for quantitative determination of enzyme activity in single cells was developed by scanning a microelectrode (ME) over a nitrocellulose film-covered microreactor with micropores by means of a scanning electrochemical microscope (SECM). Peroxidase (PO) in neutrophils was chosen as the model system. The microreactor consisted of a microwell with a solution and a nitrocellulose film with micropores. A single cell perforated by digitonin was injected into the microwell. After the perforated cell was lysed and allowed to dry, physiological buffer saline (PBS) containing hydroquinone (H2Q) and H2O2 as substrates of the enzyme-catalyzed reaction was added in the microwell. The microwell containing the extract of the lysed cell and the enzyme substrates was covered with Parafilm to prevent evaporation. The solution in the microwell was incubated for 20 min. In this case, the released PO from the cell converted H2Q into benzoquinone (BQ). Then, the Parafilm was replaced by a nitrocellulose film with micropores to fabricate the microreactor. The microreactor was placed in an electrochemical cell containing PBS, H2Q, and H2O2. After a 10-microm-radius Au ME was inserted into the electrochemical cell and approached down to the microreactor, the ME was scanned along the central line across the microreactor by means of a SECM. The scan curve with a peak was obtained by detecting BQ that diffused out from the microreactor through the micropores on the nitrocellulose film. PO activity could be quantified on the basis of the peak current on the scan curve using a calibration curve. This method had two obvious advantages: no electrode fouling and no oxygen interference. PMID:17263362

  6. Single cell optical transfection.

    PubMed

    Stevenson, David J; Gunn-Moore, Frank J; Campbell, Paul; Dholakia, Kishan

    2010-06-01

    The plasma membrane of a eukaryotic cell is impermeable to most hydrophilic substances, yet the insertion of these materials into cells is an extremely important and universal requirement for the cell biologist. To address this need, many transfection techniques have been developed including viral, lipoplex, polyplex, capillary microinjection, gene gun and electroporation. The current discussion explores a procedure called optical injection, where a laser field transiently increases the membrane permeability to allow species to be internalized. If the internalized substance is a nucleic acid, such as DNA, RNA or small interfering RNA (siRNA), then the process is called optical transfection. This contactless, aseptic, single cell transfection method provides a key nanosurgical tool to the microscopist-the intracellular delivery of reagents and single nanoscopic objects. The experimental possibilities enabled by this technology are only beginning to be realized. A review of optical transfection is presented, along with a forecast of future applications of this rapidly developing and exciting technology. PMID:20064901

  7. Thermomicrocapillaries as temperature biosensors in single cells

    NASA Astrophysics Data System (ADS)

    Herth, Simone; Giesguth, Miriam; Wedel, Waldemar; Reiss, Günther; Dietz, Karl-Josef

    2013-03-01

    Temperature is an important physical parameter in biology and its deviation from optimum can cause damage in biosystems. Thermocouples based on the Seebeck effect can be structured on glass microcapillaries to obtain thermomicrocapillaries (TMCs) usable in a micromanipulation setup. The suitability of the setup was proven by monitoring the temperature increase upon illumination of leaves and single cells following insertion of the TMC. The increase was 1.5 K in green tissue and 0.75 K in white leaf sections due to lower absorption. In single cells of trichomes, the increase was 0.5 K due to heat dissipation to the surrounding air.

  8. Voltage clamp measurements of the hyperpolarization-activated inward current I(f) in single cells from rabbit sino-atrial node.

    PubMed Central

    van Ginneken, A C; Giles, W

    1991-01-01

    1. The kinetics and ion transfer characteristics of the hyperpolarization-activated inward current, I(f), have been studied in single cells obtained by enzymatic dispersion from the rabbit sino-atrial (S-A) node. These experiments were done to assess the role of I(f) in the generation of the pacemaker depolarization in the S-A node. 2. The activation and the deactivation of I(f) in these single cells are accompanied by significant conductance increases and decreases respectively, confirming earlier findings from multicellular man-made strips of rabbit S-A node, and from mammalian Purkinje fibres. 3. The steady-state activation of I(f) lies between -40 and -120 mV, and its voltage dependence can be described by a Boltzmann relation with the half-activation point at approximately -70 mV. 4. The delay or sigmoidicity in both the onset of I(f) and the deactivation of the tail currents can be accounted for semi-quantitatively by using a second-order Hodgkin-Huxley kinetic scheme. 5. The reversal potential for I(f) is -24 +/- 2 mV (mean +/- S.E.M., n = 6). It does not change significantly as a function of the amount of I(f) which is activated, indicating that ion accumulation or depletion phenomena are not important variables controlling the time course of I(f), or its selectivity. 6. The fully-activated current-voltage relationship for I(f) is approximately linear with a slope conductance of 12.0 +/- 0.88 nS per cell (mean +/- S.E.M., n = 6). 7. A simple mathematical model based on the measured values of maximum conductance, reversal potential, and kinetics of I(f) has been developed to simulate the size and time course of I(f) during typical spontaneous pacemaker activity in rabbit sino-atrial node cells. The calculations show that I(f) can change significantly during pacing and suggest that this current change is, at least in part, responsible for the pacemaker depolarization. Images Fig. 1 PMID:1708824

  9. Monitoring of Single-Cell Responses in the Optic Tectum of Adult Zebrafish with Dextran-Coupled Calcium Dyes Delivered via Local Electroporation

    PubMed Central

    Kassing, Vanessa

    2013-01-01

    The zebrafish (Danio rerio) has become one of the major animal models for in vivo examination of sensory and neuronal computation. Similar to Xenopus tadpoles neural activity in the optic tectum, the major region controlling visually guided behavior, can be examined in zebrafish larvae by optical imaging. Prerequisites of these approaches are usually the transparency of larvae up to a certain age and the use of two-photon microscopy. This principle of fluorescence excitation was necessary to suppress crosstalk between signals from individual neurons, which is a critical issue when using membrane-permeant dyes. This makes the equipment to study neuronal processing costly and limits the approach to the study of larvae. Thus there is lack of knowledge about the properties of neurons in the optic tectum of adult animals. We established a procedure to circumvent these problems, enabling in vivo calcium imaging in the optic tectum of adult zebrafish. Following local application of dextran-coupled dyes single-neuron activity of adult zebrafish can be monitored with conventional widefield microscopy, because dye labeling remains restricted to tens of neurons or less. Among the neurons characterized with our technique we found neurons that were selective for a certain pattern orientation as well as neurons that responded in a direction-selective way to visual motion. These findings are consistent with previous studies and indicate that the functional integrity of neuronal circuits in the optic tectum of adult zebrafish is preserved with our staining technique. Overall, our protocol for in vivo calcium imaging provides a useful approach to monitor visual responses of individual neurons in the optic tectum of adult zebrafish even when only widefield microscopy is available. This approach will help to obtain valuable insight into the principles of visual computation in adult vertebrates and thus complement previous work on developing visual circuits. PMID:23667529

  10. Single-cell mass cytometry of TCR signaling: amplification of small initial differences results in low ERK activation in NOD mice.

    PubMed

    Mingueneau, Michael; Krishnaswamy, Smita; Spitzer, Matthew H; Bendall, Sean C; Stone, Erica L; Hedrick, Stephen M; Pe'er, Dana; Mathis, Diane; Nolan, Garry P; Benoist, Christophe

    2014-11-18

    Signaling from the T-cell receptor (TCR) conditions T-cell differentiation and activation, requiring exquisite sensitivity and discrimination. Using mass cytometry, a high-dimensional technique that can probe multiple signaling nodes at the single-cell level, we interrogate TCR signaling dynamics in control C57BL/6 and autoimmunity-prone nonobese diabetic (NOD) mice, which show ineffective ERK activation after TCR triggering. By quantitating signals at multiple steps along the signaling cascade and parsing the phosphorylation level of each node as a function of its predecessors, we show that a small impairment in initial pCD3ζ activation resonates farther down the signaling cascade and results in larger defects in activation of the ERK1/2-S6 and IκBα modules. This nonlinear property of TCR signaling networks, which magnifies small initial differences during signal propagation, also applies in cells from B6 mice activated at different levels of intensity. Impairment in pCD3ζ and pSLP76 is not a feedback consequence of a primary deficiency in ERK activation because no proximal signaling defect was observed in Erk2 KO T cells. These defects, which were manifest at all stages of T-cell differentiation from early thymic pre-T cells to memory T cells, may condition the imbalanced immunoregulation and tolerance in NOD T cells. More generally, this amplification of small initial differences in signal intensity may explain how T cells discriminate between closely related ligands and adopt strongly delineated cell fates. PMID:25362052

  11. Single-cell mass cytometry of TCR signaling: Amplification of small initial differences results in low ERK activation in NOD mice

    PubMed Central

    Mingueneau, Michael; Krishnaswamy, Smita; Spitzer, Matthew H.; Bendall, Sean C.; Stone, Erica L.; Hedrick, Stephen M.; Pe'er, Dana; Mathis, Diane; Nolan, Garry P.; Benoist, Christophe

    2014-01-01

    Signaling from the T-cell receptor (TCR) conditions T-cell differentiation and activation, requiring exquisite sensitivity and discrimination. Using mass cytometry, a high-dimensional technique that can probe multiple signaling nodes at the single-cell level, we interrogate TCR signaling dynamics in control C57BL/6 and autoimmunity-prone nonobese diabetic (NOD) mice, which show ineffective ERK activation after TCR triggering. By quantitating signals at multiple steps along the signaling cascade and parsing the phosphorylation level of each node as a function of its predecessors, we show that a small impairment in initial pCD3ζ activation resonates farther down the signaling cascade and results in larger defects in activation of the ERK1/2–S6 and IκBα modules. This nonlinear property of TCR signaling networks, which magnifies small initial differences during signal propagation, also applies in cells from B6 mice activated at different levels of intensity. Impairment in pCD3ζ and pSLP76 is not a feedback consequence of a primary deficiency in ERK activation because no proximal signaling defect was observed in Erk2 KO T cells. These defects, which were manifest at all stages of T-cell differentiation from early thymic pre-T cells to memory T cells, may condition the imbalanced immunoregulation and tolerance in NOD T cells. More generally, this amplification of small initial differences in signal intensity may explain how T cells discriminate between closely related ligands and adopt strongly delineated cell fates. PMID:25362052

  12. A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells

    PubMed Central

    van Unen, Jakobus; Stumpf, Anette D.; Schmid, Benedikt; Reinhard, Nathalie R.; Hordijk, Peter L.; Hoffmann, Carsten; Gadella, Theodorus W. J.; Goedhart, Joachim

    2016-01-01

    G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation. PMID:26799488

  13. A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells.

    PubMed

    van Unen, Jakobus; Stumpf, Anette D; Schmid, Benedikt; Reinhard, Nathalie R; Hordijk, Peter L; Hoffmann, Carsten; Gadella, Theodorus W J; Goedhart, Joachim

    2016-01-01

    G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation. PMID:26799488

  14. Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

    PubMed

    Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

    2014-10-01

    Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells. PMID:25086649

  15. Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex

    PubMed Central

    Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay AA

    2014-01-01

    Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships, but require efficient methods for cell capture and mRNA sequencing1–4. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths5, the limitations of shallow sequencing have not been directly investigated. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In developing cortex we identify diverse cell types including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells. PMID:25086649

  16. Chemical Analysis of Single Cells

    NASA Astrophysics Data System (ADS)

    Borland, Laura M.; Kottegoda, Sumith; Phillips, K. Scott; Allbritton, Nancy L.

    2008-07-01

    Chemical analysis of single cells requires methods for quickly and quantitatively detecting a diverse array of analytes from extremely small volumes (femtoliters to nanoliters) with very high sensitivity and selectivity. Microelectrophoretic separations, using both traditional capillary electrophoresis and emerging microfluidic methods, are well suited for handling the unique size of single cells and limited numbers of intracellular molecules. Numerous analytes, ranging from small molecules such as amino acids and neurotransmitters to large proteins and subcellular organelles, have been quantified in single cells using microelectrophoretic separation techniques. Microseparation techniques, coupled to varying detection schemes including absorbance and fluorescence detection, electrochemical detection, and mass spectrometry, have allowed researchers to examine a number of processes inside single cells. This review also touches on a promising direction in single cell cytometry: the development of microfluidics for integrated cellular manipulation, chemical processing, and separation of cellular contents.

  17. Technologies for Single-Cell Isolation

    PubMed Central

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

    2015-01-01

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

  18. Technologies for Single-Cell Isolation.

    PubMed

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

    2015-01-01

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

  19. Single-cell transcriptomics for microbial eukaryotes.

    PubMed

    Kolisko, Martin; Boscaro, Vittorio; Burki, Fabien; Lynn, Denis H; Keeling, Patrick J

    2014-11-17

    One of the greatest hindrances to a comprehensive understanding of microbial genomics, cell biology, ecology, and evolution is that most microbial life is not in culture. Solutions to this problem have mainly focused on whole-community surveys like metagenomics, but these analyses inevitably loose information and present particular challenges for eukaryotes, which are relatively rare and possess large, gene-sparse genomes. Single-cell analyses present an alternative solution that allows for specific species to be targeted, while retaining information on cellular identity, morphology, and partitioning of activities within microbial communities. Single-cell transcriptomics, pioneered in medical research, offers particular potential advantages for uncultivated eukaryotes, but the efficiency and biases have not been tested. Here we describe a simple and reproducible method for single-cell transcriptomics using manually isolated cells from five model ciliate species; we examine impacts of amplification bias and contamination, and compare the efficacy of gene discovery to traditional culture-based transcriptomics. Gene discovery using single-cell transcriptomes was found to be comparable to mass-culture methods, suggesting single-cell transcriptomics is an efficient entry point into genomic data from the vast majority of eukaryotic biodiversity. PMID:25458215

  20. Capillary electrophoresis strategy to monitor the released and remaining nitric oxide from the same single cell using a specially designed water-soluble fluorescent probe.

    PubMed

    Zhang, Zi-Xing; Guo, Xiao-Feng; Wang, Hong; Zhang, Hua-Shan

    2015-04-01

    Gasotransmitters including nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) have attracted more and more attention in the past decades due to their unique signaling and functions. However, as a fundamental issue in the investigations of gasotransmitters, the cell membrane permeability and release behavior of them is controversial in reports because of the lack of an efficient approach to determine gasotransmitters released out of and remaining in the same cells simultaneously. To solve such problem, taking NO as representative, a robust and facile strategy has been reported based on a completely water-soluble fluorescent probe and a commercially available capillary electrophoresis system. A specially designed boron-dipyrromethene (BODIPY)-based fluorescent probe with two sulfonate groups, disodium 2,6-disulfonate-1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl) difluoroboradiaza-s-indance (TMDSDAB), has been developed. As a turn-on fluorescent probe, TMDSDAB can react with NO promptly in aqueous media, and 540-fold enhancement of fluorescence is obtained. Using TMDSDAB, the trapping and quantification of NO released out of and remaining in the same single cell was achieved by capillary electrophoresis with laser-induced fluorescence detection. The limit of detection is 0.5 nM for NO. The proposed method has been applied to estimate the release behavior of single macrophages, and the results indicated that the cell membrane should be a barrier to NO diffusion. PMID:25707954

  1. PCR-free and label-free fluorescent detection of telomerase activity at single-cell level based on triple amplification.

    PubMed

    Gao, Yanfang; Xu, Jing; Li, Baoxin; Jin, Yan

    2016-07-15

    As a universal biomarker for cancer diagnostics and cancer therapeutics, telomerase has attracted extensive attention concerning its detection and discovery of its inhibitors. Herein, we developed a PCR-free and label-free fluorescent strategy for facile, reliable and highly sensitive assay of human telomerase activity from crude cancer cell extracts. A G-quadruplex-selective fluorescent dye, N-methyl mesoporphyrin IX (NMM), was utilized as signal probe. Two hairpin probes with hidden G-quadruplex strand in their stem were designed as assembly components of strand displacement reaction (SDR). In this strategy, one telomerase elongation product contains several hexamer repeats which can hybridize with numerous assistant DNA to release a lot of trigger DNA (T-DNA) of SDR for achieving first step amplification. Then, strand displacement reaction led to the formation of G-quadruplex at the both end of two hairpin DNA probes for realizing second step amplification. Finally, the re-released T-DNA initiated another cycle of SDR, resulting in a significant increase in the fluorescence intensity of NMM. By taking advantage of triple signal amplification, the telomerase activity in the HeLa extracts equivalent to 1-3000 cells was detected in homogeneous solution. Telomerase activities of different cell lines, including cancer cells and normal cell, were also successfully evaluated. Meanwhile, the inhibition effect of 3'-azido-3'-deoxythymidine (AZT) was also investigated. Therefore, it offers a simple and reliable method for detecting telomerase activity at single-cell level without complex pre-modification of probe and enzyme auxiliary signal amplification, which has the merits of simplicity, rapid response, low cost and high reliability. PMID:26999622

  2. Assessment of nutritional value of single-cell protein from waste-activated sludge as a protein supplement in poultry feed.

    PubMed

    Nkhalambayausi-Chirwa, Evans M; Lebitso, Moses T

    2012-12-01

    The amount of protein wasted through sludge in Gauteng, South Africa, amounts to 95 000 metric tonne/yr, with the order of magnitude of the national protein requirement of approximately 145 000 metric tonne/yr. Waste-activated sludge (WAS) from wastewater treatment plants (WWTPs) that treat domestic wastewater contains protein in a ratio of 2:1 against fishmeal. This protein source has not been utilized because of the high content of toxic heavy metals and other potential carcinogenic pollutants in the sludge. In this study, a pretreatment method of modified aqua regia dilute acid wash was used to lower the metal content by approximately 60%. However, this resulted in a 33% loss of amino acids in the acid-washed WAS. A feed substitution test in poultry with different fishmeal-sludge ratios (0%, 25%, 50%, 75%, and 100% WAS as percent substitution of fishmeal) showed no impact of sludge single-cell protein (SCP) on mortality rate. However, sludge substitution in the feed yielded weight gains and cost savings up to 46%. PMID:23342942

  3. Functional single-cell analysis of T-cell activation by supported lipid bilayer-tethered ligands on arrays of nanowells

    PubMed Central

    Torres, Alexis J.; Contento, Rita Lucia; Gordo, Susana; Wucherpfennig, Kai W.; Love, J. Christopher

    2012-01-01

    Supported lipid bilayers are an important biomolecular tool for characterizing immunological synapses. Immobilized bilayers presenting tethered ligands on planar substrates have yielded both spatio-temporal and structural insights into how T cell receptors (TCRs) reorganize during the initial formation of synapses upon recognition of peptide antigen bound to major histocompatibility complex (MHC) molecules. The prototypical configuration of these assays, however, limits the extent to which the kinetics and structure of the supramolecular activation clusters of the synapse (that occur in seconds or minutes) can be related to subsequent complex cellular responses, such as cytokine secretion and proliferation, occurring over hours to days. Here we describe a new method that allows correlative measures of both attributes with single-cell resolution by using immobilized lipid bilayers and tethered ligands on the surface of dense arrays of subnanoliter wells. This modification allows each nanowell to function as an artificial antigen-presenting cell (APC), and the synapses formed upon contact can be imaged by fluorescence microscopy. We show that the lipid bilayers remain stable and mobile on the surface of the PDMS, and that modifying the ligands tethered to the bilayer alters the structure of the resulting synapses in expected ways. Finally, we demonstrate that this approach allows the subsequent characterization of secreted cytokines from the activated human T cell clones by microengraving in both antigen- and pan-specific manners. This new technique should allow detailed investigations on how biophysical and structural aspects of the synapse influence the activation of individual T cells and their subsequent complex functional responses. PMID:23070211

  4. Functional single-cell analysis of T-cell activation by supported lipid bilayer-tethered ligands on arrays of nanowells.

    PubMed

    Torres, Alexis J; Contento, Rita Lucia; Gordo, Susana; Wucherpfennig, Kai W; Love, J Christopher

    2013-01-01

    Supported lipid bilayers are an important biomolecular tool for characterizing immunological synapses. Immobilized bilayers presenting tethered ligands on planar substrates have yielded both spatio-temporal and structural insights into how T cell receptors (TCRs) reorganize during the initial formation of synapses upon recognition of peptide antigens bound to major histocompatibility complex (MHC) molecules. The prototypical configuration of these assays, however, limits the extent to which the kinetics and structure of the supramolecular activation clusters of the synapse (that occur in seconds or minutes) can be related to subsequent complex cellular responses, such as cytokine secretion and proliferation, occurring over hours to days. Here we describe a new method that allows correlative measures of both attributes with single-cell resolution by using immobilized lipid bilayers and tethered ligands on the surface of dense arrays of subnanoliter wells. This modification allows each nanowell to function as an artificial antigen-presenting cell (APC), and the synapses formed upon contact can be imaged by fluorescence microscopy. We show that the lipid bilayers remain stable and mobile on the surface of the PDMS, and that modifying the ligands tethered to the bilayer alters the structure of the resulting synapses in expected ways. Finally, we demonstrate that this approach allows the subsequent characterization of secreted cytokines from the activated human T cell clones by microengraving in both antigen- and pan-specific manners. This new technique should allow detailed investigations on how biophysical and structural aspects of the synapse influence the activation of individual T cells and their complex functional responses. PMID:23070211

  5. Monitoring active volcanoes

    USGS Publications Warehouse

    Tilling, Robert I.

    1987-01-01

    One of the most spectacular, awesomely beautiful, and at times destructive displays of natural energy is an erupting volcano, belching fume and ash thousands of meters into the atmosphere and pouring out red-hot molten lava in fountains and streams. Countless eruptions in the geologic past have produced volcanic rocks that form much of the Earth's present surface. The gradual disintegration and weathering of these rocks have yielded some of the richest farmlands in the world, and these fertile soils play a significant role in sustaining our large and growing population. Were it not for volcanic activity, the Hawaiian Islands with their sugar cane and pineapple fields and magnificent landscapes and seascapes would not exist to support their residents and to charm their visitors. Yet, the actual eruptive processes are catastrophic and can claim life and property.

  6. Core-shell self-assembly triggered via a thiol-disulfide exchange reaction for reduced glutathione detection and single cells monitoring.

    PubMed

    Zhang, Zhen; Jiao, Yuting; Wang, Yuanyuan; Zhang, Shusheng

    2016-01-01

    A novel core-shell DNA self-assembly catalyzed by thiol-disulfide exchange reactions was proposed, which could realize GSH-initiated hybridization chain reaction (HCR) for signal amplification and molecules gathering. Significantly, these self-assembled products via electrostatic interaction could accumulate into prominent and clustered fluorescence-bright spots in single cancer cells for reduced glutathione monitoring, which will effectively drive cell monitoring into a new era. PMID:27412605

  7. Core-shell self-assembly triggered via a thiol-disulfide exchange reaction for reduced glutathione detection and single cells monitoring

    PubMed Central

    Zhang, Zhen; Jiao, Yuting; Wang, Yuanyuan; Zhang, Shusheng

    2016-01-01

    A novel core-shell DNA self-assembly catalyzed by thiol-disulfide exchange reactions was proposed, which could realize GSH-initiated hybridization chain reaction (HCR) for signal amplification and molecules gathering. Significantly, these self-assembled products via electrostatic interaction could accumulate into prominent and clustered fluorescence-bright spots in single cancer cells for reduced glutathione monitoring, which will effectively drive cell monitoring into a new era. PMID:27412605

  8. Single Cell Analysis of a Bacterial Sender-Receiver System.

    PubMed

    Ramalho, Tiago; Meyer, Andrea; Mückl, Andrea; Kapsner, Korbinian; Gerland, Ulrich; Simmel, Friedrich C

    2016-01-01

    Monitoring gene expression dynamics on the single cell level provides important information on cellular heterogeneity and stochasticity, and potentially allows for more accurate quantitation of gene expression processes. We here study bacterial senders and receivers genetically engineered with components of the quorum sensing system derived from Aliivibrio fischeri on the single cell level using microfluidics-based bacterial chemostats and fluorescence video microscopy. We track large numbers of bacteria over extended periods of time, which allows us to determine bacterial lineages and filter out subpopulations within a heterogeneous population. We quantitatively determine the dynamic gene expression response of receiver bacteria to varying amounts of the quorum sensing inducer N-3-oxo-C6-homoserine lactone (AHL). From this we construct AHL response curves and characterize gene expression dynamics of whole bacterial populations by investigating the statistical distribution of gene expression activity over time. The bacteria are found to display heterogeneous induction behavior within the population. We therefore also characterize gene expression in a homogeneous bacterial subpopulation by focusing on single cell trajectories derived only from bacteria with similar induction behavior. The response at the single cell level is found to be more cooperative than that obtained for the heterogeneous total population. For the analysis of systems containing both AHL senders and receiver cells, we utilize the receiver cells as 'bacterial sensors' for AHL. Based on a simple gene expression model and the response curves obtained in receiver-only experiments, the effective AHL concentration established by the senders and their 'sending power' is determined. PMID:26808777

  9. Single Cell Analysis of a Bacterial Sender-Receiver System

    PubMed Central

    Mückl, Andrea; Kapsner, Korbinian; Gerland, Ulrich; Simmel, Friedrich C.

    2016-01-01

    Monitoring gene expression dynamics on the single cell level provides important information on cellular heterogeneity and stochasticity, and potentially allows for more accurate quantitation of gene expression processes. We here study bacterial senders and receivers genetically engineered with components of the quorum sensing system derived from Aliivibrio fischeri on the single cell level using microfluidics-based bacterial chemostats and fluorescence video microscopy. We track large numbers of bacteria over extended periods of time, which allows us to determine bacterial lineages and filter out subpopulations within a heterogeneous population. We quantitatively determine the dynamic gene expression response of receiver bacteria to varying amounts of the quorum sensing inducer N-3-oxo-C6-homoserine lactone (AHL). From this we construct AHL response curves and characterize gene expression dynamics of whole bacterial populations by investigating the statistical distribution of gene expression activity over time. The bacteria are found to display heterogeneous induction behavior within the population. We therefore also characterize gene expression in a homogeneous bacterial subpopulation by focusing on single cell trajectories derived only from bacteria with similar induction behavior. The response at the single cell level is found to be more cooperative than that obtained for the heterogeneous total population. For the analysis of systems containing both AHL senders and receiver cells, we utilize the receiver cells as ‘bacterial sensors’ for AHL. Based on a simple gene expression model and the response curves obtained in receiver-only experiments, the effective AHL concentration established by the senders and their ‘sending power’ is determined. PMID:26808777

  10. Single cell elemental analysis using nuclear microscopy

    NASA Astrophysics Data System (ADS)

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

    1999-04-01

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

  11. Active Job Monitoring in Pilots

    NASA Astrophysics Data System (ADS)

    Kuehn, Eileen; Fischer, Max; Giffels, Manuel; Jung, Christopher; Petzold, Andreas

    2015-12-01

    Recent developments in high energy physics (HEP) including multi-core jobs and multi-core pilots require data centres to gain a deep understanding of the system to monitor, design, and upgrade computing clusters. Networking is a critical component. Especially the increased usage of data federations, for example in diskless computing centres or as a fallback solution, relies on WAN connectivity and availability. The specific demands of different experiments and communities, but also the need for identification of misbehaving batch jobs, requires an active monitoring. Existing monitoring tools are not capable of measuring fine-grained information at batch job level. This complicates network-aware scheduling and optimisations. In addition, pilots add another layer of abstraction. They behave like batch systems themselves by managing and executing payloads of jobs internally. The number of real jobs being executed is unknown, as the original batch system has no access to internal information about the scheduling process inside the pilots. Therefore, the comparability of jobs and pilots for predicting run-time behaviour or network performance cannot be ensured. Hence, identifying the actual payload is important. At the GridKa Tier 1 centre a specific tool is in use that allows the monitoring of network traffic information at batch job level. This contribution presents the current monitoring approach and discusses recent efforts and importance to identify pilots and their substructures inside the batch system. It will also show how to determine monitoring data of specific jobs from identified pilots. Finally, the approach is evaluated.

  12. Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-α-induced apoptosis in vivo

    PubMed Central

    Simmons, Alan J; Banerjee, Amrita; McKinley, Eliot T; Scurrah, Cherie' R; Herring, Charles A; Gewin, Leslie S; Masuzaki, Ryota; Karp, Seth J; Franklin, Jeffrey L; Gerdes, Michael J; Irish, Jonathan M; Coffey, Robert J; Lau, Ken S

    2015-01-01

    Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single-cell resolution. However, probing signaling in epithelial tissues using cytometry-based single-cell analysis has been confounded by the necessity of single-cell dissociation, where disrupting cell-to-cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue—DISSECT) that preserves native signaling for Cytometry Time-of-Flight (CyTOF) and fluorescent flow cytometry applications. A 21-plex CyTOF analysis encompassing core signaling and cell-identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor-alpha (TNF-α) stimulation. Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF-α-induced apoptosis in the seemingly homogeneous enterocyte population. Specifically, p-ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact-dependent survival. Our novel single-cell approach can broadly be applied, using both CyTOF and multi-parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues. PMID:26519361

  13. Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-α-induced apoptosis in vivo.

    PubMed

    Simmons, Alan J; Banerjee, Amrita; McKinley, Eliot T; Scurrah, Cherie' R; Herring, Charles A; Gewin, Leslie S; Masuzaki, Ryota; Karp, Seth J; Franklin, Jeffrey L; Gerdes, Michael J; Irish, Jonathan M; Coffey, Robert J; Lau, Ken S

    2015-10-01

    Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single-cell resolution. However, probing signaling in epithelial tissues using cytometry-based single-cell analysis has been confounded by the necessity of single-cell dissociation, where disrupting cell-to-cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue-DISSECT) that preserves native signaling for Cytometry Time-of-Flight (CyTOF) and fluorescent flow cytometry applications. A 21-plex CyTOF analysis encompassing core signaling and cell-identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor-alpha (TNF-α) stimulation. Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF-α-induced apoptosis in the seemingly homogeneous enterocyte population. Specifically, p-ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact-dependent survival. Our novel single-cell approach can broadly be applied, using both CyTOF and multi-parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues. PMID:26519361

  14. Monitoring tectal neuronal activities and motor behavior in zebrafish larvae.

    PubMed

    Sumbre, Germán; Poo, Mu-Ming

    2013-09-01

    To understand how visuomotor behaviors are controlled by the nervous system, it is necessary to monitor the activity of large populations of neurons with single-cell resolution over a large area of the brain in a relatively simple, behaving organism. The zebrafish larva, a small lower vertebrate with transparent skin, serves as an excellent model for this purpose. Immediately after the larva hatches, it needs to catch prey and avoid predators. This strong evolutionary pressure leads to the rapid development of functional sensory systems, particularly vision. By 5 d postfertilization (dpf), tectal cells show distinct visually evoked patterns of activation, and the larvae are able to perform a variety of visuomotor behaviors. During the early larval stage, zebrafish breathe mainly through the skin and can be restrained under the microscope using a drop of low-melting-point agarose, without the use of anesthetics. Moreover, the transparency of the skin, the small diameter of the neurons (4-5 µm), and the high-neuronal density enable the use of in vivo noninvasive imaging techniques to monitor neuronal activities of up to ∼500 cells within the central nervous system, still with single-cell resolution. This article describes a method for simultaneously monitoring spontaneous and visually evoked activities of large populations of neurons in the optic tectum of the zebrafish larva, using a synthetic calcium dye (Oregon Green BAPTA-1 AM) and a conventional confocal or two-photon scanning fluorescence microscope, together with a method for measuring the tail motor behavior of the head-immobilized zebrafish larva. PMID:24003199

  15. Quantification of Circadian Rhythms in Single Cells

    PubMed Central

    Westermark, Pål O.; Welsh, David K.; Okamura, Hitoshi; Herzel, Hanspeter

    2009-01-01

    Bioluminescence techniques allow accurate monitoring of the circadian clock in single cells. We have analyzed bioluminescence data of Per gene expression in mouse SCN neurons and fibroblasts. From these data, we extracted parameters such as damping rate and noise intensity using two simple mathematical models, one describing a damped oscillator driven by noise, and one describing a self-sustained noisy oscillator. Both models describe the data well and enabled us to quantitatively characterize both wild-type cells and several mutants. It has been suggested that the circadian clock is self-sustained at the single cell level, but we conclude that present data are not sufficient to determine whether the circadian clock of single SCN neurons and fibroblasts is a damped or a self-sustained oscillator. We show how to settle this question, however, by testing the models' predictions of different phases and amplitudes in response to a periodic entrainment signal (zeitgeber). PMID:19956762

  16. Continuous cultivation of fission yeast: analysis of single-cell protein synthesis kinetics

    SciTech Connect

    Agar, D.W.; Bailey, J.E.

    1981-01-01

    A fundamental problem in microbial reactor analysis is identification of the relation between environment and individual cell metabolic activity. Population balance equations provide a link between experimental measurements of composition frequency functions in microbial populations on the one hand and macromolecule synthesis kinetics and cell division control parameters for single cells on the other. Flow microfluorometry measurements of frequency functions for single-cell protein content in Schizosaccharomyces pombe in balanced exponential growth were analyzed by 2 different methods. One approach utilizes the integrated form of the population balance equation known as the Collins-Richmond equation, and the other method involves optimization of parameters in assumed kinetic and cell division functional forms to fit measured frequency functions with corresponding model solutions. Both data interpretation techniques indicate that rates of protein synthesis increase most in low-protein-content cells as the population specific growth rate increases, leading to parabolic single-cell protein synthesis kinetics at large specific growth rates. Utilization of frequency function data for an asynchronous population is in this case a far more sensitive method for determination of single-cell kinetics than is monitoring the metabolic dynamics of a single cell or, equivalently, synchronous culture analyses.

  17. Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

    2016-06-01

    Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10–100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity.

  18. Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

    PubMed Central

    Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

    2016-01-01

    Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10–100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity. PMID:27283981

  19. Microfluidic techniques for high throughput single cell analysis.

    PubMed

    Reece, Amy; Xia, Bingzhao; Jiang, Zhongliang; Noren, Benjamin; McBride, Ralph; Oakey, John

    2016-08-01

    The microfabrication of microfluidic control systems and the development of increasingly sensitive molecular amplification tools have enabled the miniaturization of single cells analytical platforms. Only recently has the throughput of these platforms increased to a level at which populations can be screened at the single cell level. Techniques based upon both active and passive manipulation are now capable of discriminating between single cell phenotypes for sorting, diagnostic or prognostic applications in a variety of clinical scenarios. The introduction of multiphase microfluidics enables the segmentation of single cells into biochemically discrete picoliter environments. The combination of these techniques are enabling a class of single cell analytical platforms within great potential for data driven biomedicine, genomics and transcriptomics. PMID:27032065

  20. UV Decontamination of MDA Reagents for Single Cell Genomics

    SciTech Connect

    Lee, Janey; Tighe, Damon; Sczyrba, Alexander; Malmatrom, Rex; Clingenpeel, Scott; Malfatti, Stephanie; Rinke, Christian; Wang, Zhong; Stepanauskas, Ramunas; Cheng, Jan-Fang; Woyke, Tanja

    2011-03-18

    Single cell genomics, the amplification and sequencing of genomes from single cells, can provide a glimpse into the genetic make-up and thus life style of the vast majority of uncultured microbial cells, making it an immensely powerful and increasingly popular tool. This is accomplished by use of multiple displacement amplification (MDA), which can generate billions of copies of a single bacterial genome producing microgram-range DNA required for shotgun sequencing. Here, we address a key challenge inherent to this approach and propose a solution for the improved recovery of single cell genomes. While DNA-free reagents for the amplification of a single cell genome are a prerequisite for successful single cell sequencing and analysis, DNA contamination has been detected in various reagents, which poses a considerable challenge. Our study demonstrates the effect of UV irradiation in efficient elimination of exogenous contaminant DNA found in MDA reagents, while maintaining Phi29 activity. Consequently, we also find that increased UV exposure to Phi29 does not adversely affect genome coverage of MDA amplified single cells. While additional challenges in single cell genomics remain to be resolved, the proposed methodology is relatively quick and simple and we believe that its application will be of high value for future single cell sequencing projects.

  1. Digital microfluidic immunocytochemistry in single cells.

    PubMed

    Ng, Alphonsus H C; Dean Chamberlain, M; Situ, Haozhong; Lee, Victor; Wheeler, Aaron R

    2015-01-01

    We report a new technique called Digital microfluidic Immunocytochemistry in Single Cells (DISC). DISC automates protocols for cell culture, stimulation and immunocytochemistry, enabling the interrogation of protein phosphorylation on pulsing with stimulus for as little as 3 s. DISC was used to probe the phosphorylation states of platelet-derived growth factor receptor (PDGFR) and the downstream signalling protein, Akt, to evaluate concentration- and time-dependent effects of stimulation. The high time resolution of the technique allowed for surprising new observations-for example, a 10 s pulse stimulus of a low concentration of PDGF is sufficient to cause >30% of adherent fibroblasts to commit to Akt activation. With the ability to quantitatively probe signalling events with high time resolution at the single-cell level, we propose that DISC may be an important new technique for a wide range of applications, especially for screening signalling responses of a heterogeneous cell population. PMID:26104298

  2. Anatahan Activity and Monitoring, 2005

    NASA Astrophysics Data System (ADS)

    Lockhart, A.; White, R.; Koyanagi, S.; Trusdell, F.; Kauahikaua, J.; Marso, J.; Ewert, J.

    2005-12-01

    Anatahan volcano began erupting in 2003 and continued with a second eruptive phase in 2004. In January 2005 the volcano began a sequence of eruptions and unrest that continues as of September 2005. The activity has been characterized by punctuated episodes of very steamy strombolian activity and vigorous ash emission. Some of the ash emissions have reached 50,000-foot elevations, with VOG and ash occasionally reaching the Philippines and southernmost Japan, over 1000 miles away. Vigorous ash emission has been almost continuous since June 2005. A M4.8 long-period earthquake (LP) occurred in mid-August, one of the largest LPs recorded on the planet in the last quarter-century. Real-time monitoring consisting of a few telemetered short-period seismometers and acoustic sensors has been severely hampered by ashfall on the small island. Monitoring efforts have been focused on the aircraft/ash hazard, with the goal of providing the FAA and airline industry with rapid notice of seismic signatures that may indicate ash columns rising to the altitude of airline traffic, or nominally above 20,000-30,000 ft.

  3. A Single-Cell Assay for Time Lapse Studies of Exosome Secretion and Cell Behaviors.

    PubMed

    Chiu, Yu-Jui; Cai, Wei; Shih, Yu-Ru V; Lian, Ian; Lo, Yu-Hwa

    2016-07-01

    To understand the inhomogeneity of cells in biological systems, there is a growing demand on the capability of characterizing the properties of individual single cells. Since single-cell studies require continuous monitoring of the cell behaviors, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and, proliferation of single cells and convenient, noninvasive tests of single-cell behaviors from molecular markers. Here, a highly versatile single-cell assay is presented that can accommodate different cellular types, enable easy and efficient single-cell loading and culturing, and be suitable for the study of effects of in vitro environmental factors in combination with drug screening. One salient feature of the assay is the noninvasive collection and surveying of single-cell secretions at different time points, producing unprecedented insight of single-cell behaviors based on the biomarker signals from individual cells under given perturbations. Above all, the acquired information is quantitative, for example, measured by the number of exosomes each single-cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single-cell properties. PMID:27254278

  4. Superposition of Individual Activities: Urea-Mediated Suppression of Nitrate Uptake in the Dinoflagellate Prorocentrum minimum Revealed at the Population and Single-Cell Levels.

    PubMed

    Matantseva, Olga; Skarlato, Sergei; Vogts, Angela; Pozdnyakov, Ilya; Liskow, Iris; Schubert, Hendrik; Voss, Maren

    2016-01-01

    Dinoflagellates readily use diverse inorganic and organic compounds as nitrogen sources, which is advantageous in eutrophied coastal areas exposed to high loads of anthropogenic nutrients, e.g., urea, one of the most abundant organic nitrogen substrates in seawater. Cell-to-cell variability in nutritional physiology can further enhance the diversity of metabolic strategies among dinoflagellates of the same species, but it has not been studied in free-living microalgae. We applied stable isotope tracers, isotope ratio mass spectrometry and nanoscale secondary ion mass spectrometry (NanoSIMS) to investigate the response of cultured nitrate-acclimated dinoflagellates Prorocentrum minimum to a sudden input of urea and the effect of urea on the concurrent nitrate uptake at the population and single-cell levels. We demonstrate that inputs of urea lead to suppression of nitrate uptake by P. minimum, and urea uptake exceeds the concurrent uptake of nitrate. Individual dinoflagellate cells within a population display significant heterogeneity in the rates of nutrient uptake and extent of the urea-mediated inhibition of the nitrate uptake, thus forming several groups characterized by different modes of nutrition. We conclude that urea originating from sporadic sources is rapidly utilized by dinoflagellates and can be used in biosynthesis or stored intracellularly depending on the nutrient status; therefore, sudden urea inputs can represent one of the factors triggering or supporting harmful algal blooms. Significant physiological heterogeneity revealed at the single-cell level is likely to play a role in alleviation of intra-population competition for resources and can affect the dynamics of phytoplankton populations and their maintenance in natural environments. PMID:27610101

  5. Superposition of Individual Activities: Urea-Mediated Suppression of Nitrate Uptake in the Dinoflagellate Prorocentrum minimum Revealed at the Population and Single-Cell Levels

    PubMed Central

    Matantseva, Olga; Skarlato, Sergei; Vogts, Angela; Pozdnyakov, Ilya; Liskow, Iris; Schubert, Hendrik; Voss, Maren

    2016-01-01

    Dinoflagellates readily use diverse inorganic and organic compounds as nitrogen sources, which is advantageous in eutrophied coastal areas exposed to high loads of anthropogenic nutrients, e.g., urea, one of the most abundant organic nitrogen substrates in seawater. Cell-to-cell variability in nutritional physiology can further enhance the diversity of metabolic strategies among dinoflagellates of the same species, but it has not been studied in free-living microalgae. We applied stable isotope tracers, isotope ratio mass spectrometry and nanoscale secondary ion mass spectrometry (NanoSIMS) to investigate the response of cultured nitrate-acclimated dinoflagellates Prorocentrum minimum to a sudden input of urea and the effect of urea on the concurrent nitrate uptake at the population and single-cell levels. We demonstrate that inputs of urea lead to suppression of nitrate uptake by P. minimum, and urea uptake exceeds the concurrent uptake of nitrate. Individual dinoflagellate cells within a population display significant heterogeneity in the rates of nutrient uptake and extent of the urea-mediated inhibition of the nitrate uptake, thus forming several groups characterized by different modes of nutrition. We conclude that urea originating from sporadic sources is rapidly utilized by dinoflagellates and can be used in biosynthesis or stored intracellularly depending on the nutrient status; therefore, sudden urea inputs can represent one of the factors triggering or supporting harmful algal blooms. Significant physiological heterogeneity revealed at the single-cell level is likely to play a role in alleviation of intra-population competition for resources and can affect the dynamics of phytoplankton populations and their maintenance in natural environments. PMID:27610101

  6. Single-cell RNA-seq reveals activation of unique gene groups as a consequence of stem cell-parenchymal cell fusion.

    PubMed

    Freeman, Brian T; Jung, Jangwook P; Ogle, Brenda M

    2016-01-01

    Fusion of donor mesenchymal stem cells with parenchymal cells of the recipient can occur in the brain, liver, intestine and heart following transplantation. The therapeutic benefit or detriment of resultant hybrids is unknown. Here we sought a global view of phenotypic diversification of mesenchymal stem cell-cardiomyocyte hybrids and associated time course. Using single-cell RNA-seq, we found hybrids consistently increase ribosome components and decrease genes associated with the cell cycle suggesting an increase in protein production and decrease in proliferation to accommodate the fused state. But in the case of most other gene groups, hybrids were individually distinct. In fact, though hybrids can express a transcriptome similar to individual fusion partners, approximately one-third acquired distinct expression profiles in a single day. Some hybrids underwent reprogramming, expressing pluripotency and cardiac precursor genes latent in parental cells and associated with developmental and morphogenic gene groups. Other hybrids expressed genes associated with ontologic cancer sets and two hybrids of separate experimental replicates clustered with breast cancer cells, expressing critical oncogenes and lacking tumor suppressor genes. Rapid transcriptional diversification of this type garners consideration in the context of cellular transplantation to damaged tissues, those with viral infection or other microenvironmental conditions that might promote fusion. PMID:26997336

  7. Single-cell RNA-seq reveals activation of unique gene groups as a consequence of stem cell-parenchymal cell fusion

    PubMed Central

    Freeman, Brian T.; Jung, Jangwook P.; Ogle, Brenda M.

    2016-01-01

    Fusion of donor mesenchymal stem cells with parenchymal cells of the recipient can occur in the brain, liver, intestine and heart following transplantation. The therapeutic benefit or detriment of resultant hybrids is unknown. Here we sought a global view of phenotypic diversification of mesenchymal stem cell-cardiomyocyte hybrids and associated time course. Using single-cell RNA-seq, we found hybrids consistently increase ribosome components and decrease genes associated with the cell cycle suggesting an increase in protein production and decrease in proliferation to accommodate the fused state. But in the case of most other gene groups, hybrids were individually distinct. In fact, though hybrids can express a transcriptome similar to individual fusion partners, approximately one-third acquired distinct expression profiles in a single day. Some hybrids underwent reprogramming, expressing pluripotency and cardiac precursor genes latent in parental cells and associated with developmental and morphogenic gene groups. Other hybrids expressed genes associated with ontologic cancer sets and two hybrids of separate experimental replicates clustered with breast cancer cells, expressing critical oncogenes and lacking tumor suppressor genes. Rapid transcriptional diversification of this type garners consideration in the context of cellular transplantation to damaged tissues, those with viral infection or other microenvironmental conditions that might promote fusion. PMID:26997336

  8. Lhcb transcription is coordinated with cell size and chlorophyll accumulation. Studies on fluorescence-activated, cell-sorter-purified single cells from wild-type and immutans Arabidopsis thaliana

    SciTech Connect

    Meehan, L.; Harkins, K.; Rodermel, S.

    1996-11-01

    To study the mechanisms that integrate pigment and chlorophyll a/b-binding apoprotein biosynthesis during light-harvesting complex II assembly, we have examined {beta}-glucuronidase (GUS) enzyme activities, cell-sorting-separated single cells sizes in fluorescence activated, cell-sorting-separated single cells from transgenic Arabidopsis thaliana wild-type and immutans variegation mutant plants that express an Lhcb (photosystem II chlorophyll a/b-binding polypeptide gene)/GUS promoter fusion. We found that GUS activities are positively correlated with chlorophyll content and cell size in green cells from the control and immutans plants, indicating that Lhcb gene transcription is coordinated with cell size in this species. Compared with the control plants, however, chlorophyll production is enhanced in the green cells of immutans; this may represent part of a strategy to maximize photosynthesis in the white sectors of the mutant. Lhcb transcription is significantly higher in pure-white cells of the transgenic immutans plants than in pure-white cells from norflurazon-treated, photooxidized A. thaliana leaves. This suggests that immutans partially uncouples Lhcb transcription from its normal dependence on chlorophyll accumulation and chloroplast development. We conclude that immutans may play a role in regulating Lhcb transcription, and may be a key component in the signal transduction pathways that control chloroplast biogenesis. 58 refs., 5 figs., 2 tabs.

  9. Advances in High-Throughput Single-Cell Microtechnologies

    PubMed Central

    Weaver, Westbrook M.; Tseng, Peter; Kunze, Anja; Masaeli, Mahdohkht; Chung, Aram J.; Dudani, Jaideep S.; Kittur, Harsha; Kulkarni, Rajan P.; Di Carlo, Dino

    2013-01-01

    Micro-scale biological tools that have allowed probing of individual cells - from the genetic, to proteomic, to phenotypic level - have revealed important contributions of single cells to direct normal and diseased body processes. In analyzing single cells, sample heterogeneity between and within specific cell types drives the need for high-throughput and quantitative measurement of cellular parameters. In recent years, high-throughput single-cell analysis platforms have revealed rare genetic subpopulations in growing tumors, begun to uncover the mechanisms of antibiotic resistance in bacteria, and described the cell-to-cell variations in stem cell differentiation and immune cell response to activation by pathogens. This review surveys these recent technologies, presenting their strengths and contributions to the field, and identifies needs still unmet toward the development of high-throughput single-cell analysis tools to benefit life science research and clinical diagnostics. PMID:24484889

  10. Prediction and monitoring of volcanic activities

    SciTech Connect

    Sudradjat, A.

    1986-07-01

    This paper summarizes the state of the art for predicting and monitoring volcanic activities, and it emphasizes the experience obtained by the Volcanological Survey Indonesia for active volcanoes. The limited available funds, the large number of active volcanoes to monitor, and the high population density of the volcanic area are the main problems encountered. Seven methods of volcano monitoring are applied to the active volcanoes of Indonesia: seismicity, ground deformation, gravity and magnetic studies, self-potential studies, petrochemistry, gas monitoring, and visual observation. Seismic monitoring augmented by gas monitoring has proven to be effective, particularly for predicting individual eruptions at the after-initial phase. However, the success of the prediction depends on the characteristics of each volcano. In general, the initial eruption phase is the most difficult phenomenon to predict. The preparation of hazard maps and the continuous awareness of the volcanic eruption are the most practical ways to mitigate volcanic danger.

  11. Inside Single Cells: Quantitative Analysis with Advanced Optics and Nanomaterials

    PubMed Central

    Cui, Yi; Irudayaraj, Joseph

    2014-01-01

    Single cell explorations offer a unique window to inspect molecules and events relevant to mechanisms and heterogeneity constituting the central dogma of biology. A large number of nucleic acids, proteins, metabolites and small molecules are involved in determining and fine-tuning the state and function of a single cell at a given time point. Advanced optical platforms and nanotools provide tremendous opportunities to probe intracellular components with single-molecule accuracy, as well as promising tools to adjust single cell activity. In order to obtain quantitative information (e.g. molecular quantity, kinetics and stoichiometry) within an intact cell, achieving the observation with comparable spatiotemporal resolution is a challenge. For single cell studies both the method of detection and the biocompatibility are critical factors as they determine the feasibility, especially when considering live cell analysis. Although a considerable proportion of single cell methodologies depend on specialized expertise and expensive instruments, it is our expectation that the information content and implication will outweigh the costs given the impact on life science enabled by single cell analysis. PMID:25430077

  12. Inside single cells: quantitative analysis with advanced optics and nanomaterials.

    PubMed

    Cui, Yi; Irudayaraj, Joseph

    2015-01-01

    Single-cell explorations offer a unique window to inspect molecules and events relevant to mechanisms and heterogeneity constituting the central dogma of biology. A large number of nucleic acids, proteins, metabolites, and small molecules are involved in determining and fine-tuning the state and function of a single cell at a given time point. Advanced optical platforms and nanotools provide tremendous opportunities to probe intracellular components with single-molecule accuracy, as well as promising tools to adjust single-cell activity. To obtain quantitative information (e.g., molecular quantity, kinetics, and stoichiometry) within an intact cell, achieving the observation with comparable spatiotemporal resolution is a challenge. For single-cell studies, both the method of detection and the biocompatibility are critical factors as they determine the feasibility, especially when considering live-cell analysis. Although a considerable proportion of single-cell methodologies depend on specialized expertise and expensive instruments, it is our expectation that the information content and implication will outweigh the costs given the impact on life science enabled by single-cell analysis. PMID:25430077

  13. A microfluidic approach to parallelized transcriptional profiling of single cells

    PubMed Central

    Sun, Hao; Olsen, Timothy; Zhu, Jing; Tao, Jianguo; Ponnaiya, Brian; Amundson, Sally A.; Brenner, David J.; Lin, Qiao

    2016-01-01

    The ability to correlate single-cell genetic information with cellular phenotypes is of great importance to biology and medicine, as it holds the potential to gain insight into disease pathways that is unavailable from ensemble measurements. We present a microfluidic approach to parallelized, rapid, quantitative analysis of messenger RNA from single cells via RT-qPCR. The approach leverages an array of single-cell RT-qPCR analysis units formed by a set of parallel microchannels concurrently controlled by elastomeric pneumatic valves, thereby enabling parallelized handling and processing of single cells in a drastically simplified operation procedure using a relatively small number of microvalves. All steps for single-cell RT-qPCR, including cell isolation and immobilization, cell lysis, mRNA purification, reverse transcription and qPCR, are integrated on a single chip, eliminating the need for off-chip manual cell and reagent transfer and qPCR amplification as commonly used in existing approaches. Additionally, the approach incorporates optically transparent microfluidic components to allow monitoring of single-cell trapping without the need for molecular labeling that can potentially alter the targeted gene expression and utilizes a polycarbonate film as a barrier against evaporation to minimize the loss of reagents at elevated temperatures during the analysis. We demonstrate the utility of the approach by the transcriptional profiling for the induction of the cyclin-dependent kinase inhibitor 1a and the glyceraldehyde 3-phosphate dehydrogenase in single cells from the MCF-7 breast cancer cell line. Furthermore, the methyl methanesulfonate is employed to allow measurement of the expression of the genes in individual cells responding to a genotoxic stress. PMID:27194954

  14. A microfluidic platform for regulating signal transduction in single cells

    NASA Astrophysics Data System (ADS)

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

    2004-11-01

    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.

  15. 7 CFR 800.216 - Activities that shall be monitored.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... merchandising activities identified in this section shall be monitored in accordance with the instructions. (b) Grain merchandising activities. Grain merchandising activities subject to monitoring for compliance with...) Recordkeeping activities. Elevator and merchandising recordkeeping activities subject to monitoring...

  16. 7 CFR 800.216 - Activities that shall be monitored.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... merchandising activities identified in this section shall be monitored in accordance with the instructions. (b) Grain merchandising activities. Grain merchandising activities subject to monitoring for compliance with...) Recordkeeping activities. Elevator and merchandising recordkeeping activities subject to monitoring...

  17. Overview of single-cell elastic light scattering techniques.

    PubMed

    Kinnunen, Matti; Karmenyan, Artashes

    2015-05-01

    We present and discuss several modern optical methods based on elastic light scattering (ELS), along with their technical features and applications in biomedicine and life sciences. In particular, we review some ELS experiments at the single-cell level and explore new directions of applications. Due to recent developments in experimental systems (as shown in the literature), ELS lends itself to useful applications in the life sciences. Of the developed methods, we cover elastic scattering spectroscopy, optical tweezer-assisted measurement, goniometers, Fourier transform light scattering (FTLS), and microscopic methods. FTLS significantly extends the potential analysis of single cells by allowing monitoring of dynamical changes at the single-cell level. The main aim of our review is to demonstrate developments in the experimental investigation of ELS in single cells including issues related to theoretical “representations” and modeling of biological systems (cells, cellular systems, tissues, and so on). Goniometric measurements of ELS from optically trapped single cells are shown and the importance of the experimental verification of theoretical models of ELS in the context of biomedical applications is discussed. PMID:25760756

  18. Real-Time Monitoring of Active Landslides

    USGS Publications Warehouse

    Reid, Mark E.; LaHusen, Richard G.; Ellis, William L.

    1999-01-01

    Landslides threaten lives and property in every State in the Nation. To reduce the risk from active landslides, the U.S. Geological Survey (USGS) develops and uses real-time landslide monitoring systems. Monitoring can detect early indications of rapid, catastrophic movement. Up-to-the-minute or real-time monitoring provides immediate notification of landslide activity, potentially saving lives and property. Continuous information from real-time monitoring also provides a better understanding of landslide behavior, enabling engineers to create more effective designs for halting landslide movement.

  19. Airborne chemistry single cell level

    NASA Astrophysics Data System (ADS)

    Nilsson, Staffan; Viberg, Peter; Spegel, Peter; Santesson, Sabina; Cedergren, Eila; Degerman, Eva; Johansson, Tomas; Nilsson, Johan

    2002-11-01

    A miniaturized analysis system for the studying of living cells and biochemical reactions in microdrops was developed. Cell studies were performed using single adipocytes in 250-nL drops. Continuous flow-through droplet dispensers, developed in-house, were used for additions to the levitated droplet. Addition of b-adrenergic agonists stimulates the lipolysis in the adipocytes, leading to free fatty acid release and a consequent pH decrease of the surrounding buffer, a change that can be easily followed using a pH-dependent fluorophore continuously monitored by fluorescence imaging detection. An analytical method using capillary electrophoresis and nanospray mass spectrometry for measurement of the cAMP level in activated single adipocytes are now being developed for future use in combination with the levitation technique. The levitation approach was also employed for the screening of nucleation conditions for macromolecules. Here, the acoustic levitator offers a simplified way to determine the main features of the phase diagram (i.e., precipitation diagram). Using the droplet dispensers, different types and amounts of precipitation agents are injected into the levitated drop, allowing a systematic search for nucleation conditions that is not possible using standard crystallization methods. Once the precipitation diagram has been obtained, optimization using standard methods is employed to grow the crystals.

  20. A new toolbox for assessing single cells.

    PubMed

    Tsioris, Konstantinos; Torres, Alexis J; Douce, Thomas B; Love, J Christopher

    2014-01-01

    Unprecedented access to the biology of single cells is now feasible, enabled by recent technological advancements that allow us to manipulate and measure sparse samples and achieve a new level of resolution in space and time. This review focuses on advances in tools to study single cells for specific areas of biology. We examine both mature and nascent techniques to study single cells at the genomics, transcriptomics, and proteomics level. In addition, we provide an overview of tools that are well suited for following biological responses to defined perturbations with single-cell resolution. Techniques to analyze and manipulate single cells through soluble and chemical ligands, the microenvironment, and cell-cell interactions are provided. For each of these topics, we highlight the biological motivation, applications, methods, recent advances, and opportunities for improvement. The toolbox presented in this review can function as a starting point for the design of single-cell experiments. PMID:24910919

  1. An automated programmable platform enabling multiplex dynamic stimuli delivery and cellular response monitoring for high-throughput suspension single-cell signaling studies† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4lc01070a Click here for additional data file.

    PubMed Central

    He, Luye; Kniss, Ariel; San-Miguel, Adriana; Rouse, Tel; Kemp, Melissa L.

    2015-01-01

    Cell signaling events are orchestrated by dynamic external biochemical cues. By rapidly perturbing cells with dynamic inputs and examining the output from these systems, one could study the structure and dynamic properties of a cellular signaling network. Conventional experimental techniques limit the implementation of these systematic approaches due to the lack of sophistication in manipulating individual cells and the fluid microenvironment around them; existing microfluidic technologies thus far are mainly targeting adherent cells. In this paper we present an automated platform to interrogate suspension cells with dynamic stimuli while simultaneously monitoring cellular responses in a high-throughput manner at single-cell resolution. We demonstrate the use of this platform in an experiment to measure Jurkat T cells in response to distinct dynamic patterns of stimuli; we find cells exhibit highly heterogeneous responses under each stimulation condition. More interestingly, these cells act as low-pass filters, only entrained to the low frequency stimulus signals. We also demonstrate that this platform can be easily programmed to actively generate arbitrary dynamic signals. We envision our platform to be useful in other contexts to study cellular signaling dynamics, which may be difficult using conventional experimental methods. PMID:25609410

  2. Single-Cell Genomics for Virology.

    PubMed

    Ciuffi, Angela; Rato, Sylvie; Telenti, Amalio

    2016-01-01

    Single-cell sequencing technologies, i.e., single cell analysis followed by deep sequencing investigate cellular heterogeneity in many biological settings. It was only in the past year that single-cell sequencing analyses has been applied in the field of virology, providing new ways to explore viral diversity and cell response to viral infection, which are summarized in the present review. PMID:27153082

  3. Single-Cell Genomics for Virology

    PubMed Central

    Ciuffi, Angela; Rato, Sylvie; Telenti, Amalio

    2016-01-01

    Single-cell sequencing technologies, i.e., single cell analysis followed by deep sequencing investigate cellular heterogeneity in many biological settings. It was only in the past year that single-cell sequencing analyses has been applied in the field of virology, providing new ways to explore viral diversity and cell response to viral infection, which are summarized in the present review. PMID:27153082

  4. The niche in single-cell technologies.

    PubMed

    Donati, Giacomo

    2016-03-01

    The niche is the microenvironment in which each cell exists and is able to keep its own peculiar characteristics. The importance of the niche has been intensively studied especially in the context of stem cells, as it is responsible for both the maintenance of stemness and activation of differentiation. In the past few years, a variety of single-cell technologies have shed light on the extraordinary variability that characterizes different stem cell populations both in vitro and in vivo, but in most cases positional information is lost. Recent developments of new technologies aim to integrate both the transcriptomic profiling of cells and their spatial location. In this review I will discuss the state of the art of these technologies and the integration with others approaches that will be important in the study of stem cell populations. PMID:26620629

  5. Single cell analysis of Vibrio harveyi uncovers functional heterogeneity in response to quorum sensing signals

    PubMed Central

    2012-01-01

    Background Vibrio harveyi and closely related species are important pathogens in aquaculture. A complex quorum sensing cascade involving three autoinducers controls bioluminescence and several genes encoding virulence factors. Single cell analysis of a V. harveyi population has already indicated intercellular heterogeneity in the production of bioluminescence. This study was undertaken to analyze the expression of various autoinducer-dependent genes in individual cells. Results Here we used reporter strains bearing promoter::gfp fusions to monitor the induction/repression of three autoinducer-regulated genes in wild type conjugates at the single cell level. Two genes involved in pathogenesis - vhp and vscP, which code for an exoprotease and a component of the type III secretion system, respectively, and luxC (the first gene in the lux operon) were chosen for analysis. The lux operon and the exoprotease gene are induced, while vscP is repressed at high cell density. As controls luxS and recA, whose expression is not dependent on autoinducers, were examined. The responses of the promoter::gfp fusions in individual cells from the same culture ranged from no to high induction. Importantly, simultaneous analysis of two autoinducer induced phenotypes, bioluminescence (light detection) and exoproteolytic activity (fluorescence of a promoter::gfp fusion), in single cells provided evidence for functional heterogeneity within a V. harveyi population. Conclusions Autoinducers are not only an indicator for cell density, but play a pivotal role in the coordination of physiological activities within the population. PMID:22985329

  6. Integrated Electrowetting Nanoinjector for Single Cell Transfection

    PubMed Central

    Shekaramiz, Elaheh; Varadarajalu, Ganeshkumar; Day, Philip J.; Wickramasinghe, H. Kumar

    2016-01-01

    Single cell transfection techniques are essential to understand the heterogeneity between cells. We have developed an integrated electrowetting nanoinjector (INENI) to transfect single cells. The high transfection efficiency, controlled dosage delivery and ease of INENI fabrication promote the widespread application of the INENI in cell transfection assays. PMID:27374766

  7. Tunable Single-Cell Extraction for Molecular Analyses.

    PubMed

    Guillaume-Gentil, Orane; Grindberg, Rashel V; Kooger, Romain; Dorwling-Carter, Livie; Martinez, Vincent; Ossola, Dario; Pilhofer, Martin; Zambelli, Tomaso; Vorholt, Julia A

    2016-07-14

    Because of cellular heterogeneity, the analysis of endogenous molecules from single cells is of significant interest and has major implications. While micromanipulation or cell sorting followed by cell lysis is already used for subsequent molecular examinations, approaches to directly extract the content of living cells remain a challenging but promising alternative to achieving non-destructive sampling and cell-context preservation. Here, we demonstrate the quantitative extraction from single cells with spatiotemporal control using fluidic force microscopy. We further present a comprehensive analysis of the soluble molecules withdrawn from the cytoplasm or the nucleus, including the detection of enzyme activities and transcript abundances. This approach has uncovered the ability of cells to withstand extraction of up to several picoliters and opens opportunities to study cellular dynamics and cell-cell communication under physiological conditions at the single-cell level. PMID:27419874

  8. Apoptosis induction-related cytosolic calcium responses revealed by the dual FRET imaging of calcium signals and caspase-3 activation in a single cell.

    PubMed

    Miyamoto, Akitoshi; Miyauchi, Hiroshi; Kogure, Takako; Miyawaki, Atsushi; Michikawa, Takayuki; Mikoshiba, Katsuhiko

    2015-04-24

    Stimulus-induced changes in the intracellular Ca(2+) concentration control cell fate decision, including apoptosis. However, the precise patterns of the cytosolic Ca(2+) signals that are associated with apoptotic induction remain unknown. We have developed a novel genetically encoded sensor of activated caspase-3 that can be applied in combination with a genetically encoded sensor of the Ca(2+) concentration and have established a dual imaging system that enables the imaging of both cytosolic Ca(2+) signals and caspase-3 activation, which is an indicator of apoptosis, in the same cell. Using this system, we identified differences in the cytosolic Ca(2+) signals of apoptotic and surviving DT40 B lymphocytes after B cell receptor (BCR) stimulation. In surviving cells, BCR stimulation evoked larger initial Ca(2+) spikes followed by a larger sustained elevation of the Ca(2+) concentration than those in apoptotic cells; BCR stimulation also resulted in repetitive transient Ca(2+) spikes, which were mediated by the influx of Ca(2+) from the extracellular space. Our results indicate that the observation of both Ca(2+) signals and cells fate in same cell is crucial to gain an accurate understanding of the function of intracellular Ca(2+) signals in apoptotic induction. PMID:25998736

  9. Monitoring Biological Activity at Geothermal Power Plants

    SciTech Connect

    Peter Pryfogle

    2005-09-01

    The economic impact of microbial growth in geothermal power plants has been estimated to be as high as $500,000 annually for a 100 MWe plant. Many methods are available to monitor biological activity at these facilities; however, very few plants have any on-line monitoring program in place. Metal coupon, selective culturing (MPN), total organic carbon (TOC), adenosine triphosphate (ATP), respirometry, phospholipid fatty acid (PLFA), and denaturing gradient gel electrophoresis (DGGE) characterizations have been conducted using water samples collected from geothermal plants located in California and Utah. In addition, the on-line performance of a commercial electrochemical monitor, the BIoGEORGE?, has been evaluated during extended deployments at geothermal facilities. This report provides a review of these techniques, presents data on their application from laboratory and field studies, and discusses their value in characterizing and monitoring biological activities at geothermal power plants.

  10. Active personal radiation monitor for lunar EVA

    NASA Astrophysics Data System (ADS)

    Straume, Tore; Borak, Tom; Braby, L. A.; Lusby, Terry; Semones, Edward J.; Vazquez, Marcelo E.

    As astronauts return to the Moon-and this time, work for extended periods-there will be a critical need for crew personnel radiation monitoring as they operate lunar rovers or otherwise perform a myriad of extravehicular activities (EVAs). Our focus is on development of a small personal radiation monitor for lunar EVA that responds to the complex radiation quality and changing dose rates on the Moon. Of particular concern are active monitoring capabilities that provide both early warning and radiation dosimetry information during solar particle events (SPEs). To accomplish this, we are developing small detectors integrated with modern high speed, low power microelectronics to measure dose-rate and dose-mean lineal energy in real time. The monitor is designed to perform over the range of dose rates and LETs expected from both GCR and SPE radiations during lunar EVA missions. The monitor design provides simultaneous measurement of dose-equivalent rates at two tissue-equivalent depths simulating skin and marrow. The compact personal monitor is estimated to be the size of a cell phone and would fit on an EVA spacesuit (e.g., in backpack) or in a toolbox. The four-year development effort (which began December 2007) will result in a prototype radiation monitor field tested and characterized for the major radiations expected on the surface of the Moon. We acknowledge support from NSBRI through grants to NASA Ames Research Center (T. Straume, PI) and Colorado State University (T. Borak, PI).

  11. Cellular Stress Responses and Monitored Cellular Activities.

    PubMed

    Sawa, Teiji; Naito, Yoshifumi; Kato, Hideya; Amaya, Fumimasa

    2016-08-01

    To survive, organisms require mechanisms that enable them to sense changes in the outside environment, introduce necessary responses, and resist unfavorable distortion. Consequently, through evolutionary adaptation, cells have become equipped with the apparatus required to monitor their fundamental intracellular processes and the mechanisms needed to try to offset malfunction without receiving any direct signals from the outside environment. It has been shown recently that eukaryotic cells are equipped with a special mechanism that monitors their fundamental cellular functions and that some pathogenic proteobacteria can override this monitoring mechanism to cause harm. The monitored cellular activities involved in the stressed intracellular response have been researched extensively in Caenorhabditis elegans, where discovery of an association between key mitochondrial activities and innate immune responses was named "cellular associated detoxification and defenses (cSADD)." This cellular surveillance pathway (cSADD) oversees core cellular activities such as mitochondrial respiration and protein transport into mitochondria, detects xenobiotics and invading pathogens, and activates the endocrine pathways controlling behavior, detoxification, and immunity. The cSADD pathway is probably associated with cellular responses to stress in human inflammatory diseases. In the critical care field, the pathogenesis of lethal inflammatory syndromes (e.g., respiratory distress syndromes and sepsis) involves the disturbance of mitochondrial respiration leading to cell death. Up-to-date knowledge about monitored cellular activities and cSADD, especially focusing on mitochondrial involvement, can probably help fill a knowledge gap regarding the pathogenesis of lethal inflammatory syndromes in the critical care field. PMID:26954943

  12. Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia.

    PubMed Central

    Jonas, S. K.; Benedetto, C.; Flatman, A.; Hammond, R. H.; Micheletti, L.; Riley, C.; Riley, P. A.; Spargo, D. J.; Zonca, M.; Slater, T. F.

    1992-01-01

    The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected]. Images Figure 1 PMID:1637668

  13. Automated Single Cell Data Decontamination Pipeline

    SciTech Connect

    Tennessen, Kristin; Pati, Amrita

    2014-03-21

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

  14. Active Acoustic Monitoring of Aquatic Life.

    PubMed

    Stein, Peter J; Edson, Patrick

    2016-01-01

    Active acoustic monitoring (AAM) can be used to study the behavioral response of marine life and to mitigate harm during high-danger anthropogenic activities. This has been done in fish studies for many decades, and there are now case studies in which AAM has been used for marine mammal monitoring as well. This includes monitoring where the ranges, AAM frequency of operation, and species are such that the AAM operation is completely outside the hearing range of the animals. However, it also includes AAM operations within the hearing range of marine life, although this does not necessarily that imply AAM is not a suitable tool. It is just not always possible to have a sufficient detection and tracking range and operate at a frequency outside the marine life hearing range. Likely, the best and most important application of AAM is when the anthropogenic activity to be conducted is temporary and presents a clear danger to aquatic life. PMID:26611075

  15. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1990-10-01

    DOE Order 5820.2A requires that low-level waste (LLW) disposal sites active on or after September 1988 and all transuranic (TRU) waste storage sites be monitored periodically to assure that radioactive contamination does not escape from the waste sites and pose a threat to the public or to the environment. This plan describes such a monitoring program for the active LLW disposal sites in SWSA 6 and the TRU waste storage sites in SWSA 5 North. 14 refs., 8 figs.

  16. Efficient Synergistic Single-Cell Genome Assembly.

    PubMed

    Movahedi, Narjes S; Embree, Mallory; Nagarajan, Harish; Zengler, Karsten; Chitsaz, Hamidreza

    2016-01-01

    As the vast majority of all microbes are unculturable, single-cell sequencing has become a significant method to gain insight into microbial physiology. Single-cell sequencing methods, currently powered by multiple displacement genome amplification (MDA), have passed important milestones such as finishing and closing the genome of a prokaryote. However, the quality and reliability of genome assemblies from single cells are still unsatisfactory due to uneven coverage depth and the absence of scattered chunks of the genome in the final collection of reads caused by MDA bias. In this work, our new algorithm Hybrid De novo Assembler (HyDA) demonstrates the power of coassembly of multiple single-cell genomic data sets through significant improvement of the assembly quality in terms of predicted functional elements and length statistics. Coassemblies contain significantly more base pairs and protein coding genes, cover more subsystems, and consist of longer contigs compared to individual assemblies by the same algorithm as well as state-of-the-art single-cell assemblers SPAdes and IDBA-UD. Hybrid De novo Assembler (HyDA) is also able to avoid chimeric assemblies by detecting and separating shared and exclusive pieces of sequence for input data sets. By replacing one deep single-cell sequencing experiment with a few single-cell sequencing experiments of lower depth, the coassembly method can hedge against the risk of failure and loss of the sample, without significantly increasing sequencing cost. Application of the single-cell coassembler HyDA to the study of three uncultured members of an alkane-degrading methanogenic community validated the usefulness of the coassembly concept. HyDA is open source and publicly available at http://chitsazlab.org/software.html, and the raw reads are available at http://chitsazlab.org/research.html. PMID:27243002

  17. Efficient Synergistic Single-Cell Genome Assembly

    PubMed Central

    Movahedi, Narjes S.; Embree, Mallory; Nagarajan, Harish; Zengler, Karsten; Chitsaz, Hamidreza

    2016-01-01

    As the vast majority of all microbes are unculturable, single-cell sequencing has become a significant method to gain insight into microbial physiology. Single-cell sequencing methods, currently powered by multiple displacement genome amplification (MDA), have passed important milestones such as finishing and closing the genome of a prokaryote. However, the quality and reliability of genome assemblies from single cells are still unsatisfactory due to uneven coverage depth and the absence of scattered chunks of the genome in the final collection of reads caused by MDA bias. In this work, our new algorithm Hybrid De novo Assembler (HyDA) demonstrates the power of coassembly of multiple single-cell genomic data sets through significant improvement of the assembly quality in terms of predicted functional elements and length statistics. Coassemblies contain significantly more base pairs and protein coding genes, cover more subsystems, and consist of longer contigs compared to individual assemblies by the same algorithm as well as state-of-the-art single-cell assemblers SPAdes and IDBA-UD. Hybrid De novo Assembler (HyDA) is also able to avoid chimeric assemblies by detecting and separating shared and exclusive pieces of sequence for input data sets. By replacing one deep single-cell sequencing experiment with a few single-cell sequencing experiments of lower depth, the coassembly method can hedge against the risk of failure and loss of the sample, without significantly increasing sequencing cost. Application of the single-cell coassembler HyDA to the study of three uncultured members of an alkane-degrading methanogenic community validated the usefulness of the coassembly concept. HyDA is open source and publicly available at http://chitsazlab.org/software.html, and the raw reads are available at http://chitsazlab.org/research.html. PMID:27243002

  18. Measurement of Larval Activity in the Drosophila Activity Monitor

    PubMed Central

    McParland, Aidan L.; Follansbee, Taylor L.; Ganter, Geoffrey K.

    2016-01-01

    Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals’ positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish. PMID:25993121

  19. Long Wavelength Monitoring of Protein Kinase Activity

    PubMed Central

    Oien, Nathan P.; Nguyen, Luong T.; Jernigan, Finith E.; Priestman, Melanie A.

    2014-01-01

    A family of long wavelength protein kinase fluorescent reporters is described in which the probing wavelength is pre-programmed using readily available fluorophores. These agents can assess protein kinase activity within the optical window of tissue, as exemplified by monitoring endogenous cAMP-dependent protein kinase activity (1) in erythrocyte lysates and (2) in intact erythrocytes using a light-activatable reporter. PMID:24604833

  20. PHASE I SINGLE CELL ELECTROLYZER TEST RESULTS

    SciTech Connect

    Steimke, J; Timothy Steeper, T

    2008-08-05

    This document reports the results of Phase I Single Cell testing of an SO{sub 2}-Depolarized Water Electrolyzer. Testing was performed primarily during the first quarter of FY 2008 at the Savannah River National Laboratory (SRNL) using an electrolyzer cell designed and built at SRNL. Other facility hardware were also designed and built at SRNL. This test further advances this technology for which work began at SRNL in 2005. This research is valuable in achieving the ultimate goal of an economical hydrogen production process based on the Hybrid Sulfur (HyS) Cycle. The focus of this work was to conduct single cell electrolyzer tests to further develop the technology of SO{sub 2}-depolarized electrolysis as part of the HyS Cycle. The HyS Cycle is a hybrid thermochemical cycle that may be used in conjunction with advanced nuclear reactors or centralized solar receivers to produce hydrogen by water-splitting. Like all other sulfur-based cycles, HyS utilizes the high temperature thermal decomposition of sulfuric acid to produce oxygen and regenerate sulfur dioxide. The unique aspect of HyS is the generation of hydrogen in a water electrolyzer that is operated under conditions where dissolved sulfur dioxide depolarizes the anodic reaction, resulting in substantial voltage reduction. Low cell voltage is essential for both thermodynamic efficiency and hydrogen cost. Sulfur dioxide is oxidized at the anode, producing sulfuric acid that is sent to the high temperature acid decomposition portion of the cycle. The electrolyzer cell uses the membrane electrode assembly (MEA) concept. The anode and cathode are formed by spraying platinum containing catalyst on both sides of a Proton Exchange Membrane (PEM). In most testing the material of the PEM was NafionR. The electrolyzer cell active area can be as large as 54.8 cm{sup 2}. Feed to the anode of the electrolyzer is a sulfuric acid solution containing sulfur dioxide. The partial pressure of sulfur dioxide could be varied in the

  1. Single cell correlation fractal dimension of chromatin

    PubMed Central

    Récamier, Vincent; Izeddin, Ignacio; Bosanac, Lana; Dahan, Maxime; Proux, Florence; Darzacq, Xavier

    2014-01-01

    Chromatin is a major nuclear component, and it is an active matter of debate to understand its different levels of spatial organization, as well as its implication in gene regulation. Measurements of nuclear chromatin compaction were recently used to understand how DNA is folded inside the nucleus and to detect cellular dysfunctions such as cancer. Super-resolution imaging opens new possibilities to measure chromatin organization in situ. Here, we performed a direct measure of chromatin compaction at the single cell level. We used histone H2B, one of the 4 core histone proteins forming the nucleosome, as a chromatin density marker. Using photoactivation localization microscopy (PALM) and adaptive optics, we measured the three-dimensional distribution of H2B with nanometric resolution. We computed the distribution of distances between every two points of the chromatin structure, namely the Ripley K(r) distribution. We found that the K(r) distribution of H2B followed a power law, leading to a precise measurement of the correlation fractal dimension of chromatin of 2.7. Moreover, using photoactivable GFP fused to H2B, we observed dynamic evolution of chromatin sub-regions compaction. As a result, the correlation fractal dimension of chromatin reported here can be interpreted as a dynamically maintained non-equilibrium state. PMID:24637833

  2. Research highlights: microfluidic-enabled single-cell epigenetics.

    PubMed

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

    2015-11-01

    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. PMID:26405849

  3. Computational analysis of signaling patterns in single cells

    PubMed Central

    Davis, Denise M.; Purvis, Jeremy E.

    2014-01-01

    Signaling proteins are flexible in both form and function. They can bind to multiple molecular partners and integrate diverse types of cellular information. When imaged by time-lapse microscopy, many signaling proteins show complex patterns of activity or localization that vary from cell to cell. This heterogeneity is so prevalent that it has spurred the development of new computational strategies to analyze single-cell signaling patterns. A collective observation from these analyses is that cells appear less heterogeneous when their responses are normalized to, or synchronized with, other single-cell measurements. In many cases, these transformed signaling patterns show distinct dynamical trends that correspond with predictable phenotypic outcomes. When signaling mechanisms are unclear, computational models can suggest putative molecular interactions that are experimentally testable. Thus, computational analysis of single-cell signaling has not only provided new ways to quantify the responses of individual cells, but has helped resolve longstanding questions surrounding many well-studied human signaling proteins including NF-κB, p53, ERK1/2, and CDK2. A number of specific challenges lie ahead for single-cell analysis such as quantifying the contribution of non-cell autonomous signaling as well as the characterization of protein signaling dynamics in vivo. PMID:25263011

  4. Monitoring Malware Activity on the LAN Network

    NASA Astrophysics Data System (ADS)

    Skrzewski, Mirosław

    Many security related organizations periodically publish current network and systems security information, with the lists of top malware programs. These lists raises the question how these threats spreads out, if the worms (the only threat with own communication abilities) are low or missing on these lists. The paper discuss the research on malware network activity, aimed to deliver the answer to the question, what is the main infection channel of modern malware, done with the usage of virtual honeypot systems on dedicated, unprotected network. Systems setup, network and systems monitoring solutions, results of over three months of network traffic and malware monitoring are presented, along with the proposed answer to our research question.

  5. Reporters to monitor cellular MMP12 activity

    NASA Astrophysics Data System (ADS)

    Cobos-Correa, Amanda; Mall, Marcus A.; Schultz, Carsten

    2010-02-01

    Macrophage elastase, also called MMP12, belongs to a family of proteolytic enzymes whose best known physiological function is the remodeling of the extracellular matrix. Under certain pathological conditions, including inflammation, chronic overexpression of MMP12 has been observed and its elevated proteolytic activity has been suggested to be the cause of pulmonary emphysema. However, it was until recently impossible to monitor the activity of MMP12 under disease conditions, mainly due to a lack of detection methods. Recent development of new reporters for monitoring MMP12 activity in living cells, such as LaRee1, provided novel insights into the pathobiology of MMP12 in pulmonary inflammation.1 In the future, these reporters might contribute to improved diagnosis and in finding better treatments for chronic inflammatory lung diseases and emphysema. Our approach for visualizing MMP12 activity is based on peptidic, membrane-targeted FRET (Foerster Resonance Energy Transfer) reporters. Here we describe a set of new reporters containing different fluorophore pairs as well as modifications in the membrane-targeting lipid moiety. We studied the influence of these modifications on reporter performance and the reporter mobility on live cell membranes by FRAP (fluorescence recovery after photobleaching). Finally, we generated several new fluorescently labeled MMP inhibitors based on the peptidic reporter structures as prototypes for future tools to inhibit and monitor MMP activity at the same time.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

    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.

  7. Multiwavelength Monitoring of Active Galactic Nuclei

    NASA Technical Reports Server (NTRS)

    Peterson, Bradley M.

    2001-01-01

    By intensive monitoring of AGN variability over a large range in wavelength, we can probe the structure and physics of active galactic nuclei on microarcsecond angular scales. For example, multi-wavelength variability data allow us (a) to establish causal relationships between variations in different wavebands, and thus determine which physical processes are primary and which spectral changes are induced by variations at other wavelengths, and (b) through reverberation mapping of the UV/optical emission lines, to determine the structure and kinematics of the line-emitting region, and thus accurately determine the central masses in AGNs. Multiwavelength monitoring is resource-intensive, and is difficult to implement with general-purpose facilities. As a result, virtually all programs undertaken to date have been either sparsely sampled, or short in duration, or both. The potentially high return on this type of investigation, however, argues for dedicated facilities for multiwavelength monitoring programs.

  8. Quantification noise in single cell experiments

    PubMed Central

    Reiter, M.; Kirchner, B.; Müller, H.; Holzhauer, C.; Mann, W.; Pfaffl, M. W.

    2011-01-01

    In quantitative single-cell studies, the critical part is the low amount of nucleic acids present and the resulting experimental variations. In addition biological data obtained from heterogeneous tissue are not reflecting the expression behaviour of every single-cell. These variations can be derived from natural biological variance or can be introduced externally. Both have negative effects on the quantification result. The aim of this study is to make quantitative single-cell studies more transparent and reliable in order to fulfil the MIQE guidelines at the single-cell level. The technical variability introduced by RT, pre-amplification, evaporation, biological material and qPCR itself was evaluated by using RNA or DNA standards. Secondly, the biological expression variances of GAPDH, TNFα, IL-1β, TLR4 were measured by mRNA profiling experiment in single lymphocytes. The used quantification setup was sensitive enough to detect single standard copies and transcripts out of one solitary cell. Most variability was introduced by RT, followed by evaporation, and pre-amplification. The qPCR analysis and the biological matrix introduced only minor variability. Both conducted studies impressively demonstrate the heterogeneity of expression patterns in individual cells and showed clearly today's limitation in quantitative single-cell expression analysis. PMID:21745823

  9. Single-cell transcriptome analysis of endometrial tissue

    PubMed Central

    Krjutškov, K.; Katayama, S.; Saare, M.; Vera-Rodriguez, M.; Lubenets, D.; Samuel, K.; Laisk-Podar, T.; Teder, H.; Einarsdottir, E.; Salumets, A.; Kere, J.

    2016-01-01

    STUDY QUESTION How can we study the full transcriptome of endometrial stromal and epithelial cells at the single-cell level? SUMMARY ANSWER By compiling and developing novel analytical tools for biopsy, tissue cryopreservation and disaggregation, single-cell sorting, library preparation, RNA sequencing (RNA-seq) and statistical data analysis. WHAT IS KNOWN ALREADY Although single-cell transcriptome analyses from various biopsied tissues have been published recently, corresponding protocols for human endometrium have not been described. STUDY DESIGN, SIZE, DURATION The frozen-thawed endometrial biopsies were fluorescence-activated cell sorted (FACS) to distinguish CD13-positive stromal and CD9-positive epithelial cells and single-cell transcriptome analysis performed from biopsied tissues without culturing the cells. We studied gene transcription, applying a modern and efficient RNA-seq protocol. In parallel, endometrial stromal cells were cultured and global expression profiles were compared with uncultured cells. PARTICIPANTS/MATERIALS, SETTING, METHODS For method validation, we used two endometrial biopsies, one from mid-secretory phase (Day 21, LH+8) and another from late-secretory phase (Day 25). The samples underwent single-cell FACS sorting, single-cell RNA-seq library preparation and Illumina sequencing. MAIN RESULTS AND THE ROLE OF CHANCE Here we present a complete pipeline for single-cell gene-expression studies, from clinical sampling to statistical data analysis. Tissue manipulation, starting from disaggregation and cell-type-specific labelling and ending with single-cell automated sorting, is managed within 90 min at low temperature to minimize changes in the gene expression profile. The single living stromal and epithelial cells were sorted using CD13- and CD9-specific antibodies, respectively. Of the 8622 detected genes, 2661 were more active in cultured stromal cells than in biopsy cells. In the comparison of biopsy versus cultured cells, 5603

  10. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  11. MicroBioRobots for single cell manipulation

    NASA Astrophysics Data System (ADS)

    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

  12. CalQuo: automated, simultaneous single-cell and population-level quantification of global intracellular Ca2+ responses

    PubMed Central

    Fritzsche, Marco; Fernandes, Ricardo A.; Colin-York, Huw; Santos, Ana M.; Lee, Steven F.; Lagerholm, B. Christoffer; Davis, Simon J.; Eggeling, Christian

    2015-01-01

    Detecting intracellular calcium signaling with fluorescent calcium indicator dyes is often coupled with microscopy techniques to follow the activation state of non-excitable cells, including lymphocytes. However, the analysis of global intracellular calcium responses both at the single-cell level and in large ensembles simultaneously has yet to be automated. Here, we present a new software package, CalQuo (Calcium Quantification), which allows the automated analysis and simultaneous monitoring of global fluorescent calcium reporter-based signaling responses in up to 1000 single cells per experiment, at temporal resolutions of sub-seconds to seconds. CalQuo quantifies the number and fraction of responding cells, the temporal dependence of calcium signaling and provides global and individual calcium-reporter fluorescence intensity profiles. We demonstrate the utility of the new method by comparing the calcium-based signaling responses of genetically manipulated human lymphocytic cell lines. PMID:26563585

  13. Regenerable activated bauxite adsorbent alkali monitor probe

    DOEpatents

    Lee, Sheldon H. D.

    1992-01-01

    A regenerable activated bauxite adsorber alkali monitor probe for field applications to provide reliable measurement of alkali-vapor concentration in combustion gas with special emphasis on pressurized fluidized-bed combustion (PFBC) off-gas. More particularly, the invention relates to the development of a easily regenerable bauxite adsorbent for use in a method to accurately determine the alkali-vapor content of PFBC exhaust gases.

  14. Regenerable activated bauxite adsorbent alkali monitor probe

    DOEpatents

    Lee, S.H.D.

    1992-12-22

    A regenerable activated bauxite adsorber alkali monitor probe for field applications to provide reliable measurement of alkali-vapor concentration in combustion gas with special emphasis on pressurized fluidized-bed combustion (PFBC) off-gas. More particularly, the invention relates to the development of a easily regenerable bauxite adsorbent for use in a method to accurately determine the alkali-vapor content of PFBC exhaust gases. 6 figs.

  15. Defining cell types and states with single-cell genomics

    PubMed Central

    Trapnell, Cole

    2015-01-01

    A revolution in cellular measurement technology is under way: For the first time, we have the ability to monitor global gene regulation in thousands of individual cells in a single experiment. Such experiments will allow us to discover new cell types and states and trace their developmental origins. They overcome fundamental limitations inherent in measurements of bulk cell population that have frustrated efforts to resolve cellular states. Single-cell genomics and proteomics enable not only precise characterization of cell state, but also provide a stunningly high-resolution view of transitions between states. These measurements may finally make explicit the metaphor that C.H. Waddington posed nearly 60 years ago to explain cellular plasticity: Cells are residents of a vast “landscape” of possible states, over which they travel during development and in disease. Single-cell technology helps not only locate cells on this landscape, but illuminates the molecular mechanisms that shape the landscape itself. However, single-cell genomics is a field in its infancy, with many experimental and computational advances needed to fully realize its full potential. PMID:26430159

  16. Activity monitor accuracy in persons using canes.

    PubMed

    Wendland, Deborah Michael; Sprigle, Stephen H

    2012-01-01

    The StepWatch activity monitor has not been validated on multiple indoor and outdoor surfaces in a population using ambulation aids. The aims of this technical report are to report on strategies to configure the StepWatch activity monitor on subjects using a cane and to report the accuracy of both leg-mounted and cane-mounted StepWatch devices on people ambulating over different surfaces while using a cane. Sixteen subjects aged 67 to 85 yr (mean 75.6) who regularly use a cane for ambulation participated. StepWatch calibration was performed by adjusting sensitivity and cadence. Following calibration optimization, accuracy was tested on both the leg-mounted and cane-mounted devices on different surfaces, including linoleum, sidewalk, grass, ramp, and stairs. The leg-mounted device had an accuracy of 93.4% across all surfaces, while the cane-mounted device had an aggregate accuracy of 84.7% across all surfaces. Accuracy of the StepWatch on the stairs was significantly less accurate (p < 0.001) when comparing surfaces using repeated measures analysis of variance. When monitoring community mobility, placement of a StepWatch on a person and his/her ambulation aid can accurately document both activity and device use. PMID:23341318

  17. Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

    PubMed Central

    2011-01-01

    Background Single cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively. Results Here different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied. Conclusions It was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development. PMID:21496219

  18. Linking Microbial Phylogeny to Metabolic Activity at the Single-Cell Level by Using Enhanced Element Labeling-Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (EL-FISH) and NanoSIMS▿ †

    PubMed Central

    Behrens, Sebastian; Lösekann, Tina; Pett-Ridge, Jennifer; Weber, Peter K.; Ng, Wing-On; Stevenson, Bradley S.; Hutcheon, Ian D.; Relman, David A.; Spormann, Alfred M.

    2008-01-01

    To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with 13C-carbon and 15N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities. PMID:18359832

  19. NEMix: single-cell nested effects models for probabilistic pathway stimulation.

    PubMed

    Siebourg-Polster, Juliane; Mudrak, Daria; Emmenlauer, Mario; Rämö, Pauli; Dehio, Christoph; Greber, Urs; Fröhlich, Holger; Beerenwinkel, Niko

    2015-04-01

    Nested effects models have been used successfully for learning subcellular networks from high-dimensional perturbation effects that result from RNA interference (RNAi) experiments. Here, we further develop the basic nested effects model using high-content single-cell imaging data from RNAi screens of cultured cells infected with human rhinovirus. RNAi screens with single-cell readouts are becoming increasingly common, and they often reveal high cell-to-cell variation. As a consequence of this cellular heterogeneity, knock-downs result in variable effects among cells and lead to weak average phenotypes on the cell population level. To address this confounding factor in network inference, we explicitly model the stimulation status of a signaling pathway in individual cells. We extend the framework of nested effects models to probabilistic combinatorial knock-downs and propose NEMix, a nested effects mixture model that accounts for unobserved pathway activation. We analyzed the identifiability of NEMix and developed a parameter inference scheme based on the Expectation Maximization algorithm. In an extensive simulation study, we show that NEMix improves learning of pathway structures over classical NEMs significantly in the presence of hidden pathway stimulation. We applied our model to single-cell imaging data from RNAi screens monitoring human rhinovirus infection, where limited infection efficiency of the assay results in uncertain pathway stimulation. Using a subset of genes with known interactions, we show that the inferred NEMix network has high accuracy and outperforms the classical nested effects model without hidden pathway activity. NEMix is implemented as part of the R/Bioconductor package 'nem' and available at www.cbg.ethz.ch/software/NEMix. PMID:25879530

  20. NEMix: Single-cell Nested Effects Models for Probabilistic Pathway Stimulation

    PubMed Central

    Siebourg-Polster, Juliane; Mudrak, Daria; Emmenlauer, Mario; Rämö, Pauli; Dehio, Christoph; Greber, Urs; Fröhlich, Holger; Beerenwinkel, Niko

    2015-01-01

    Nested effects models have been used successfully for learning subcellular networks from high-dimensional perturbation effects that result from RNA interference (RNAi) experiments. Here, we further develop the basic nested effects model using high-content single-cell imaging data from RNAi screens of cultured cells infected with human rhinovirus. RNAi screens with single-cell readouts are becoming increasingly common, and they often reveal high cell-to-cell variation. As a consequence of this cellular heterogeneity, knock-downs result in variable effects among cells and lead to weak average phenotypes on the cell population level. To address this confounding factor in network inference, we explicitly model the stimulation status of a signaling pathway in individual cells. We extend the framework of nested effects models to probabilistic combinatorial knock-downs and propose NEMix, a nested effects mixture model that accounts for unobserved pathway activation. We analyzed the identifiability of NEMix and developed a parameter inference scheme based on the Expectation Maximization algorithm. In an extensive simulation study, we show that NEMix improves learning of pathway structures over classical NEMs significantly in the presence of hidden pathway stimulation. We applied our model to single-cell imaging data from RNAi screens monitoring human rhinovirus infection, where limited infection efficiency of the assay results in uncertain pathway stimulation. Using a subset of genes with known interactions, we show that the inferred NEMix network has high accuracy and outperforms the classical nested effects model without hidden pathway activity. NEMix is implemented as part of the R/Bioconductor package ‘nem’ and available at www.cbg.ethz.ch/software/NEMix. PMID:25879530

  1. Capillary Electrophoretic Technologies for Single Cell Metabolomics

    ERIC Educational Resources Information Center

    Lapainis, Theodore E.

    2009-01-01

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

  2. Localized, macromolecular transport for thin, adherent, single cells via an automated, single cell electroporation biomanipulator.

    PubMed

    Sakaki, Kelly; Esmaeilsabzali, Hadi; Massah, Shabnam; Prefontaine, Gratien G; Dechev, Nikolai; Burke, Robert D; Park, Edward J

    2013-11-01

    Single cell electroporation (SCE), via microcapillary, is an effective method for molecular, transmembrane transport used to gain insight on cell processes with minimal preparation. Although possessing great potential, SCE is difficult to execute and the technology spans broad fields within cell biology and engineering. The technical complexities, the focus and expertise demanded during manual operation, and the lack of an automated SCE platform limit the widespread use of this technique, thus the potential of SCE has not been realized. In this study, an automated biomanipulator for SCE is presented. Our system is capable of delivering molecules into the cytoplasm of extremely thin cellular features of adherent cells. The intent of the system is to abstract the technical challenges and exploit the accuracy and repeatability of automated instrumentation, leaving only the focus of the experimental design to the operator. Each sequence of SCE including cell and SCE site localization, tip-membrane contact detection, and SCE has been automated. Positions of low-contrast cells are localized and "SCE sites" for microcapillary tip placement are determined using machine vision. In addition, new milestones within automated cell manipulation have been achieved. The system described herein has the capability of automated SCE of "thin" cell features less than 10 μm in thickness. Finally, SCE events are anticipated using visual feedback, while monitoring fluorescing dye entering the cytoplasm of a cell. The execution is demonstrated by inserting a combination of a fluorescing dye and a reporter gene into NIH/3T3 fibroblast cells. PMID:23771309

  3. Laser tweezers Raman spectroscopy of single cells

    NASA Astrophysics Data System (ADS)

    Chen, De

    Raman scattering is an inelastic collision between the vibrating molecules inside the sample and the incident photons. During this process, energy exchange takes place between the photon and the scattering molecule. By measuring the energy change of the photon, the molecular vibration mode can be probed. The vibrational spectrum contains valuable information about the disposition of atomic nuclei and chemical bonds within a molecule, the chemical compositions and the interactions between the molecule and its surroundings. In this dissertation, laser tweezers Raman spectroscopy (LTRS) technique is applied for the analysis of biological cells and human cells at single cell level. In LTRS, an individual cell is trapped in aqueous medium with laser tweezers, and Raman scattering spectra from the trapped cell are recorded in real-time. The Raman spectra of these cells can be used to reveal the dynamical processes of cell growth, cell response to environment changes, and can be used as the finger print for the identification of a bacterial cell species. Several biophysical experiments were carried out using LTRS: (1) the dynamic germination process of individual spores of Bacillus thuringiensis was detected via Ca-DPA, a spore-specific biomarker molecule; (2) inactivation and killing of Bacillus subtilis spores by microwave irradiation and wet heat were studied at single cell level; (3) the heat shock activation process of single B. subtilis spores were analyzed, in which the reversible transition from glass-like state at low temperature to liquid-like state at high temperature in spore was revealed at the molecular level; (4) the kinetic processes of bacterial cell lysis of E. coli by lysozyme and by temperature induction of lambda phage were detected real-time; (5) the fixation and rehydration of human platelets were quantitatively evaluated and characterized with Raman spectroscopy method, which provided a rapid way to quantify the quality of freeze-dried therapeutic

  4. Studying bacterial quorum-sensing at the single cell level

    NASA Astrophysics Data System (ADS)

    Delfino Perez, Pablo; Pelakh, Leslie; Young, Jonathan; Johnson, Elaine; Hagen, Stephen

    2010-03-01

    Like many bacterial species, Vibrio fischeri can detect its own population density through a quorum sensing (QS) mechanism. The bacterium releases a signal molecule (AI, autoinducer), which accumulates at high population density and triggers a genetic switch. In V.fischeri this leads to bioluminescence. Little is known about how stochastic gene expression affects QS at the level of single cells. We are imaging the luminescence of individual V.fischeri cells in a flow chamber and directly measuring the intercell variability in AI activation of the QS circuit. Our single-cell luminescence experiments allow us to track cells over time and characterize variations in their response to AI levels. We find heterogeneous response to the external signal: at a given AI concentration some cells may be strongly luminescent while others are virtually dark. The analysis of noise in the individual cell response can eventually lead to a better understanding of how cells use QS to gather information about their environment.

  5. Single-cell approaches for molecular classification of endocrine tumors

    PubMed Central

    Koh, James; Allbritton, Nancy L.; Sosa, Julie A.

    2015-01-01

    Purpose of review In this review, we summarize recent developments in single-cell technologies that can be employed for the functional and molecular classification of endocrine cells in normal and neoplastic tissue. Recent findings The emergence of new platforms for the isolation, analysis, and dynamic assessment of individual cell identity and reactive behavior enables experimental deconstruction of intratumoral heterogeneity and other contexts, where variability in cell signaling and biochemical responsiveness inform biological function and clinical presentation. These tools are particularly appropriate for examining and classifying endocrine neoplasias, as the clinical sequelae of these tumors are often driven by disrupted hormonal responsiveness secondary to compromised cell signaling. Single-cell methods allow for multidimensional experimental designs incorporating both spatial and temporal parameters with the capacity to probe dynamic cell signaling behaviors and kinetic response patterns dependent upon sequential agonist challenge. Summary Intratumoral heterogeneity in the provenance, composition, and biological activity of different forms of endocrine neoplasia presents a significant challenge for prognostic assessment. Single-cell technologies provide an array of powerful new approaches uniquely well suited for dissecting complex endocrine tumors. Studies examining the relationship between clinical behavior and tumor compositional variations in cellular activity are now possible, providing new opportunities to deconstruct the underlying mechanisms of endocrine neoplasia. PMID:26632769

  6. Spatially selective sampling of single cells using optically trapped fusogenic emulsion droplets: a new single-cell proteomic tool

    PubMed Central

    Lanigan, Peter M.P.; Chan, Karen; Ninkovic, Tanya; Templer, Richard H.; French, P.M.W.; de Mello, A.J.; Willison, K.R.; Parker, P.J.; Neil, M.A.A.; Ces, Oscar; Klug, D.R.

    2008-01-01

    We present a platform for the spatially selective sampling of the plasma membrane of single cells. Optically trapped lipid-coated oil droplets (smart droplet microtools, SDMs), typically 0.5–5 μm in size, composed of a hexadecane hydrocarbon core and fusogenic lipid outer coating (mixture of 1,2-dioleoyl-phosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) were brought into controlled contact with target colon cancer cells leading to the formation of connecting membrane tethers. Material transfer from the cell to the SDM across the membrane tether was monitored by tracking membrane-localized enhanced green fluorescent protein. PMID:18664432

  7. System and method for monitoring cellular activity

    NASA Technical Reports Server (NTRS)

    Bearman, Gregory H. (Inventor); Fraser, Scott E. (Inventor); Lansford, Russell D. (Inventor)

    2004-01-01

    A system and method for monitoring cellular activity in a cellular specimen. According to one embodiment, a plurality of excitable markers are applied to the specimen. A multi-photon laser microscope is provided to excite a region of the specimen and cause fluorescence to be radiated from the region. The radiating fluorescence is processed by a spectral analyzer to separate the fluorescence into respective wavelength bands. The respective bands of fluorescence are then collected by an array of detectors, with each detector receiving a corresponding one of the wavelength bands.

  8. System and method for monitoring cellular activity

    NASA Technical Reports Server (NTRS)

    Bearman, Gregory H. (Inventor); Fraser, Scott E. (Inventor); Lansford, Russell D. (Inventor)

    2002-01-01

    A system and method for monitoring cellular activity in a cellular specimen. According to one embodiment, a plurality of excitable markers are applied to the specimen. A multi-photon laser microscope is provided to excite a region of the specimen and cause fluorescence to be radiated from the region. The radiating fluorescence is processed by a spectral analyzer to separate the fluorescence into respective wavelength bands. The respective bands of fluorescence are then collected by an array of detectors, with each detector receiving a corresponding one of the wavelength bands.

  9. Single-Cell Analysis of [18F]Fluorodeoxyglucose Uptake by Droplet Radiofluidics

    PubMed Central

    Türkcan, Silvan; Nguyen, Julia; Vilalta, Marta; Shen, Bin; Chin, Frederick T.; Pratx, Guillem; Abbyad, Paul

    2015-01-01

    Radiolabels can be used to detect small biomolecules with high sensitivity and specificity, and without interfering with the biochemical activity of the labeled molecule. For instance, the radiolabeled glucose analogue, [18F]fluorodeoxyglucose (FDG), is routinely used in positron emission tomography (PET) scans for cancer diagnosis, staging and monitoring. However, despite their widespread usage, conventional radionuclide techniques are unable to measure the variability and modulation of FDG uptake in single cells. We present here a novel microfluidic technique, dubbed droplet radiofluidics, that can measure radiotracer uptake for single cells encapsulated into an array of microdroplets. The advantages of this approach are multiple. First, droplets can be quickly and easily positioned in a predetermined pattern for optimal imaging throughput. Second, droplet encapsulation reduces cell efflux as a confounding factor, because any effluxed radionuclide is trapped in the droplet. Last, multiplexed measurements can be performed using fluorescent labels. In this new approach, intracellular radiotracers are imaged on a conventional fluorescence microscope by capturing individual flashes of visible light that are produced as individual positrons, emitted during radioactive decay, traverse a scintillator plate placed below the cells. This method is used to measure the cell-to-cell heterogeneity in the uptake of tracers such as FDG in cell lines and cultured primary cells. The capacity of the platform to perform multiplexed measurements was demonstrated by measuring differential FDG uptake in single cells subjected to different incubation conditions and expressing different types of glucose transporters. This method opens many new avenues of research in basic cell biology and human disease by capturing the full range of stochastic variations in highly heterogeneous cell populations in a repeatable and high-throughput manner. PMID:26035453

  10. Single-Cell Analysis of [18F]Fluorodeoxyglucose Uptake by Droplet Radiofluidics.

    PubMed

    Türkcan, Silvan; Nguyen, Julia; Vilalta, Marta; Shen, Bin; Chin, Frederick T; Pratx, Guillem; Abbyad, Paul

    2015-07-01

    Radiolabels can be used to detect small biomolecules with high sensitivity and specificity without interfering with the biochemical activity of the labeled molecule. For instance, the radiolabeled glucose analogue, [18F]fluorodeoxyglucose (FDG), is routinely used in positron emission tomography (PET) scans for cancer diagnosis, staging, and monitoring. However, despite their widespread usage, conventional radionuclide techniques are unable to measure the variability and modulation of FDG uptake in single cells. We present here a novel microfluidic technique, dubbed droplet radiofluidics, that can measure radiotracer uptake for single cells encapsulated into an array of microdroplets. The advantages of this approach are multiple. First, droplets can be quickly and easily positioned in a predetermined pattern for optimal imaging throughput. Second, droplet encapsulation reduces cell efflux as a confounding factor, because any effluxed radionuclide is trapped in the droplet. Last, multiplexed measurements can be performed using fluorescent labels. In this new approach, intracellular radiotracers are imaged on a conventional fluorescence microscope by capturing individual flashes of visible light that are produced as individual positrons, emitted during radioactive decay, traverse a scintillator plate placed below the cells. This method is used to measure the cell-to-cell heterogeneity in the uptake of tracers such as FDG in cell lines and cultured primary cells. The capacity of the platform to perform multiplexed measurements was demonstrated by measuring differential FDG uptake in single cells subjected to different incubation conditions and expressing different types of glucose transporters. This method opens many new avenues of research in basic cell biology and human disease by capturing the full range of stochastic variations in highly heterogeneous cell populations in a repeatable and high-throughput manner. PMID:26035453

  11. 21 CFR 884.2730 - Home uterine activity monitor.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Home uterine activity monitor. 884.2730 Section... Devices § 884.2730 Home uterine activity monitor. (a) Identification. A home uterine activity monitor (HUAM) is an electronic system for at home antepartum measurement of uterine contractions,...

  12. 21 CFR 884.2730 - Home uterine activity monitor.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Home uterine activity monitor. 884.2730 Section... Devices § 884.2730 Home uterine activity monitor. (a) Identification. A home uterine activity monitor (HUAM) is an electronic system for at home antepartum measurement of uterine contractions,...

  13. 21 CFR 884.2730 - Home uterine activity monitor.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Home uterine activity monitor. 884.2730 Section... Devices § 884.2730 Home uterine activity monitor. (a) Identification. A home uterine activity monitor (HUAM) is an electronic system for at home antepartum measurement of uterine contractions,...

  14. 21 CFR 884.2730 - Home uterine activity monitor.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Home uterine activity monitor. 884.2730 Section... Devices § 884.2730 Home uterine activity monitor. (a) Identification. A home uterine activity monitor (HUAM) is an electronic system for at home antepartum measurement of uterine contractions,...

  15. 21 CFR 884.2730 - Home uterine activity monitor.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Home uterine activity monitor. 884.2730 Section... Devices § 884.2730 Home uterine activity monitor. (a) Identification. A home uterine activity monitor (HUAM) is an electronic system for at home antepartum measurement of uterine contractions,...

  16. Single cell dissection of early kidney development: multilineage priming.

    PubMed

    Brunskill, Eric W; Park, Joo-Seop; Chung, Eunah; Chen, Feng; Magella, Bliss; Potter, S Steven

    2014-08-01

    We used a single cell RNA-seq strategy to create an atlas of gene expression patterns in the developing kidney. At several stages of kidney development, histologically uniform populations of cells give rise to multiple distinct lineages. We performed single cell RNA-seq analysis of total mouse kidneys at E11.5 and E12.5, as well as the renal vesicles at P4. We define an early stage of progenitor cell induction driven primarily by gene repression. Surprising stochastic expression of marker genes associated with differentiated cell types was observed in E11.5 progenitors. We provide a global view of the polarized gene expression already present in the renal vesicle, the first epithelial precursor of the nephron. We show that Hox gene read-through transcripts can be spliced to produce intergenic homeobox swaps. We also identify a surprising number of genes with partially degraded noncoding RNA. Perhaps most interesting, at early developmental times single cells often expressed genes related to several developmental pathways. This provides powerful evidence that initial organogenesis involves a process of multilineage priming. This is followed by a combination of gene repression, which turns off the genes associated with most possible lineages, and the activation of increasing numbers of genes driving the chosen developmental direction. PMID:25053437

  17. Single cell dissection of early kidney development: multilineage priming

    PubMed Central

    Brunskill, Eric W.; Park, Joo-Seop; Chung, Eunah; Chen, Feng; Magella, Bliss; Potter, S. Steven

    2014-01-01

    We used a single cell RNA-seq strategy to create an atlas of gene expression patterns in the developing kidney. At several stages of kidney development, histologically uniform populations of cells give rise to multiple distinct lineages. We performed single cell RNA-seq analysis of total mouse kidneys at E11.5 and E12.5, as well as the renal vesicles at P4. We define an early stage of progenitor cell induction driven primarily by gene repression. Surprising stochastic expression of marker genes associated with differentiated cell types was observed in E11.5 progenitors. We provide a global view of the polarized gene expression already present in the renal vesicle, the first epithelial precursor of the nephron. We show that Hox gene read-through transcripts can be spliced to produce intergenic homeobox swaps. We also identify a surprising number of genes with partially degraded noncoding RNA. Perhaps most interesting, at early developmental times single cells often expressed genes related to several developmental pathways. This provides powerful evidence that initial organogenesis involves a process of multilineage priming. This is followed by a combination of gene repression, which turns off the genes associated with most possible lineages, and the activation of increasing numbers of genes driving the chosen developmental direction. PMID:25053437

  18. Visualizing Wnt Palmitoylation in Single Cells.

    PubMed

    Gao, Xinxin; Hannoush, Rami N

    2016-01-01

    Wnt palmitoylation regulates its secretion and signaling activity in cells. Methods to monitor cellular Wnt palmitoylation are instrumental in investigating Wnt activity, secretion, and its interaction with cellular membrane compartments. This protocol describes a method we have recently developed to detect cellular Wnt palmitoylation. The method, combining click chemistry, bio-orthogonal fatty acid probes, and proximity ligation assay (PLA), provides high sensitivity and subcellular resolution for detection of Wnt palmitoylation. It is also compatible with multiple imaging platforms, and is applicable to detecting palmitoylated forms of other fatty acylated proteins. PMID:27590146

  19. Tree inference for single-cell data.

    PubMed

    Jahn, Katharina; Kuipers, Jack; Beerenwinkel, Niko

    2016-01-01

    Understanding the mutational heterogeneity within tumors is a keystone for the development of efficient cancer therapies. Here, we present SCITE, a stochastic search algorithm to identify the evolutionary history of a tumor from noisy and incomplete mutation profiles of single cells. SCITE comprises a flexible Markov chain Monte Carlo sampling scheme that allows the user to compute the maximum-likelihood mutation history, to sample from the posterior probability distribution, and to estimate the error rates of the underlying sequencing experiments. Evaluation on real cancer data and on simulation studies shows the scalability of SCITE to present-day single-cell sequencing data and improved reconstruction accuracy compared to existing approaches. PMID:27149953

  20. Automated micropipette aspiration of single cells.

    PubMed

    Shojaei-Baghini, Ehsan; Zheng, Yi; Sun, Yu

    2013-06-01

    This paper presents a system for mechanically characterizing single cells using automated micropipette aspiration. Using vision-based control and position control, the system controls a micromanipulator, a motorized translation stage, and a custom-built pressure system to position a micropipette (4 μm opening) to approach a cell, form a seal, and aspirate the cell into the micropipette for quantifying the cell's elastic and viscoelastic parameters as well as viscosity. Image processing algorithms were developed to provide controllers with real-time visual feedback and to accurately measure cell deformation behavior on line. Experiments on both solid-like and liquid-like cells demonstrated that the system is capable of efficiently performing single-cell micropipette aspiration and has low operator skill requirements. PMID:23508635

  1. Mass Cytometry: Single Cells, Many Features.

    PubMed

    Spitzer, Matthew H; Nolan, Garry P

    2016-05-01

    Technology development in biological research often aims to either increase the number of cellular features that can be surveyed simultaneously or enhance the resolution at which such observations are possible. For decades, flow cytometry has balanced these goals to fill a critical need by enabling the measurement of multiple features in single cells, commonly to examine complex or hierarchical cellular systems. Recently, a format for flow cytometry has been developed that leverages the precision of mass spectrometry. This fusion of the two technologies, termed mass cytometry, provides measurement of over 40 simultaneous cellular parameters at single-cell resolution, significantly augmenting the ability of cytometry to evaluate complex cellular systems and processes. In this Primer, we review the current state of mass cytometry, providing an overview of the instrumentation, its present capabilities, and methods of data analysis, as well as thoughts on future developments and applications. PMID:27153492

  2. Single cell-resolution western blotting.

    PubMed

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

    2016-08-01

    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. PMID:27466711

  3. Single cell microfluidics for systems oncology

    NASA Astrophysics Data System (ADS)

    Fan, Rong

    2012-02-01

    The singular term ``cancer'' is never one kind of disease, but deceivingly encompasses a large number of heterogeneous disease states, which makes it impossible to completely treat cancer using a generic approach. Rather systems approaches are urgently required to assess cancer heterogeneity, stratify patients and enable the most effective, individualized treatment. The heterogeneity of tumors at the single cell level is reflected by the hierarchical complexity of the tumor microenvironment. To identify all the cellular components, including both tumor and infiltrating immune cells, and to delineate the associated cell-to-cell signaling network that dictates tumor initiation, progression and metastasis, we developed a single cell microfluidics chip that can analyze a panel of proteins that are potentially associated inter-cellular signaling network in tumor microenvironment from hundreds of single cells in parallel. This platform integrates two advanced technologies -- microfluidic single cell handling and ultra-high density protein array. This device was first tested for highly multiplexed profiling of secreted proteins including tumor-immune signaling molecules from monocytic leukemia cells. We observed profound cellular heterogeneity with all functional phenotypes quantitatively identified. Correlation analysis further indicated the existence of an intercellular cytokine network in which TNFα-induced secondary signaling cascades further increased functional cellular diversity. It was also exploited to evaluate polyfunctionality of tumor antigen-specific T cells from melanoma patients being treated with adoptive T cell transfer immunotherapy. This platform could be further extended to analyze both solid tumor cells (e.g. human lung carcinoma cells) and infiltrating immune cells (e.g. macrophages) so as to enable systems analysis of the complex tumor microenvironment from small amounts of clinical specimens, e.g. skinny needle biopsies. Thus, it could potentially

  4. Computing tumor trees from single cells.

    PubMed

    Davis, Alexander; Navin, Nicholas E

    2016-01-01

    Computational methods have been developed to reconstruct evolutionary lineages from tumors using single-cell genomic data. The resulting tumor trees have important applications in cancer research and clinical oncology.Please see related Research articles: http://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0929-9 and http://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0936-x . PMID:27230879

  5. ATP Consumption of Eukaryotic Flagella Measured at a Single-Cell Level

    NASA Astrophysics Data System (ADS)

    Chen, Daniel T. N.; Heymann, Michael; Fraden, Seth; Nicastro, Daniela; Dogic, Zvonimir

    2015-12-01

    The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. Here, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axonemes ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ~2.3e5 ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights into the beating mechanism of flagella and a powerful tool for future studies.

  6. ATP Consumption of Eukaryotic Flagella Measured at a Single-Cell Level.

    PubMed

    Chen, Daniel T N; Heymann, Michael; Fraden, Seth; Nicastro, Daniela; Dogic, Zvonimir

    2015-12-15

    The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural, and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. In this study, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axoneme's ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ∼2.3 × 10(5) ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights, to our knowledge, into the beating mechanism of flagella and a powerful tool for future studies. PMID:26682814

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

    PubMed Central

    Ren, Lihui; Su, Xiaoquan; Wang, Yun; Xu, Jian

    2014-01-01

    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. PMID:25024908

  8. Quality Assurance Project Plan for Facility Effluent Monitoring Plan activities

    SciTech Connect

    Frazier, T.P.

    1994-10-20

    This Quality Assurance Project Plan addresses the quality assurance requirements for the activities associated with the Facility Effluent Monitoring Plans, which are part of the overall Hanford Site Environmental Protection Plan. This plan specifically applies to the sampling and analysis activities and continuous monitoring performed for all Facility Effluent Monitoring Plan activities conducted by Westinghouse Hanford Company. It is generic in approach and will be implemented in conjunction with the specific requirements of the individual Facility Effluent Monitoring Plans.

  9. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-06-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis.

  10. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    PubMed Central

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-01-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis. PMID:27292795

  11. Accelerometer based calf muscle pump activity monitoring.

    PubMed

    O'Donovan, Karol J; O'Keeffe, Derek T; Grace, Pierce A; Lyons, Gerard M

    2005-10-01

    Long distance travel is associated with increased risk of deep vein thrombosis (DVT). There is an increased risk of travel related DVT in passengers with a predisposition to thrombosis. Assisting blood circulation in the lower limb will reduce the risk of DVT. Leg exercises are recommended as a DVT preventative measure while flying but this fails to account for a passenger who is distracted by in flight entertainment or who falls asleep for an extended period. A method for monitoring calf muscle pump activity using accelerometers has been developed and evaluated. The proposed technique could be used to alert the traveller that there is a need to exercise their calf muscle, thus reducing the risk of DVT. PMID:16139770

  12. Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis

    PubMed Central

    Lo, Shih-Jie; Yao, Da-Jeng

    2015-01-01

    This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell. PMID:26213918

  13. In vivo lipidomics using single-cell Raman spectroscopy

    PubMed Central

    Wu, Huawen; Volponi, Joanne V.; Oliver, Ann E.; Parikh, Atul N.; Simmons, Blake A.; Singh, Seema

    2011-01-01

    We describe a method for direct, quantitative, in vivo lipid profiling of oil-producing microalgae using single-cell laser-trapping Raman spectroscopy. This approach is demonstrated in the quantitative determination of the degree of unsaturation and transition temperatures of constituent lipids within microalgae. These properties are important markers for determining engine compatibility and performance metrics of algal biodiesel. We show that these factors can be directly measured from a single living microalgal cell held in place with an optical trap while simultaneously collecting Raman data. Cellular response to different growth conditions is monitored in real time. Our approach circumvents the need for lipid extraction and analysis that is both slow and invasive. Furthermore, this technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of impermeability, toxicity, and specificity of the fluorescent probes common in currently used protocols. Although the single-cell Raman spectroscopy demonstrated here is focused on the study of the microalgal lipids with biofuel applications, the analytical capability and quantitation algorithms demonstrated are applicable to many different organisms and should prove useful for a diverse range of applications in lipidomics. PMID:21310969

  14. Nanosecond fluorescence microscopy of single cells

    NASA Astrophysics Data System (ADS)

    Keating, Susan M.; Wensel, Theodore G.

    1990-05-01

    A microscope based time-correlated single photon counting instrument has been used to measure nanosecond fluorescence decays from single cells. The excitation source for the instrument is a frequency doubled train of picosecond pulses from the cavity dumped output of a synchronously pumped dye laser. The dye laser is pumped by a mode-locked argon ion laser. In the microscope, the sample is excited and the emission collected using epi-illumination optics before being transmitted through an adjustable diaphragm, which can be closed to 10 μm in diameter. A Hamamatsu R928 photomultiplier is used to collect the fluorescence which is then analyzed using a non-linear least squares procedure. The microscope has been used to measure the intensity decays of model probes to determine the instrument performance and sensitivity. In addition, intensity and anisotropy decays collected from fura-2 loaded into single adherent rat basophilic leukemia cells were measured to demonstrate that the nanosecond fluorescence microscope can be used to obtain information about the environment and mobility of fluorescent probes in single cells.

  15. Single Cell Chromatography, LDRD Feasibility Study

    SciTech Connect

    Knize, M G; Bailey, C G

    2007-02-22

    A limitation in the mass spectrometry of biological materials is the reduced ion formation caused by sample complexity. We proposed to develop an enabling technology, single cell planar chromatography, which will greatly increase the amount of chemical information that can be obtained from single biological cells when using imaging mass spectrometry or other surface analysis methods. The sample preparation methods were developed for the time-of-flight secondary mass spectrometer (ToF-SIMS) at LLNL. This instrument has a measured zeptomole (10{sup -21} mole, 600 atoms) limit-of-detection for a molecule with a mass to charge ratio of 225[1]. Our goal was to use planar chromatographic separation to approach similar low limits of detection even with the chemically complex contents of a single cell. The process was proposed to reduce ion suppression and at the same time expose more of the cell contents to the ion beam. The method of work was to deposit biological cells on a silicon chip with suitable chromatographic and electrical properties, dissolve the cell with a droplet of solvent, allow the solvent to evaporate, and then allow the movement of cell contents laterally by immersing an edge of the chip in to a chromatographic solvent, that then moves through the chromatographic matrix allowing the components to interact with, and be separated by, the chromatographic substrate. This process is a miniaturized version of thin layer chromatography with detection by surface mass spectrometry.

  16. Optical manipulation for single-cell studies.

    PubMed

    Ramser, Kerstin; Hanstorp, Dag

    2010-04-01

    In the last decade optical manipulation has evolved from a field of interest for physicists to a versatile tool widely used within life sciences. This has been made possible in particular due to the development of a large variety of imaging techniques that allow detailed information to be gained from investigations of single cells. The use of multiple optical traps has high potential within single-cell analysis since parallel measurements provide good statistics. Multifunctional optical tweezers are, for instance, used to study cell heterogeneity in an ensemble, and force measurements are used to investigate the mechanical properties of individual cells. Investigations of molecular motors and forces on the single-molecule level have led to discoveries that would have been difficult to make with other techniques. Optical manipulation has prospects within the field of cell signalling and tissue engineering. When combined with microfluidic systems the chemical environment of cells can be precisely controlled. Hence the influence of pH, salt concentration, drugs and temperature can be investigated in real time. Fast advancing technical developments of automated and user-friendly optical manipulation tools and cross-disciplinary collaboration will contribute to the routinely use of optical manipulation techniques within the life sciences. PMID:19718682

  17. Single Molecule and Single Cell Epigenomics

    PubMed Central

    Hyun, Byung-Ryool; McElwee, John L.; Soloway, Paul D.

    2014-01-01

    Dynamically regulated changes in chromatin states are vital for normal development and can produce disease when they go awry. Accordingly, much effort has been devoted to characterizing these states under normal and pathological conditions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most widely used method to characterize where in the genome transcription factors, modified histones, modified nucleotides and chromatin binding proteins are found; bisulfite sequencing (BS-seq) and its variants are commonly used to characterize the locations of DNA modifications. Though very powerful, these methods are not without limitations. Notably, they are best at characterizing one chromatin feature at a time, yet chromatin features arise and function in combination. Investigators commonly superimpose separate ChIP-seq or BS-seq datasets, and then infer where chromatin features are found together. While these inferences might be correct, they can be misleading when the chromatin source has distinct cell types, or when a given cell type exhibits any cell to cell variation in chromatin state. These ambiguities can be eliminated by robust methods that directly characterize the existence and genomic locations of combinations of chromatin features in very small inputs of cells or ideally, single cells. Here we review single molecule epigenomic methods under development to overcome these limitations, the technical challenges associated with single molecule methods and their potential application to single cells. PMID:25204781

  18. Nanofountain Probe Electroporation of Single Cells

    PubMed Central

    Kang, Wonmo; Yavari, Fazel; Minary-Jolandan, Majid; Giraldo-Vela, Juan P.; Safi, Asmahan; McNaughton, Rebecca L.; Parpoil, Victor; Espinosa, Horacio D.

    2013-01-01

    The ability to precisely deliver molecules into single cells is of great interest to biotechnology researchers for advancing applications in therapeutics, diagnostics, and drug delivery toward the promise of personalized medicine. The use of bulk electroporation techniques for cell transfection has increased significantly in the last decade, but the technique is nonspecific and requires high voltage, resulting in variable efficiency and low cell viability. We have developed a new tool for electroporation using nanofountain probe (NFP) technology, which can deliver molecules into cells in a manner that is highly efficient and gentler to cells than bulk electroporation or microinjection. Here we demonstrate NFP electroporation (NFP-E) of single HeLa cells within a population by transfecting them with fluorescently labeled dextran and imaging the cells to evaluate the transfection efficiency and cell viability. Our theoretical analysis of the mechanism of NFP-E reveals that application of the voltage creates a localized electric field between the NFP cantilever tip and the region of the cell membrane in contact with the tip. Therefore, NFP-E can deliver molecules to a target cell with minimal effect of the electric potential on the cell. Our experiments on HeLa cells confirm that NFP-E offers single cell selectivity, high transfection efficiency (>95%), qualitative dosage control, and very high viability (92%) of transfected cells. PMID:23650871

  19. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq.

    PubMed

    Tirosh, Itay; Izar, Benjamin; Prakadan, Sanjay M; Wadsworth, Marc H; Treacy, Daniel; Trombetta, John J; Rotem, Asaf; Rodman, Christopher; Lian, Christine; Murphy, George; Fallahi-Sichani, Mohammad; Dutton-Regester, Ken; Lin, Jia-Ren; Cohen, Ofir; Shah, Parin; Lu, Diana; Genshaft, Alex S; Hughes, Travis K; Ziegler, Carly G K; Kazer, Samuel W; Gaillard, Aleth; Kolb, Kellie E; Villani, Alexandra-Chloé; Johannessen, Cory M; Andreev, Aleksandr Y; Van Allen, Eliezer M; Bertagnolli, Monica; Sorger, Peter K; Sullivan, Ryan J; Flaherty, Keith T; Frederick, Dennie T; Jané-Valbuena, Judit; Yoon, Charles H; Rozenblatt-Rosen, Orit; Shalek, Alex K; Regev, Aviv; Garraway, Levi A

    2016-04-01

    To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies. PMID:27124452

  20. A fast solution switching system with temperature control for single cell measurements

    PubMed Central

    Koh, Duk-Su; Chen, Liangyi; Ufret-Vincenty, Carmen A.; Jung, Seung-Ryoung

    2011-01-01

    This article describes a perfusion system for biophysical single cell experiments at the physiological temperature. Our system regulates temperature of test solutions using a small heat exchanger that includes several capillaries. Water circulating inside the heat exchanger warms or cools test solutions flowing inside the capillaries. Temperature-controlled solutions are delivered directly to a single cell(s) through a multibarreled manifold that switches solutions bathing a cell in less than 1 s. This solution exchange is optimal for patch clamp, single-cell microamperometry, and microfluorometry experiments. Using this system, we demonstrate that exocytosis from pancreatic β cells and activation of TRPV1 channels are temperature sensitive. We also discuss how to measure local temperature near a single cell under investigation. PMID:21536068

  1. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq

    PubMed Central

    Tirosh, Itay; Izar, Benjamin; Prakadan, Sanjay M.; Wadsworth, Marc H.; Treacy, Daniel; Trombetta, John J.; Rotem, Asaf; Rodman, Christopher; Lian, Christine; Murphy, George; Fallahi-Sichani, Mohammad; Dutton-Regester, Ken; Lin, Jia-Ren; Cohen, Ofir; Shah, Parin; Lu, Diana; Genshaft, Alex S.; Hughes, Travis K.; Ziegler, Carly G. K.; Kazer, Samuel W.; Gaillard, Aleth; Kolb, Kellie E.; Villani, Alexandra-Chloé; Johannessen, Cory M.; Andreev, Aleksandr Y.; Van Allen, Eliezer M.; Bertagnolli, Monica; Sorger, Peter K.; Sullivan, Ryan J.; Flaherty, Keith T.; Frederick, Dennie T.; Jané-Valbuena, Judit; Yoon, Charles H.; Rozenblatt-Rosen, Orit; Shalek, Alex K.; Regev, Aviv; Garraway, Levi A.

    2016-01-01

    To explore the distinct genotypic and phenotypic states of melanoma tumors we applied single-cell RNA-seq to 4,645 single cells isolated from 19 patients, profiling malignant, immune, stromal and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that “MITF-high” tumors also contained “AXL-high” tumor cells. Single-cell analyses suggested distinct tumor micro-environmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and to clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single cell genomics offers insights with implications for both targeted and immune therapies. PMID:27124452

  2. Infiltration rate measurement by active perfluorocarbon monitoring

    SciTech Connect

    Menzies, K.T.; Pong, C.M.; Randel, M.A. )

    1987-01-01

    The rate of air infiltration in homes and buildings is a significant factor affecting the magnitude of human exposure to air pollutants in the indoor environment. Several techniques have been utilized for the determination of air infiltration. These include building pressurization and tracer analysis, e.g., SF/sub 6/. Dietz and Cote at Brookhaven National Laboratory (BNL) have developed a simple, steady-state tracer kit that can be utilized by homeowners. This kit includes a source(s) of perfluorocarbon, i.e., perfluoromethylcyclohexane (PMCH) or perfluorodimethylcyclohexane (PDCH), and a passive sampling tube containing Ambersorb XE-347. Typically, the sampling tube is deployed for several days and then returned to a laboratory for analysis by thermal desorption/gas chromatography/electron capture detection. The authors developed an alternative sampling and analysis technique for PMCH/PDCH in homes. In order to facilitate monitoring of short-term infiltration rates (i.e., less than one day) they developed an active sorbent sampling method and solvent desorption/gas chromatography/electron capture detection analytical method. The method is based on the collection of PMCH on charcoal. The method validation, which is discussed in this article, includes analytical method development, selection of a solid sorbent, determination of desorption efficiency, analysis of breakthrough, testing of storage stability, and assessment of precision and accuracy in both the laboratory and field environment.

  3. Active Seismic Monitoring for Earthquake Forecasting

    NASA Astrophysics Data System (ADS)

    Artamonova, M.; Korneev, V.

    2005-12-01

    Earthquake prediction remains high priority issue for disaster prevention. Study of the M6.0 2004 Parkfield and M7.0 1989 Loma Prieta strike-slip earthquakes on the San Andreas Fault (SAF) reveal seismicity peaks in the surrounding crust several months prior to the main events. Earthquakes directly within the SAF zone were intentionally excluded from the analysis because they manifest stress-release processes rather than stress accumulation. The observed increase in seismicity is interpreted as a signature of the increasing stress level in the surrounding crust, while the peak that occurs several months prior to the main event and the subsequent decrease in seismicity are attributed to damage-induced softening processes. Furthermore, in both cases there is a distinctive zone of low seismic activity that surrounds the epicentral region in the pre-event period. The increase of seismicity in the crust surrounding a potential future event and the development of a low-seismicity epicentral zone can be regarded as promising precursory information that could help signal the arrival of large earthquakes. We modeled the seismicity precursor phenomena using finite-element 2D model capable to replicate non-linear breaking of elastic rock. The distinctive seismicity peak was observed for a model simulating SAF properties at Park field. Such peaks are likely to be a good mid-term precursors allowing to declare alerts several months before earthquakes and pointing on their epicenter regions. The short tern alerts require use of active sources and their proper placement in order to monitor the developments of rock softening processes.

  4. Ahead with Cairo. Monitoring country activities.

    PubMed

    Danguilan, M; Wainer, J; Widyantoro, N; Capoor, I; Huq, N; Ashino, Y; Sadasivam, B; Le Thi Nham Tuyet

    1995-04-01

    In the aftermath of the 1994 UN Conference on Population and Development (ICPD) in Cairo, countries are proceeding with their implementation of the plan of action adopted at the conference. A brief description is given of some actions taken by specific countries toward plan implementation. In the Philippines meetings were held immediately after the conference in October on the implications for the Management, Family Planning, and Nongovernmental Organizations programs. The issues of concern were identified as the need for regular consultative meetings among relevant agencies, consultations with women's groups, and a responsive adolescents program. In Australia the program thrust was to focus on the implications for immigration. Monitoring of the plans of action will be undertaken by nongovernmental organizations (NGOs). In Malaysia committees are preparing a program of action suitable for implementation in Malaysia. A regional women's NGO organized a forum on the implications of ICPD for women's reproductive health, women's rights, and empowerment in Malaysia. In Vietnam, press conferences are used to communicate conference results. An NGO translated relevant ICPD materials into Vietnamese. In Indonesia, several ministries convened meetings among donors, NGOs, women's groups, and experts. In India, the government held a national conference. One view was that population issues should be discussed in the context of gender equality and empowerment of women. Another issue was the importance of placing reproductive health in the larger context of health and primary health services. Health personnel at all levels were considered in need of sensitization on gender issues. Problems such as anemia have not been successfully addressed in existing programs. The government agreed to remove in phases target driven programs and the sterilization emphasis. In Bangladesh, a national committee was formed, and NGOs are actively distributing information. In Japan, the Family Planning

  5. Nanoparticle-Aided Amplification of Fluorescence Polarization for Ultrasensitively Monitoring Activity of Telomerase.

    PubMed

    Gao, Yanfang; Xu, Jing; Li, Baoxin; Jin, Yan

    2016-06-01

    To realize facile and reliable analyzing telomerase activity in homogeneous, herein, for the first time, a fluorescent polarization (FP) strategy was developed for polymerase chain reaction (PCR) free monitoring activity of human telomerase at single-cell level ground on gold nanoparticle (GNP) enhancement of FP. First, thiolated telomerase substrate (TS) primer is modified to the surface of GNP via Au-S bond. In the presence of telomerase, TS primer was extended via adding hexamer repeats (GGGTTA), leading to the formation of a long elongation DNA. Several short carboxyfluorescein (FAM)-modified complementary DNA (F-cDNA) can hybridize with the hexamer repeats, resulting in a sharp increase in FP value. Because of the GNP enhancement and self-amplification of telomerase, telomerase activity accounting to one HeLa cell can be rapidly detected in homogeneous solution. Telomerase activities of various cell lines were also favorably estimated. Meanwhile, the inhibition efficiency of telomerase inhibitor was studied, which holds great potential in screening telomerase-targeted anticancer drugs as well. So, a facile method was put forward to reliably and ultrasensitively detect telomerase activity. PMID:27184230

  6. A Canadian View of Monitoring Activities

    ERIC Educational Resources Information Center

    Inhaber, Herbert

    1975-01-01

    A Canadian scientist discusses his country's environmental monitoring programs (by parameter and medium), points out their strengths and weaknesses, and indicates some possible directions for future efforts in the field of environmental monitoring at both the national and international level. (BT)

  7. Digital Microfluidics for Manipulation and Analysis of a Single Cell

    PubMed Central

    He, Jie-Long; Chen, An-Te; Lee, Jyong-Huei; Fan, Shih-Kang

    2015-01-01

    The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed. PMID:26389890

  8. Digital Microfluidics for Manipulation and Analysis of a Single Cell.

    PubMed

    He, Jie-Long; Chen, An-Te; Lee, Jyong-Huei; Fan, Shih-Kang

    2015-01-01

    The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed. PMID:26389890

  9. Single-cell transcriptome sequencing: recent advances and remaining challenges

    PubMed Central

    Liu, Serena; Trapnell, Cole

    2016-01-01

    Single-cell RNA-sequencing methods are now robust and economically practical and are becoming a powerful tool for high-throughput, high-resolution transcriptomic analysis of cell states and dynamics. Single-cell approaches circumvent the averaging artifacts associated with traditional bulk population data, yielding new insights into the cellular diversity underlying superficially homogeneous populations. Thus far, single-cell RNA-sequencing has already shown great effectiveness in unraveling complex cell populations, reconstructing developmental trajectories, and modeling transcriptional dynamics. Ongoing technical improvements to single-cell RNA-sequencing throughput and sensitivity, the development of more sophisticated analytical frameworks for single-cell data, and an increasing array of complementary single-cell assays all promise to expand the usefulness and potential applications of single-cell transcriptomic profiling. PMID:26949524

  10. Single cell protein as an occupational hazard.

    PubMed Central

    Ekenvall, L; Dölling, B; Göthe, C J; Ebbinghaus, L; von Stedingk, L V; Wasserman, J

    1983-01-01

    Single cell protein (SCP) intended for animal feed purposes was produced in a pilot plant. The SCP consisted of Methylomonas methanolica, a pseudomonas species which is an obligate methanol user. The SCP was cultured in fermenters and later dewatered and dried in a spray-drier. Seven of eight research workers had febrile reactions 6-12 hours after exposure to SCP dust. All workers had high titres of IgG and IgM antibodies against the pseudomonas species as measured with indirect ELISA and passive haemagglutination techniques. The mechanism behind the febrile reaction is judged to be a non-immunological reaction caused by endotoxins. By increasing the particle size of the SCP through using different drying procedures, a product which generated less dust was obtained. PMID:6830720

  11. JGI Genomic Single-cell Assembly Workflow

    SciTech Connect

    Trong, S.

    2011-09-16

    JIGSAW is a software package disigned to quality control and assemble genomic DNA sequences from single-cell bacterial and archaeal genomes. Amplification of singel-cell genomes using multiple displacement amplification technology presents challenges that magnify the amount of contaminants in the sample and produce non uniform depth of sequence coverage. these factors pose problems whan assembling the genomic data using currently availible short read assembles. The software addresses these problems by removing contaminants and normalizing the sequence read coverage prior to assemble. A hybrid assembly approach using two different open source genome assembly tools is then applied to piece together the DNA fragments. Additional reporting of QC metrics for the input sample and the genome assembly is provided for further analysis.

  12. Single-cell protein from waste cellulose

    NASA Technical Reports Server (NTRS)

    Dunlap, C. E.; Callihan, C. D.

    1973-01-01

    The recycle, reuse, or reclamation of single cell protein from liquid and solid agricultural waste fibers by a fermentation process is reported. It is shown that cellulose comprises the bulk of the fibers at 50% to 55% of the dry weight of the refuse and that its biodegradability is of prime importance in the choice of a substrate. The application of sodium hydroxide followed by heat and pressure serves to de-polymerize and disrupt lignin structure while swelling the cellulose to increase water uptake and pore volume. Some of the lignin, hemi-celluloses, ash, and cellulose of the material is hydrolized and solubilized. Introduction of microorganisms to the substrate fibers mixed with nutrients produces continuous fermentation of cellulose for further protein extraction and purification.

  13. Nanosensing at the single cell level✩

    PubMed Central

    Vo-Dinh, Tuan

    2013-01-01

    This article presents an overview of the development, operation, and applications of optical nanobiosensors for use in in vivo detection of biotargets in individual living cells. The nanobiosensors are equipped with immobilized bioreceptor probes (e.g., antibodies, enzyme substrate) selective to specific molecular targets. Laser excitation is transmitted into the fiber producing an evanescent field at the tip of the fiber in order to excite target molecules bound to the bioreceptors immobilized at the fiber tips. A photometric system detects the optical signal (e.g., fluorescence) originated from the analyte molecules or from the analyte–bioreceptor reaction. Examples of detection of biospecies and molecular signaling pathways of apoptosis in a living cell are discussed to illustrate the potential of the nanobiosensor technology for single cell analysis. PMID:24839348

  14. Electrodeformation for single cell mechanical characterization

    NASA Astrophysics Data System (ADS)

    Chen, Jian; Abdelgawad, Mohamed; Yu, Liming; Shakiba, Nika; Chien, Wei-Yin; Lu, Zhe; Geddie, William R.; Jewett, Michael A. S.; Sun, Yu

    2011-05-01

    This paper presents the use of electrodeformation as a method for single cell mechanical characterization in which mechanical properties of SiHa and ME180 cells (two cervical cancer cell lines) were quantified. Cells were directly placed between two microelectrodes with a rectangular ac electric field applied, and cell deformation was recorded under certain experimental conditions. Numerical simulations were performed to model cell electrodeformation based on the Maxwell stress tensor formulation. In these simulations, effects of cell electrical property variations on their electrodeformed behavior were investigated. By comparing the measured morphological changes with those obtained from numerical simulations, we were able to quantify Young's modulus of SiHa cells (601 ± 183 Pa) and ME180 cells (1463 ± 649 Pa). These values were consistent with Young's modulus values (SiHa: 400 ± 290 Pa and ME180: 1070 ± 580 Pa) obtained from conventional micropipette aspiration.

  15. JGI Genomic Single-cell Assembly Workflow

    Energy Science and Technology Software Center (ESTSC)

    2011-09-16

    JIGSAW is a software package disigned to quality control and assemble genomic DNA sequences from single-cell bacterial and archaeal genomes. Amplification of singel-cell genomes using multiple displacement amplification technology presents challenges that magnify the amount of contaminants in the sample and produce non uniform depth of sequence coverage. these factors pose problems whan assembling the genomic data using currently availible short read assembles. The software addresses these problems by removing contaminants and normalizing the sequencemore » read coverage prior to assemble. A hybrid assembly approach using two different open source genome assembly tools is then applied to piece together the DNA fragments. Additional reporting of QC metrics for the input sample and the genome assembly is provided for further analysis.« less

  16. Deformation Monitoring of AN Active Fault

    NASA Astrophysics Data System (ADS)

    Ostapchuk, A.

    2015-12-01

    The discovery of low frequency earthquakes, slow slip events and other deformation phenomena, new for geophysics, change our understanding of how the energy accumulated in the Earth's crust do release. The new geophysical data make one revise the underlying mechanism of geomechanical processes taking place in fault zones. Conditions for generating different slip modes are still unclear. The most vital question is whether a certain slip mode is intrinsic for a fault or may be controlled by external factors. This work presents the results of two and a half year deformation monitoring of a discontinuity in the zone of the Main Sayanskiy Fault. Main Sayanskiy Fault is right-lateral strike-slip fault. Observations were performed in the tunnel of Talaya seismic station (TLY), Irkutsk region, Russia. Measurements were carried out 70 m away from the entrance of the tunnel, the thickness of overlying rock was about 30 m. Inductive sensors of displacement were mounted at the both sides of a discontinuity, which recorded three components of relative fault side displacement with the accuracy of 0.2 mcm. Temperature variation inside the tunnel didn't exceed 0.5oC during the all period of observations. Important information about deformation properties of an active fault was obtained. A pronounced seasonality of deformation characteristics of discontinuity is observed in the investigated segment of rock. A great number of slow slip events with durations from several hours to several weeks were registered. Besides that alterations of fault deformation characteristics before the megathrust earthquake M9.0 Tohoku Oki 11 March 2011 and reaction to the event itself were detected. The work was supported by the Russian Science Foundation (grant no. 14-17-00719).

  17. Active sites environmental monitoring Program - Program Plan: Revision 2

    SciTech Connect

    Morrissey, C.M.; Hicks, D.S.; Ashwood, T.L.; Cunningham, G.R.

    1994-05-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of active low-level-waste (LLW) and transuranic (TRU) waste facilities at Oak Ridge National Laboratory (ORNL). Several changes have recently occurred in regard to the sites that are currently used for waste storage and disposal. These changes require a second set of revisions to the ASEMP program plan. This document incorporates those revisions. This program plan presents the organization and procedures for monitoring the active sites. The program plan also provides internal reporting levels to guide the evaluation of monitoring results.

  18. 7 CFR 800.216 - Activities that shall be monitored.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 800.216 Agriculture Regulations of the Department of Agriculture (Continued) GRAIN INSPECTION, PACKERS AND STOCKYARD ADMINISTRATION (FEDERAL GRAIN INSPECTION SERVICE), DEPARTMENT OF AGRICULTURE GENERAL...) Grain merchandising activities. Grain merchandising activities subject to monitoring for compliance...

  19. 7 CFR 800.216 - Activities that shall be monitored.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 800.216 Agriculture Regulations of the Department of Agriculture (Continued) GRAIN INSPECTION, PACKERS AND STOCKYARD ADMINISTRATION (FEDERAL GRAIN INSPECTION SERVICE), DEPARTMENT OF AGRICULTURE GENERAL...) Grain merchandising activities. Grain merchandising activities subject to monitoring for compliance...

  20. 7 CFR 800.216 - Activities that shall be monitored.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 800.216 Agriculture Regulations of the Department of Agriculture (Continued) GRAIN INSPECTION, PACKERS AND STOCKYARD ADMINISTRATION (FEDERAL GRAIN INSPECTION SERVICE), DEPARTMENT OF AGRICULTURE GENERAL...) Grain merchandising activities. Grain merchandising activities subject to monitoring for compliance...

  1. Instructional physical activity monitor video in english and spanish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ActiGraph activity monitor is a widely used method for assessing physical activity. Compliance with study procedures in critical. A common procedure is for the research team to meet with participants and demonstrate how and when to attach and remove the monitor and convey how many wear-days are ...

  2. Monitoring metal concentrations in tissues and single cells using ultramicrosensors.

    PubMed Central

    Malinski, T; Grunfeld, S; Taha, Z; Tomboulian, P

    1994-01-01

    Intercellular and extracellular metal concentrations were measured using carbon fiber ultramicrosensors plated with mercury or with polymeric porphyrinic p-type semiconductors. Concentrations of unbound nickel and lead ions were studied within individual BC3H-1 myocytes, and H4-11-C3 rat hepatoma cells. Unbound ions are predominantly solvated inorganic ions not coordinated to biological cellular components. Fabrication of ultramicrosensors appropriate for the cells under investigation is described, including procedures for sharpening and waxing the microsensors in order to control the shape, area, and dimensions of the electroactive surface. Metal ion movement through cell membranes and intracellular ion diffusion in aorta tissue were studied. Images Figure 2. PMID:7843090

  3. Tracing haematopoietic stem cell formation at single-cell resolution.

    PubMed

    Zhou, Fan; Li, Xianlong; Wang, Weili; Zhu, Ping; Zhou, Jie; He, Wenyan; Ding, Meng; Xiong, Fuyin; Zheng, Xiaona; Li, Zhuan; Ni, Yanli; Mu, Xiaohuan; Wen, Lu; Cheng, Tao; Lan, Yu; Yuan, Weiping; Tang, Fuchou; Liu, Bing

    2016-05-26

    Haematopoietic stem cells (HSCs) are derived early from embryonic precursors, such as haemogenic endothelial cells and pre-haematopoietic stem cells (pre-HSCs), the molecular identity of which still remains elusive. Here we use potent surface markers to capture the nascent pre-HSCs at high purity, as rigorously validated by single-cell-initiated serial transplantation. Then we apply single-cell RNA sequencing to analyse endothelial cells, CD45(-) and CD45(+) pre-HSCs in the aorta-gonad-mesonephros region, and HSCs in fetal liver. Pre-HSCs show unique features in transcriptional machinery, arterial signature, metabolism state, signalling pathway, and transcription factor network. Functionally, activation of mechanistic targets of rapamycin (mTOR) is shown to be indispensable for the emergence of HSCs but not haematopoietic progenitors. Transcriptome data-based functional analysis reveals remarkable heterogeneity in cell-cycle status of pre-HSCs. Finally, the core molecular signature of pre-HSCs is identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating stepwise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical applications. PMID:27225119

  4. High-Content Quantification of Single-Cell Immune Dynamics

    PubMed Central

    Junkin, Michael; Kaestli, Alicia J.; Cheng, Zhang; Jordi, Christian; Albayrak, Cem; Hoffmann, Alexander; Tay, Savaş

    2016-01-01

    Summary Cells receive time-varying signals from the environment and generate functional responses by secreting their own signaling molecules. Characterizing dynamic input-output relationships in single cells is crucial for understanding and modeling cellular systems. We developed an automated microfluidic system that delivers precisely defined dynamical inputs to individual living cells and simultaneously measures key immune parameters dynamically. Our system combines nanoliter immunoassays, microfluidic input generation, and time-lapse microscopy, enabling study of previously untestable aspects of immunity by measuring time-dependent cytokine secretion and transcription factor activity from single cells stimulated with dynamic inflammatory inputs. Employing this system to analyze macrophage signal processing under pathogen inputs, we found that the dynamics of TNF secretion are highly heterogeneous and surprisingly uncorrelated with the dynamics of NF-κB, the transcription factor controlling TNF production. Computational modeling of the LPS/TLR4 pathway shows that post-transcriptional regulation by TRIF is a key determinant of noisy and uncorrelated TNF secretion dynamics in single macrophages. PMID:27050527

  5. Kinetics of small molecule interactions with membrane proteins in single cells measured with mechanical amplification

    PubMed Central

    Guan, Yan; Shan, Xiaonan; Zhang, Fenni; Wang, Shaopeng; Chen, Hong-Yuan; Tao, Nongjian

    2015-01-01

    Measuring small molecule interactions with membrane proteins in single cells is critical for understanding many cellular processes and for screening drugs. However, developing such a capability has been a difficult challenge. We show that molecular interactions with membrane proteins induce a mechanical deformation in the cellular membrane, and real-time monitoring of the deformation with subnanometer resolution allows quantitative analysis of small molecule–membrane protein interaction kinetics in single cells. This new strategy provides mechanical amplification of small binding signals, making it possible to detect small molecule interactions with membrane proteins. This capability, together with spatial resolution, also allows the study of the heterogeneous nature of cells by analyzing the interaction kinetics variability between different cells and between different regions of a single cell. PMID:26601298

  6. Quality Assurance Project Plan for Facility Effluent Monitoring Plan activities

    SciTech Connect

    Nickels, J.M.

    1991-06-01

    This Quality Assurance Project Plan addresses the quality assurance requirements for the Facility Monitoring Plans of the overall site-wide environmental monitoring plan. This plan specifically applies to the sampling and analysis activities and continuous monitoring performed for all Facility Effluent Monitoring Plan activities conducted by Westinghouse Hanford Company. It is generic in approach and will be implemented in conjunction with the specific requirements of individual Facility Effluent Monitoring Plans. This document is intended to be a basic road map to the Facility Effluent Monitoring Plan documents (i.e., the guidance document for preparing Facility Effluent Monitoring Plans, Facility Effluent Monitoring Plan determinations, management plan, and Facility Effluent Monitoring Plans). The implementing procedures, plans, and instructions are appropriate for the control of effluent monitoring plans requiring compliance with US Department of Energy, US Environmental Protection Agency, state, and local requirements. This Quality Assurance Project Plan contains a matrix of organizational responsibilities, procedural resources from facility or site manuals used in the Facility Effluent Monitoring Plans, and a list of the analytes of interest and analytical methods for each facility preparing a Facility Effluent Monitoring Plan. 44 refs., 1 figs., 2 tabs.

  7. Update on nutrition monitoring activities in the United States.

    PubMed

    Kuczmarski, M F; Moshfegh, A; Briefel, R

    1994-07-01

    This article provides an overview of planned and proposed nutrition monitoring activities of the National Nutrition Monitoring and Related Research (NNMRR) Program. Key provisions of the NNMRR Act of 1990 are described, including the roles and responsibilities of the Interagency Board of Nutrition Monitoring and Related Research (IBNMRR) and the National Nutrition Monitoring Advisory Council and the development of the Ten-Year Comprehensive Plan. The Plan, which was developed under the guidance of the IBNMRR and reviewed by the National Nutrition Monitoring Advisory Council, is the basis for planning and coordinating the monitoring activities of 22 federal agencies. Also discussed are the resources generated from nutrition monitoring activities, from publications to conferences, that are available to dietitians and nutritionists. Professionals view the scientific reports that describe the nutritional status of the US population and the directories of federal and state monitoring activities as valuable resources. Suggestions from users of nutrition monitoring data related to their information and research needs have been extremely helpful to federal agencies in the development of future monitoring publications and the Ten-Year Comprehensive Plan. Continued communication between dietitians and the federal agencies responsible for the NNMRR Program is important. PMID:8021417

  8. Live Single-Cell Plant Hormone Analysis by Video-Mass Spectrometry.

    PubMed

    Shimizu, Takafumi; Miyakawa, Shinya; Esaki, Tsuyoshi; Mizuno, Hajime; Masujima, Tsutomu; Koshiba, Tomokazu; Seo, Mitsunori

    2015-07-01

    Studies have indicated that endogenous concentrations of plant hormones are regulated very locally within plants. To understand the mechanisms underlying hormone-mediated physiological processes, it is indispensable to know the exact hormone concentrations at cellular levels. In the present study, we established a system to determine levels of ABA and jasmonoyl-isoleucine (JA-Ile) from single cells. Samples taken from a cell of Vicia faba leaves using nano-electrospray ionization (ESI) tips under a microscope were directly introduced into mass spectrometers by infusion and subjected to tandem mass spectrometry (MS/MS) analysis. Stable isotope-labeled [D(6)]ABA or [(13)C(6)]JA-Ile was used as an internal standard to compensate ionization efficiencies, which determine the amount of ions introduced into mass spectrometers. We detected ABA and JA-Ile from single cells of water- and wound-stressed leaves, whereas they were almost undetectable in non-stressed single cells. The levels of ABA and JA-Ile found in the single-cell analysis were compared with levels found by analysis of purified extracts with liquid chromatography-tandem mass spectrometry (LC-MS/MS). These results demonstrated that stress-induced accumulation of ABA and JA-Ile could be monitored from living single cells. PMID:25759328

  9. New Approach to Investigate the Cytotoxicity of Nanomaterials Using Single Cell Mechanics

    PubMed Central

    2015-01-01

    Current in vitro methods to assess nanomaterial cytotoxicity involve various assays to monitor specific cellular dysfunction, such as metabolic imbalance or inflammation. Although high throughput, fast, and animal-free, these in vitro methods suffer from unreliability and lack of relevance to in vivo situations. New approaches, especially with the potential to reliably relate to in vivo studies directly, are in critical need. This work introduces a new approach, single cell mechanics, derived from atomic force microscopy-based single cell compression. The single cell based approach is intrinsically advantageous in terms of being able to directly correlate to in vivo investigations. Its reliability and potential to measure cytotoxicity is evaluated using known systems: zinc oxide (ZnO) and silicon dioxide (SiO2) nanoparticles (NP) on human aortic endothelial cells (HAECs). This investigation clearly indicates the reliability of single cell compression. For example, ZnO NPs cause significant changes in force vs relative deformation profiles, whereas SiO2 NPs do not. New insights into NPs–cell interactions pertaining to cytotoxicity are also revealed from this single cell mechanics approach, in addition to a qualitative cytotoxicity conclusion. The advantages and disadvantages of this approach are also compared with conventional cytotoxicity assays. PMID:24417356

  10. Remote Physical Activity Monitoring in Neurological Disease: A Systematic Review

    PubMed Central

    Block, Valerie A. J.; Pitsch, Erica; Tahir, Peggy; Cree, Bruce A. C.; Allen, Diane D.; Gelfand, Jeffrey M.

    2016-01-01

    Objective To perform a systematic review of studies using remote physical activity monitoring in neurological diseases, highlighting advances and determining gaps. Methods Studies were systematically identified in PubMed/MEDLINE, CINAHL and SCOPUS from January 2004 to December 2014 that monitored physical activity for ≥24 hours in adults with neurological diseases. Studies that measured only involuntary motor activity (tremor, seizures), energy expenditure or sleep were excluded. Feasibility, findings, and protocols were examined. Results 137 studies met inclusion criteria in multiple sclerosis (MS) (61 studies); stroke (41); Parkinson's Disease (PD) (20); dementia (11); traumatic brain injury (2) and ataxia (1). Physical activity levels measured by remote monitoring are consistently low in people with MS, stroke and dementia, and patterns of physical activity are altered in PD. In MS, decreased ambulatory activity assessed via remote monitoring is associated with greater disability and lower quality of life. In stroke, remote measures of upper limb function and ambulation are associated with functional recovery following rehabilitation and goal-directed interventions. In PD, remote monitoring may help to predict falls. In dementia, remote physical activity measures correlate with disease severity and can detect wandering. Conclusions These studies show that remote physical activity monitoring is feasible in neurological diseases, including in people with moderate to severe neurological disability. Remote monitoring can be a psychometrically sound and responsive way to assess physical activity in neurological disease. Further research is needed to ensure these tools provide meaningful information in the context of specific neurological disorders and patterns of neurological disability. PMID:27124611