Systems Analysis Initiated for All-Electric Aircraft Propulsion

NASA Technical Reports Server (NTRS)

Kohout, Lisa L.

2003-01-01

A multidisciplinary effort is underway at the NASA Glenn Research Center to develop concepts for revolutionary, nontraditional fuel cell power and propulsion systems for aircraft applications. There is a growing interest in the use of fuel cells as a power source for electric propulsion as well as an auxiliary power unit to substantially reduce or eliminate environmentally harmful emissions. A systems analysis effort was initiated to assess potential concepts in an effort to identify those configurations with the highest payoff potential. Among the technologies under consideration are advanced proton exchange membrane (PEM) and solid oxide fuel cells, alternative fuels and fuel processing, and fuel storage. Prior to this effort, the majority of fuel cell analysis done at Glenn was done for space applications. Because of this, a new suite of models was developed. These models include the hydrogen-air PEM fuel cell; internal reforming solid oxide fuel cell; balance-of-plant components (compressor, humidifier, separator, and heat exchangers); compressed gas, cryogenic, and liquid fuel storage tanks; and gas turbine/generator models for hybrid system applications. Initial mass, volume, and performance estimates of a variety of PEM systems operating on hydrogen and reformate have been completed for a baseline general aviation aircraft. Solid oxide/turbine hybrid systems are being analyzed. In conjunction with the analysis efforts, a joint effort has been initiated with Glenn s Computer Services Division to integrate fuel cell stack and component models with the visualization environment that supports the GRUVE lab, Glenn s virtual reality facility. The objective of this work is to provide an environment to assist engineers in the integration of fuel cell propulsion systems into aircraft and provide a better understanding of the interaction between system components and the resulting effect on the overall design and performance of the aircraft. Initially, three-dimensional computer-aided design (CAD) models of representative PEM fuel cell stack and components were developed and integrated into the virtual reality environment along with an Excel-based model used to calculate fuel cell electrical performance on the basis of cell dimensions (see the figure). CAD models of a representative general aviation aircraft were also developed and added to the environment. With the use of special headgear, users will be able to virtually manipulate the fuel cell s physical characteristics and its placement within the aircraft while receiving information on the resultant fuel cell output power and performance. As the systems analysis effort progresses, we will add more component models to the GRUVE environment to help us more fully understand the effect of various system configurations on the aircraft.

Probabilistic Analysis of Solid Oxide Fuel Cell Based Hybrid Gas Turbine System

NASA Technical Reports Server (NTRS)

Gorla, Rama S. R.; Pai, Shantaram S.; Rusick, Jeffrey J.

2003-01-01

The emergence of fuel cell systems and hybrid fuel cell systems requires the evolution of analysis strategies for evaluating thermodynamic performance. A gas turbine thermodynamic cycle integrated with a fuel cell was computationally simulated and probabilistically evaluated in view of the several uncertainties in the thermodynamic performance parameters. Cumulative distribution functions and sensitivity factors were computed for the overall thermal efficiency and net specific power output due to the uncertainties in the thermodynamic random variables. These results can be used to quickly identify the most critical design variables in order to optimize the design and make it cost effective. The analysis leads to the selection of criteria for gas turbine performance.

Correlation of simulation/finite element analysis to the separation of intrinsically magnetic spores and red blood cells using a microfluidic magnetic deposition system.

PubMed

Sun, Jianxin; Moore, Lee; Xue, Wei; Kim, James; Zborowski, Maciej; Chalmers, Jeffrey J

2018-05-01

Magnetic separation of cells has been, and continues to be, widely used in a variety of applications, ranging from healthcare diagnostics to detection of food contamination. Typically, these technologies require cells labeled with antibody magnetic particle conjugate and a high magnetic energy gradient created in the flow containing the labeled cells (i.e., a column packed with magnetically inducible material), or dense packing of magnetic particles next to the flow cell. Such designs, while creating high magnetic energy gradients, are not amenable to easy, highly detailed, mathematic characterization. Our laboratories have been characterizing and developing analysis and separation technology that can be used on intrinsically magnetic cells or spores which are typically orders of magnitude weaker than typically immunomagnetically labeled cells. One such separation system is magnetic deposition microscopy (MDM) which not only separates cells, but deposits them in specific locations on slides for further microscopic analysis. In this study, the MDM system has been further characterized, using finite element and computational fluid mechanics software, and separation performance predicted, using a model which combines: 1) the distribution of the intrinsic magnetophoretic mobility of the cells (spores); 2) the fluid flow within the separation device; and 3) accurate maps of the values of the magnetic field (max 2.27 T), and magnetic energy gradient (max of 4.41 T 2 /mm) within the system. Guided by this model, experimental studies indicated that greater than 95% of the intrinsically magnetic Bacillus spores can be separated with the MDM system. Further, this model allows analysis of cell trajectories which can assist in the design of higher throughput systems. © 2018 Wiley Periodicals, Inc.

Feasibility study of a 110 watt per kilogram lightweight solar array system

NASA Technical Reports Server (NTRS)

Shepard, N. F.; Stahle, C. V.; Hanson, K. L.; Schneider, A.; Blomstrom, L. E.; Hansen, W. T.; Kirpich, A.

1973-01-01

The feasibility of a 10,000 watt solar array panel which has a minimum power-to-mass ratio of 110 watt/kg is discussed. The application of this ultralightweight solar array to three possible missions was investigated. With the interplanetary mission as a baseline, the constraining requirements for a geosynchronous mission and for a manned space station mission are presented. A review of existing lightweight solar array system concepts revealed that changes in the system approach are necessary to achieve the specified 110 watt/kg goal. A comprehensive review of existing component technology is presented in the areas of thin solar cells, solar cell covers, welded interconnectors, substrates and deployable booms. Advances in the state-of-the-art of solar cell and deployable boom technology were investigated. System level trade studies required to select the optimum boom bending stiffness, system aspect ratio, bus voltage level, and solar cell circuit arrangement are reported. Design analysis tasks included the thermal analysis of the solar cell blanket, thermal stress analysis of the solar cell interconnectors/substrate, and the thermostructural loading of the deployed boom.

Comparison of corneal endothelial image analysis by Konan SP8000 noncontact and Bio-Optics Bambi systems.

PubMed

Benetz, B A; Diaconu, E; Bowlin, S J; Oak, S S; Laing, R A; Lass, J H

1999-01-01

Compare corneal endothelial image analysis by Konan SP8000 and Bio-Optics Bambi image-analysis systems. Corneal endothelial images from 98 individuals (191 eyes), ranging in age from 4 to 87 years, with a normal slit-lamp examination and no history of ocular trauma, intraocular surgery, or intraocular inflammation were obtained by the Konan SP8000 noncontact specular microscope. One observer analyzed these images by using the Konan system and a second observer by using the Bio-Optics Bambi system. Three methods of analyses were used: a fixed-frame method to obtain cell density (for both Konan and Bio-Optics Bambi) and a "dot" (Konan) or "corners" (Bio-Optics Bambi) method to determine morphometric parameters. The cell density determined by the Konan fixed-frame method was significantly higher (157 cells/mm2) than the Bio-Optics Bambi fixed-frame method determination (p<0.0001). However, the difference in cell density, although still statistically significant, was smaller and reversed comparing the Konan fixed-frame method with both Konan dot and Bio-Optics Bambi comers method (-74 cells/mm2, p<0.0001; -55 cells/mm2, p<0.0001, respectively). Small but statistically significant morphometric analyses differences between Konan and Bio-Optics Bambi were seen: cell density, +19 cells/mm2 (p = 0.03); cell area, -3.0 microm2 (p = 0.008); and coefficient of variation, +1.0 (p = 0.003). There was no statistically significant difference between these two methods in the percentage of six-sided cells detected (p = 0.55). Cell densities measured by the Konan fixed-frame method were comparable with Konan and Bio-Optics Bambi's morphometric analysis, but not with the Bio-Optics Bambi fixed-frame method. The two morphometric analyses were comparable with minimal or no differences for the parameters that were studied. The Konan SP8000 endothelial image-analysis system may be useful for large-scale clinical trials determining cell loss; its noncontact system has many clinical benefits (including patient comfort, safety, ease of use, and short procedure time) and provides reliable cell-density calculations.

Artificial neural network-aided image analysis system for cell counting.

PubMed

Sjöström, P J; Frydel, B R; Wahlberg, L U

1999-05-01

In histological preparations containing debris and synthetic materials, it is difficult to automate cell counting using standard image analysis tools, i.e., systems that rely on boundary contours, histogram thresholding, etc. In an attempt to mimic manual cell recognition, an automated cell counter was constructed using a combination of artificial intelligence and standard image analysis methods. Artificial neural network (ANN) methods were applied on digitized microscopy fields without pre-ANN feature extraction. A three-layer feed-forward network with extensive weight sharing in the first hidden layer was employed and trained on 1,830 examples using the error back-propagation algorithm on a Power Macintosh 7300/180 desktop computer. The optimal number of hidden neurons was determined and the trained system was validated by comparison with blinded human counts. System performance at 50x and lO0x magnification was evaluated. The correlation index at 100x magnification neared person-to-person variability, while 50x magnification was not useful. The system was approximately six times faster than an experienced human. ANN-based automated cell counting in noisy histological preparations is feasible. Consistent histology and computer power are crucial for system performance. The system provides several benefits, such as speed of analysis and consistency, and frees up personnel for other tasks.

Manual of phosphoric acid fuel cell power plant cost model and computer program

NASA Technical Reports Server (NTRS)

Lu, C. Y.; Alkasab, K. A.

1984-01-01

Cost analysis of phosphoric acid fuel cell power plant includes two parts: a method for estimation of system capital costs, and an economic analysis which determines the levelized annual cost of operating the system used in the capital cost estimation. A FORTRAN computer has been developed for this cost analysis.

Continuous cell introduction and rapid dynamic lysis for high-throughput single-cell analysis on microfludic chips with hydrodynamic focusing.

PubMed

Xu, Chun-Xiu; Yin, Xue-Feng

2011-02-04

A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance. Copyright © 2010 Elsevier B.V. All rights reserved.

Advanced Stoichiometric Analysis of Metabolic Networks of Mammalian Systems

PubMed Central

Orman, Mehmet A.; Berthiaume, Francois; Androulakis, Ioannis P.; Ierapetritou, Marianthi G.

2013-01-01

Metabolic engineering tools have been widely applied to living organisms to gain a comprehensive understanding about cellular networks and to improve cellular properties. Metabolic flux analysis (MFA), flux balance analysis (FBA), and metabolic pathway analysis (MPA) are among the most popular tools in stoichiometric network analysis. Although application of these tools into well-known microbial systems is extensive in the literature, various barriers prevent them from being utilized in mammalian cells. Limited experimental data, complex regulatory mechanisms, and the requirement of more complex nutrient media are some major obstacles in mammalian cell systems. However, mammalian cells have been used to produce therapeutic proteins, to characterize disease states or related abnormal metabolic conditions, and to analyze the toxicological effects of some medicinally important drugs. Therefore, there is a growing need for extending metabolic engineering principles to mammalian cells in order to understand their underlying metabolic functions. In this review article, advanced metabolic engineering tools developed for stoichiometric analysis including MFA, FBA, and MPA are described. Applications of these tools in mammalian cells are discussed in detail, and the challenges and opportunities are highlighted. PMID:22196224

Small Portable PEM Fuel Cell Systems for NASA Exploration Missions

NASA Technical Reports Server (NTRS)

Burke, Kenneth A.

2005-01-01

Oxygen-Hydrogen PEM-based fuel cell systems are being examined as a portable power source alternative in addition to advanced battery technology. Fuel cell power systems have been used by the Gemini, Apollo, and Space Shuttle programs. These systems have not been portable, but have been integral parts of their spacecraft, and have used reactants from a separate cryogenic supply. These systems typically have been higher in power. They also have had significant ancillary equipment sections that perform the pumping of reactants and coolant through the fuel cell stack and the separation of the product water from the unused reactant streams. The design of small portable fuel cell systems will be a significant departure from these previous designs. These smaller designs will have very limited ancillary equipment, relying on passive techniques for reactant and thermal management, and the reactant storage will be an integral part of the fuel cell system. An analysis of the mass and volume for small portable fuel cell systems was done to evaluate and quantify areas of technological improvement. A review of current fuel cell technology as well as reactant storage and management technology was completed to validate the analysis and to identify technology challenges

GoIFISH: a system for the quantification of single cell heterogeneity from IFISH images.

PubMed

Trinh, Anne; Rye, Inga H; Almendro, Vanessa; Helland, Aslaug; Russnes, Hege G; Markowetz, Florian

2014-08-26

Molecular analysis has revealed extensive intra-tumor heterogeneity in human cancer samples, but cannot identify cell-to-cell variations within the tissue microenvironment. In contrast, in situ analysis can identify genetic aberrations in phenotypically defined cell subpopulations while preserving tissue-context specificity. GoIFISHGoIFISH is a widely applicable, user-friendly system tailored for the objective and semi-automated visualization, detection and quantification of genomic alterations and protein expression obtained from fluorescence in situ analysis. In a sample set of HER2-positive breast cancers GoIFISHGoIFISH is highly robust in visual analysis and its accuracy compares favorably to other leading image analysis methods. GoIFISHGoIFISH is freely available at www.sourceforge.net/projects/goifish/.

A Label-Free Microfluidic Biosensor for Activity Detection of Single Microalgae Cells Based on Chlorophyll Fluorescence

PubMed Central

Wang, Junsheng; Sun, Jinyang; Song, Yongxin; Xu, Yongyi; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

2013-01-01

Detection of living microalgae cells is very important for ballast water treatment and analysis. Chlorophyll fluorescence is an indicator of photosynthetic activity and hence the living status of plant cells. In this paper, we developed a novel microfluidic biosensor system that can quickly and accurately detect the viability of single microalgae cells based on chlorophyll fluorescence. The system is composed of a laser diode as an excitation light source, a photodiode detector, a signal analysis circuit, and a microfluidic chip as a microalgae cell transportation platform. To demonstrate the utility of this system, six different living and dead algae samples (Karenia mikimotoi Hansen, Chlorella vulgaris, Nitzschia closterium, Platymonas subcordiformis, Pyramidomonas delicatula and Dunaliella salina) were tested. The developed biosensor can distinguish clearly between the living microalgae cells and the dead microalgae cells. The smallest microalgae cells that can be detected by using this biosensor are 3 μm ones. Even smaller microalgae cells could be detected by increasing the excitation light power. The developed microfluidic biosensor has great potential for in situ ballast water analysis. PMID:24287532

Exergy analysis of a solid oxide fuel cell micropowerplant

NASA Astrophysics Data System (ADS)

Hotz, Nico; Senn, Stephan M.; Poulikakos, Dimos

In this paper, an analytical model of a micro solid oxide fuel cell (SOFC) system fed by butane is introduced and analyzed in order to optimize its exergetic efficiency. The micro SOFC system is equipped with a partial oxidation (POX) reformer, a vaporizer, two pre-heaters, and a post-combustor. A one-dimensional (1D) polarization model of the SOFC is used to examine the effects of concentration overpotentials, activation overpotentials, and ohmic resistances on cell performance. This 1D polarization model is extended in this study to a two-dimensional (2D) fuel cell model considering convective mass and heat transport along the fuel cell channel and from the fuel cell to the environment. The influence of significant operational parameters on the exergetic efficiency of the micro SOFC system is discussed. The present study shows the importance of an exergy analysis of the fuel cell as part of an entire thermodynamic system (transportable micropowerplant) generating electric power.

Microfluidic cell disruption system employing a magnetically actuated diaphragm.

PubMed

Huh, Yun Suk; Choi, Jong Hyun; Huh, Kyoung Ae Kim; Kim, Kyoung Ae; Park, Tae Jung; Hong, Yeon Ki; Kim, Do Hyun; Hong, Won Hi; Lee, Sang Yup

2007-12-01

A microfluidic cell lysis chip equipped with a micromixer and SPE unit was developed and used for quantitative analysis of intracellular proteins. This miniaturized sample preparation system can be employed for any purpose where cell disruption is needed to obtain intracellular constituents for the subsequent analysis. This system comprises a magnetically actuated micromixer to disrupt cells, a hydrophobic valve to manipulate the cell lysate, and a packed porous polymerized monolith chamber for SPE and filtering debris from the cell lysate. Using recombinant Escherichia coli expressing intracellular enhanced green fluorescent protein (EGFP) and lipase as model bacteria, we optimized the cell disruption condition with respect to the lysis buffer composition, mixing time, and the frequency of the diaphragm in the micromixer, which was magnetically actuated by an external magnetic stirrer in the micromixer chamber. The lysed sample prepared under the optimal condition was purified by the packed SPE in the microfluidic chip. At a frequency of 1.96 Hz, the final cell lysis efficiency and relative fluorescence intensity of EGFP after the cell disruption process were greater than 90 and 94%, respectively. Thus, this microfluidic cell disruption chip can be used for the efficient lysis of cells for further analysis of intracellular contents in many applications.

Detection of early changes in lung-cell cytology by flow-systems analysis techniques. Progress report, January 1--June 30, 1976

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinkamp, J. A.; Hansen, K. M.; Wilson, J. S.

1976-08-01

This report summarizes results of preliminary experiments to develop cytological and biochemical indicators for estimating damage to respiratory epithelium exposed to toxic agents associated with the by-products of nonnuclear energy production using advanced flow-systems cell-analysis technologies. Since initiation of the program one year ago, progress has been made in obtaining adequate numbers of exfoliated lung cells from the Syrian hamster for flow analysis; cytological techniques developed on human exfoliated gynecological samples have been adapted to hamster lung epithelium for obtaining single-cell suspensions; and lung-cell samples have been initially characterized based on DNA content, total protein, nuclear and cytoplasmic size, andmore » multiangle light-scatter measurements. Preliminary results from measurements of the above parameters which recently became available are described in this report. As the flow-systems technology is adapted further to analysis of exfoliated lung cells, measurements of changes in physical and biochemical cellular properties as a function of exposure to toxic agents will be performed.« less

Synergistic effects of arsenic trioxide combined with ascorbic acid in human osteosarcoma MG-63 cells: a systems biology analysis.

PubMed

Huang, X C; Maimaiti, X Y M; Huang, C W; Zhang, L; Li, Z B; Chen, Z G; Gao, X; Chen, T Y

2014-01-01

To further understand the synergistic mechanism of As2O3 and asscorbic acid (AA) in human osteosarcoma MG-63 cells by systems biology analysis. Human osteosarcoma MG-63 cells were treated by As2O3 (1 µmol/L), AA (62.5 µmol/L) and combined drugs (1 µmol/L As2O3 plus 62.5 µmol/L AA). Dynamic morphological characteristics were recorded by Cell-IQ system, and growth rate was calculated. Illumina beadchip assay was used to analyze the differential expression genes in different groups. Synergic effects on differential expression genes (DEGs) were analyzed by mixture linear model and singular value decomposition model. KEGG pathway annotations and GO enrichment analysis were performed to figure out the pathways involved in the synergic effects. We captured 1987 differential expression genes in combined therapy MG-63 cells. FAT1 gene was significantly upregulated in all three groups, which is a promising drug target as an important tumor suppressor analogue; meanwhile, HIST1H2BD gene was markedly downregulated in the As2O3 monotherapy group and the combined therapy group, which was found to be upregulated in prostatic cancer. These two genes might play critical roles in synergetic effects of AA and As2O3, although the exact mechanism needs further investigation. KEGG pathway analysis showed many DEGs were related with tight junction, and GO analysis also indicated that DEGs in the combined therapy cells gathered in occluding junction, apical junction complex, cell junction, and tight junction. AA potentiates the efficacy of As2O3 in MG-63 cells. Systems biology analysis showed the synergic effect on the DEGs.

Droplet-based microfluidic analysis and screening of single plant cells.

PubMed

Yu, Ziyi; Boehm, Christian R; Hibberd, Julian M; Abell, Chris; Haseloff, Jim; Burgess, Steven J; Reyna-Llorens, Ivan

2018-01-01

Droplet-based microfluidics has been used to facilitate high-throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems where phenotyping analyses typically are time-consuming and low-throughput. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 cells per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.

Generation of enteroendocrine cell diversity in midgut stem cell lineages

PubMed Central

Beehler-Evans, Ryan; Micchelli, Craig A.

2015-01-01

The endocrine system mediates long-range peptide hormone signaling to broadcast changes in metabolic status to distant target tissues via the circulatory system. In many animals, the diffuse endocrine system of the gut is the largest endocrine tissue, with the full spectrum of endocrine cell subtypes not yet fully characterized. Here, we combine molecular mapping, lineage tracing and genetic analysis in the adult fruit fly to gain new insight into the cellular and molecular mechanisms governing enteroendocrine cell diversity. Neuropeptide hormone distribution was used as a basis to generate a high-resolution cellular map of the diffuse endocrine system. Our studies show that cell diversity is seen at two distinct levels: regional and local. We find that class I and class II enteroendocrine cells can be distinguished locally by combinatorial expression of secreted neuropeptide hormones. Cell lineage tracing studies demonstrate that class I and class II cells arise from a common stem cell lineage and that peptide profiles are a stable feature of enteroendocrine cell identity during homeostasis and following challenge with the enteric pathogen Pseudomonas entomophila. Genetic analysis shows that Notch signaling controls the establishment of class II cells in the lineage, but is insufficient to reprogram extant class I cells into class II enteroendocrine cells. Thus, one mechanism by which secretory cell diversity is achieved in the diffuse endocrine system is through cell-cell signaling interactions within individual adult stem cell lineages. PMID:25670792

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

PubMed

Lo, Shih-Jie; Yao, Da-Jeng

2015-07-23

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.

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

Multicell migration tracking within angiogenic networks by deep learning-based segmentation and augmented Bayesian filtering.

PubMed

Wang, Mengmeng; Ong, Lee-Ling Sharon; Dauwels, Justin; Asada, H Harry

2018-04-01

Cell migration is a key feature for living organisms. Image analysis tools are useful in studying cell migration in three-dimensional (3-D) in vitro environments. We consider angiogenic vessels formed in 3-D microfluidic devices (MFDs) and develop an image analysis system to extract cell behaviors from experimental phase-contrast microscopy image sequences. The proposed system initializes tracks with the end-point confocal nuclei coordinates. We apply convolutional neural networks to detect cell candidates and combine backward Kalman filtering with multiple hypothesis tracking to link the cell candidates at each time step. These hypotheses incorporate prior knowledge on vessel formation and cell proliferation rates. The association accuracy reaches 86.4% for the proposed algorithm, indicating that the proposed system is able to associate cells more accurately than existing approaches. Cell culture experiments in 3-D MFDs have shown considerable promise for improving biology research. The proposed system is expected to be a useful quantitative tool for potential microscopy problems of MFDs.

Extracting archaeal populations from iron oxidizing systems

NASA Astrophysics Data System (ADS)

Whitmore, L. M.; Hutchison, J.; Chrisler, W.; Jay, Z.; Moran, J.; Inskeep, W.; Kreuzer, H.

2013-12-01

Unique environments in Yellowstone National Park offer exceptional conditions for studying microorganisms in extreme and constrained systems. However, samples from some extreme systems often contain inorganic components that pose complications during microbial and molecular analysis. Several archaeal species are found in acidic, geothermal ferric-oxyhydroxide mats; these species have been shown to adhere to mineral surfaces in flocculated colonies. For optimal microbial analysis, (microscopy, flow cytometry, genomic extractions, proteomic analysis, stable isotope analysis, and others), improved techniques are needed to better facilitate cell detachment and separation from mineral surfaces. As a requirement, these techniques must preserve cell structure while simultaneously minimizing organic carryover to downstream analysis. Several methods have been developed for removing sediments from mixed prokaryotic populations, including ultra-centrifugation, nycodenz gradient, sucrose cushions, and cell straining. In this study we conduct a comparative analysis of mechanisms used to detach archaeal cell populations from the mineral interface. Specifically, we evaluated mechanical and chemical approaches for cell separation and homogenization. Methods were compared using confocal microscopy, flow cytometry analyses, and real-time PCR detection. The methodology and approaches identified will be used to optimize biomass collection from environmental specimens or isolates grown with solid phases.

Development of Droplet Microfluidics Enabling High-Throughput Single-Cell Analysis.

PubMed

Wen, Na; Zhao, Zhan; Fan, Beiyuan; Chen, Deyong; Men, Dong; Wang, Junbo; Chen, Jian

2016-07-05

This article reviews recent developments in droplet microfluidics enabling high-throughput single-cell analysis. Five key aspects in this field are included in this review: (1) prototype demonstration of single-cell encapsulation in microfluidic droplets; (2) technical improvements of single-cell encapsulation in microfluidic droplets; (3) microfluidic droplets enabling single-cell proteomic analysis; (4) microfluidic droplets enabling single-cell genomic analysis; and (5) integrated microfluidic droplet systems enabling single-cell screening. We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on key performances of throughput, multifunctionality, and absolute quantification.

Optical cell tracking analysis using a straight-forward approach to minimize processing time for high frame rate data

NASA Astrophysics Data System (ADS)

Seeto, Wen Jun; Lipke, Elizabeth Ann

2016-03-01

Tracking of rolling cells via in vitro experiment is now commonly performed using customized computer programs. In most cases, two critical challenges continue to limit analysis of cell rolling data: long computation times due to the complexity of tracking algorithms and difficulty in accurately correlating a given cell with itself from one frame to the next, which is typically due to errors caused by cells that either come close in proximity to each other or come in contact with each other. In this paper, we have developed a sophisticated, yet simple and highly effective, rolling cell tracking system to address these two critical problems. This optical cell tracking analysis (OCTA) system first employs ImageJ for cell identification in each frame of a cell rolling video. A custom MATLAB code was written to use the geometric and positional information of all cells as the primary parameters for matching each individual cell with itself between consecutive frames and to avoid errors when tracking cells that come within close proximity to one another. Once the cells are matched, rolling velocity can be obtained for further analysis. The use of ImageJ for cell identification eliminates the need for high level MATLAB image processing knowledge. As a result, only fundamental MATLAB syntax is necessary for cell matching. OCTA has been implemented in the tracking of endothelial colony forming cell (ECFC) rolling under shear. The processing time needed to obtain tracked cell data from a 2 min ECFC rolling video recorded at 70 frames per second with a total of over 8000 frames is less than 6 min using a computer with an Intel® Core™ i7 CPU 2.80 GHz (8 CPUs). This cell tracking system benefits cell rolling analysis by substantially reducing the time required for post-acquisition data processing of high frame rate video recordings and preventing tracking errors when individual cells come in close proximity to one another.

Separation and Analysis of Adherent and Non-Adherent Cancer Cells Using a Single-Cell Microarray Chip.

PubMed

Yamamura, Shohei; Yamada, Eriko; Kimura, Fukiko; Miyajima, Kumiko; Shigeto, Hajime

2017-10-21

A new single-cell microarray chip was designed and developed to separate and analyze single adherent and non-adherent cancer cells. The single-cell microarray chip is made of polystyrene with over 60,000 microchambers of 10 different size patterns (31-40 µm upper diameter, 11-20 µm lower diameter). A drop of suspension of adherent carcinoma (NCI-H1650) and non-adherent leukocyte (CCRF-CEM) cells was placed onto the chip, and single-cell occupancy of NCI-H1650 and CCRF-CEM was determined to be 79% and 84%, respectively. This was achieved by controlling the chip design and surface treatment. Analysis of protein expression in single NCI-H1650 and CCRF-CEM cells was performed on the single-cell microarray chip by multi-antibody staining. Additionally, with this system, we retrieved positive single cells from the microchambers by a micromanipulator. Thus, this system demonstrates the potential for easy and accurate separation and analysis of various types of single cells.

Initial Design and Construction of a Mobil Regenerative Fuel Cell System

NASA Technical Reports Server (NTRS)

Colozza, Anthony J.; Maloney, Thomas; Hoberecht, Mark (Technical Monitor)

2003-01-01

The design and initial construction of a mobile regenerative power system is described. The main components of the power system consists of a photovoltaic array, regenerative fuel cell and electrolyzer. The system is mounted on a modified landscape trailer and is completely self contained. An operational analysis is also presented that shows predicted performance for the system at various times of the year. The operational analysis consists of performing an energy balance on the system based on array output and total desired operational time.

High-Throughput Single-Cell RNA Sequencing and Data Analysis.

PubMed

Sagar; Herman, Josip Stefan; Pospisilik, John Andrew; Grün, Dominic

2018-01-01

Understanding biological systems at a single cell resolution may reveal several novel insights which remain masked by the conventional population-based techniques providing an average readout of the behavior of cells. Single-cell transcriptome sequencing holds the potential to identify novel cell types and characterize the cellular composition of any organ or tissue in health and disease. Here, we describe a customized high-throughput protocol for single-cell RNA-sequencing (scRNA-seq) combining flow cytometry and a nanoliter-scale robotic system. Since scRNA-seq requires amplification of a low amount of endogenous cellular RNA, leading to substantial technical noise in the dataset, downstream data filtering and analysis require special care. Therefore, we also briefly describe in-house state-of-the-art data analysis algorithms developed to identify cellular subpopulations including rare cell types as well as to derive lineage trees by ordering the identified subpopulations of cells along the inferred differentiation trajectories.

Cryo-imaging of fluorescently labeled single cells in a mouse

NASA Astrophysics Data System (ADS)

Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

2009-02-01

We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron-scale, fluorescence, and bright field image data. Here we describe our image preprocessing, analysis, and visualization techniques. Processing improves axial resolution, reduces subsurface fluorescence by 97%, and enables single cell detection and counting. High quality 3D volume renderings enable us to evaluate cell distribution patterns. Applications include the myriad of biomedical experiments using fluorescent reporter gene and exogenous fluorophore labeling of cells in applications such as stem cell regenerative medicine, cancer, tissue engineering, etc.

Label-free enumeration, collection and downstream cytological and cytogenetic analysis of circulating tumor cells.

PubMed

Dhar, Manjima; Pao, Edward; Renier, Corinne; Go, Derek E; Che, James; Montoya, Rosita; Conrad, Rachel; Matsumoto, Melissa; Heirich, Kyra; Triboulet, Melanie; Rao, Jianyu; Jeffrey, Stefanie S; Garon, Edward B; Goldman, Jonathan; Rao, Nagesh P; Kulkarni, Rajan; Sollier-Christen, Elodie; Di Carlo, Dino

2016-10-14

Circulating tumor cells (CTCs) have a great potential as indicators of metastatic disease that may help physicians improve cancer prognostication, treatment and patient outcomes. Heterogeneous marker expression as well as the complexity of current antibody-based isolation and analysis systems highlights the need for alternative methods. In this work, we use a microfluidic Vortex device that can selectively isolate potential tumor cells from blood independent of cell surface expression. This system was adapted to interface with three protein-marker-free analysis techniques: (i) an in-flow automated image processing system to enumerate cells released, (ii) cytological analysis using Papanicolaou (Pap) staining and (iii) fluorescence in situ hybridization (FISH) targeting the ALK rearrangement. In-flow counting enables a rapid assessment of the cancer-associated large circulating cells in a sample within minutes to determine whether standard downstream assays such as cytological and cytogenetic analyses that are more time consuming and costly are warranted. Using our platform integrated with these workflows, we analyzed 32 non-small cell lung cancer (NSCLC) and 22 breast cancer patient samples, yielding 60 to 100% of the cancer patients with a cell count over the healthy threshold, depending on the detection method used: respectively 77.8% for automated, 60-100% for cytology, and 80% for immunostaining based enumeration.

Label-free enumeration, collection and downstream cytological and cytogenetic analysis of circulating tumor cells

PubMed Central

Dhar, Manjima; Pao, Edward; Renier, Corinne; Go, Derek E.; Che, James; Montoya, Rosita; Conrad, Rachel; Matsumoto, Melissa; Heirich, Kyra; Triboulet, Melanie; Rao, Jianyu; Jeffrey, Stefanie S.; Garon, Edward B.; Goldman, Jonathan; Rao, Nagesh P.; Kulkarni, Rajan; Sollier-Christen, Elodie; Di Carlo, Dino

2016-01-01

Circulating tumor cells (CTCs) have a great potential as indicators of metastatic disease that may help physicians improve cancer prognostication, treatment and patient outcomes. Heterogeneous marker expression as well as the complexity of current antibody-based isolation and analysis systems highlights the need for alternative methods. In this work, we use a microfluidic Vortex device that can selectively isolate potential tumor cells from blood independent of cell surface expression. This system was adapted to interface with three protein-marker-free analysis techniques: (i) an in-flow automated image processing system to enumerate cells released, (ii) cytological analysis using Papanicolaou (Pap) staining and (iii) fluorescence in situ hybridization (FISH) targeting the ALK rearrangement. In-flow counting enables a rapid assessment of the cancer-associated large circulating cells in a sample within minutes to determine whether standard downstream assays such as cytological and cytogenetic analyses that are more time consuming and costly are warranted. Using our platform integrated with these workflows, we analyzed 32 non-small cell lung cancer (NSCLC) and 22 breast cancer patient samples, yielding 60 to 100% of the cancer patients with a cell count over the healthy threshold, depending on the detection method used: respectively 77.8% for automated, 60–100% for cytology, and 80% for immunostaining based enumeration. PMID:27739521

More Than 1,000 Fuel Cell Units Deployed Through DOE ARRA Funding (Fact Sheet)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

This NREL Hydrogen and Fuel Cell Technical Highlight describes how early market end users are operating 1,111 fuel cell units at 301 sites in 20 states with funding from the U.S. Department of Energy Fuel Cell Technologies Program and analysis by NREL. The American Recovery and Reinvestment Act (ARRA) funded the deployment of approximately 1,000 fuel cell systems in key early markets to accelerate the commercialization and deployment of fuel cells and fuel cell manufacturing, installation, maintenance, and support services. In support of the ARRA fuel cell deployment objectives, NREL analyzes and validates the technology in real-world applications, reports onmore » the technology status, and facilitates the development of fuel cell technologies, manufacturing, and operations in strategic markets-including material handling equipment, backup power, and stationary power-where fuel cells can compete with conventional technologies. NREL is validating hydrogen and fuel cell systems in real-world settings through data collection, analysis, and reporting. The fuel cell and infrastructure analysis provides an independent, third-party assessment that focuses on fuel cell system and hydrogen infrastructure performance, operation, maintenance, use, and safety. An objective of the ARRA fuel cell project-to deploy approximately 1,000 fuel cell systems in key early markets - has been met in two years. By the end of 2011, 504 material handling equipment (MHE) fuel cell units were operating at 8 facilities and 607 backup power fuel cell units were operating at 293 sites. MHE and backup power are two markets where fuel cells are capable of meeting the operating demands, and deployments can be leveraged to accelerate fuel cell commercialization.« less

Analysis of a fuel cell on-site integrated energy system for a residential complex

NASA Technical Reports Server (NTRS)

Simons, S. N.; Maag, W. L.

1979-01-01

The energy use and costs of the on-site integrated energy system (OS/IES) which provides electric power from an on-site power plant and recovers heat that would normally be rejected to the environment is compared to a conventional system purchasing electricity from a utility and a phosphoric acid fuel cell powered system. The analysis showed that for a 500-unit apartment complex a fuel OS/IES would be about 10% more energy conservative in terms of total coal consumption than a diesel OS/IES system or a conventional system. The fuel cell OS/IES capital costs could be 30 to 55% greater than the diesel OS/IES capital costs for the same life cycle costs. The life cycle cost of a fuel cell OS/IES would be lower than that for a conventional system as long as the cost of electricity is greater than $0.05 to $0.065/kWh. An analysis of several parametric combinations of fuel cell power plant and state-of-art energy recovery systems and annual fuel requirement calculations for four locations were made. It was shown that OS/IES component choices are a major factor in fuel consumption, with the least efficient system using 25% more fuel than the most efficient. Central air conditioning and heat pumps result in minimum fuel consumption while individual air conditioning units increase it, and in general the fuel cell of highest electrical efficiency has the lowest fuel consumption.

Study on Improving Partial Load by Connecting Geo-thermal Heat Pump System to Fuel Cell Network

NASA Astrophysics Data System (ADS)

Obara, Shinya; Kudo, Kazuhiko

Hydrogen piping, the electric power line, and exhaust heat recovery piping of the distributed fuel cells are connected with network, and operational planning is carried out. Reduction of the efficiency in partial load is improved by operation of the geo-thermal heat pump linked to the fuel cell network. The energy demand pattern of the individual houses in Sapporo was introduced. And the analysis method aiming at minimization of the fuel rate by the genetic algorithm was described. The fuel cell network system of an analysis example assumed connecting the fuel cell co-generation of five houses. When geo-thermal heat pump was introduced into fuel cell network system stated in this paper, fuel consumption was reduced 6% rather than the conventional method

Quantitative biology of single neurons

PubMed Central

Eberwine, James; Lovatt, Ditte; Buckley, Peter; Dueck, Hannah; Francis, Chantal; Kim, Tae Kyung; Lee, Jaehee; Lee, Miler; Miyashiro, Kevin; Morris, Jacqueline; Peritz, Tiina; Schochet, Terri; Spaethling, Jennifer; Sul, Jai-Yoon; Kim, Junhyong

2012-01-01

The building blocks of complex biological systems are single cells. Fundamental insights gained from single-cell analysis promise to provide the framework for understanding normal biological systems development as well as the limits on systems/cellular ability to respond to disease. The interplay of cells to create functional systems is not well understood. Until recently, the study of single cells has concentrated primarily on morphological and physiological characterization. With the application of new highly sensitive molecular and genomic technologies, the quantitative biochemistry of single cells is now accessible. PMID:22915636

Development of a model system to analyze chondrogenic differentiation of mesenchymal stem cells

PubMed Central

Ruedel, Anke; Hofmeister, Simone; Bosserhoff, Anja-Katrin

2013-01-01

High-density cell culture is widely used for the analysis of cartilage development of human mesenchymal stem cells (HMSCs) in vitro. Several cell culture systems, as micromass, pellet culture and alginate culture, are applied by groups in the field to induce chondrogenic differentiation of HMSCs. A draw back of all model systems is the high amount of cells necessary for the experiments. Further, handling of large experimental approaches is difficult due to culturing e.g. in 15 ml tubes. Therefore, we aimed to develop a new model system based on “hanging drop” cultures using 10 to 100 fold less cells. Here, we demonstrate that differentiation of chondrogenic cells was induced as previously shown in other model systems. Real time RT-PCR analysis demonstrated that Collagen type II and MIA/CD-RAP were upregulated during culturing whereas for induction of hypertrophic markers like Collagen type X and AP-2 epsilon treatment with TGF beta was needed. To further test the system, siRNA against Sox9 was used and effects on chondrogenic gene expression were evaluated. In summary, the hanging drop culture system was determined to be a promising tool for in vitro chondrogenic studies. PMID:24294400

Voltage controlled nano-injection system for single-cell surgery

PubMed Central

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

2015-01-01

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

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

PubMed

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

2012-09-28

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

Study and performances analysis of fuel cell assisted vector control variable speed drive system used for electric vehicles

NASA Astrophysics Data System (ADS)

Pachauri, Rupendra Kumar; Chauhan, Yogesh K.

2017-02-01

This paper is a novel attempt to combine two important aspects of fuel cell (FC). First, it presents investigations on FC technology and its applications. A description of FC operating principles is followed by the comparative analysis of the present FC technologies together with the issues concerning various fuels. Second, this paper also proposes a model for the simulation and performances evaluation of a proton exchange membrane fuel cell (PEMFC) generation system. Furthermore, a MATLAB/Simulink-based dynamic model of PEMFC is developed and parameters of FC are so adjusted to emulate a commercially available PEMFC. The system results are obtained for the PEMFC-driven adjusted speed induction motor drive (ASIMD) system, normally used in electric vehicles and analysis is carried out for different operating conditions of FC and ASIMD system. The obtained results prove the validation of system concept and modelling.

Portable point-of-care blood analysis system for global health (Conference Presentation)

NASA Astrophysics Data System (ADS)

Dou, James J.; Aitchison, James Stewart; Chen, Lu; Nayyar, Rakesh

2016-03-01

In this paper we present a portable blood analysis system based on a disposable cartridge and hand-held reader. The platform can perform all the sample preparation, detection and waste collection required to complete a clinical test. In order to demonstrate the utility of this approach a CD4 T cell enumeration was carried out. A handheld, point-of-care CD4 T cell system was developed based on this system. In particular we will describe a pneumatic, active pumping method to control the on-chip fluidic actuation. Reagents for the CD4 T cell counting assay were dried on a reagent plug to eliminate the need for cold chain storage when used in the field. A micromixer based on the active fluidic actuation was designed to complete sample staining with fluorescent dyes that was dried on the reagent plugs. A novel image detection and analysis algorithm was developed to detect and track the flight of target particles and cells during each analysis. The handheld, point-of-care CD4 testing system was benchmarked against clinical cytometer. The experimental results demonstrated experimental results were closely matched with the flow cytometry. The same platform can be further expanded into a bead-array detection system where other types of biomolecules such as proteins can be detected using the same detection system.

Simple Electrolyzer Model Development for High-Temperature Electrolysis System Analysis Using Solid Oxide Electrolysis Cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

JaeHwa Koh; DuckJoo Yoon; Chang H. Oh

2010-07-01

An electrolyzer model for the analysis of a hydrogen-production system using a solid oxide electrolysis cell (SOEC) has been developed, and the effects for principal parameters have been estimated by sensitivity studies based on the developed model. The main parameters considered are current density, area specific resistance, temperature, pressure, and molar fraction and flow rates in the inlet and outlet. Finally, a simple model for a high-temperature hydrogen-production system using the solid oxide electrolysis cell integrated with very high temperature reactors is estimated.

A Facile Droplet-Chip-Time-Resolved Inductively Coupled Plasma Mass Spectrometry Online System for Determination of Zinc in Single Cell.

PubMed

Wang, Han; Chen, Beibei; He, Man; Hu, Bin

2017-05-02

Single cell analysis is a significant research field in recent years reflecting the heterogeneity of cells in a biological system. In this work, a facile droplet chip was fabricated and online combined with time-resolved inductively coupled plasma mass spectrometry (ICPMS) via a microflow nebulizer for the determination of zinc in single HepG2 cells. On the focusing geometric designed PDMS microfluidic chip, the aqueous cell suspension was ejected and divided by hexanol to generate droplets. The droplets encapsulated single cells remain intact during the transportation into ICP for subsequent detection. Under the optimized conditions, the frequency of droplet generation is 3-6 × 10 6 min -1 , and the injected cell number is 2500 min -1 , which can ensure the single cell encapsulation. ZnO nanoparticles (NPs) were used for the quantification of zinc in single cells, and the accuracy was validated by conventional acid digestion-ICPMS method. The ZnO NPs incubated HepG2 cells were analyzed as model samples, and the results exhibit the heterogeneity of HepG2 cells in the uptake/adsorption of ZnO NPs. The developed online droplet-chip-ICPMS analysis system achieves stable single cell encapsulation and has high throughput for single cell analysis. It has the potential in monitoring the content as well as distribution of trace elements/NPs at the single cell level.

Graph Theory-Based Analysis of the Lymph Node Fibroblastic Reticular Cell Network.

PubMed

Novkovic, Mario; Onder, Lucas; Bocharov, Gennady; Ludewig, Burkhard

2017-01-01

Secondary lymphoid organs have developed segregated niches that are able to initiate and maintain effective immune responses. Such global organization requires tight control of diverse cellular components, specifically those that regulate lymphocyte trafficking. Fibroblastic reticular cells (FRCs) form a densely interconnected network in lymph nodes and provide key factors necessary for T cell migration and retention, and foster subsequent interactions between T cells and dendritic cells. Development of integrative systems biology approaches has made it possible to elucidate this multilevel complexity of the immune system. Here, we present a graph theory-based analysis of the FRC network in murine lymph nodes, where generation of the network topology is performed using high-resolution confocal microscopy and 3D reconstruction. This approach facilitates the analysis of physical cell-to-cell connectivity, and estimation of topological robustness and global behavior of the network when it is subjected to perturbation in silico.

Systems analysis of electricity production from coal using fuel cells

NASA Technical Reports Server (NTRS)

Fleming, D. K.

1983-01-01

Gasifiers, heat transfer, gas stability, quench, water-gas shift reaction, reforming-methanation, other catalytic reactions, compressors and expanders, acid-gas removal, the fuel cell, and catalytic combustors are described. System pressure drops, efficiency of rotating power equipment, heat exchangers, chemical reactions, steam systems, and the fuel cell subsystems are discussed.

Multiway modeling and analysis in stem cell systems biology

PubMed Central

2008-01-01

Background Systems biology refers to multidisciplinary approaches designed to uncover emergent properties of biological systems. Stem cells are an attractive target for this analysis, due to their broad therapeutic potential. A central theme of systems biology is the use of computational modeling to reconstruct complex systems from a wealth of reductionist, molecular data (e.g., gene/protein expression, signal transduction activity, metabolic activity, etc.). A number of deterministic, probabilistic, and statistical learning models are used to understand sophisticated cellular behaviors such as protein expression during cellular differentiation and the activity of signaling networks. However, many of these models are bimodal i.e., they only consider row-column relationships. In contrast, multiway modeling techniques (also known as tensor models) can analyze multimodal data, which capture much more information about complex behaviors such as cell differentiation. In particular, tensors can be very powerful tools for modeling the dynamic activity of biological networks over time. Here, we review the application of systems biology to stem cells and illustrate application of tensor analysis to model collagen-induced osteogenic differentiation of human mesenchymal stem cells. Results We applied Tucker1, Tucker3, and Parallel Factor Analysis (PARAFAC) models to identify protein/gene expression patterns during extracellular matrix-induced osteogenic differentiation of human mesenchymal stem cells. In one case, we organized our data into a tensor of type protein/gene locus link × gene ontology category × osteogenic stimulant, and found that our cells expressed two distinct, stimulus-dependent sets of functionally related genes as they underwent osteogenic differentiation. In a second case, we organized DNA microarray data in a three-way tensor of gene IDs × osteogenic stimulus × replicates, and found that application of tensile strain to a collagen I substrate accelerated the osteogenic differentiation induced by a static collagen I substrate. Conclusion Our results suggest gene- and protein-level models whereby stem cells undergo transdifferentiation to osteoblasts, and lay the foundation for mechanistic, hypothesis-driven studies. Our analysis methods are applicable to a wide range of stem cell differentiation models. PMID:18625054

FLOCK cluster analysis of mast cell event clustering by high-sensitivity flow cytometry predicts systemic mastocytosis.

PubMed

Dorfman, David M; LaPlante, Charlotte D; Pozdnyakova, Olga; Li, Betty

2015-11-01

In our high-sensitivity flow cytometric approach for systemic mastocytosis (SM), we identified mast cell event clustering as a new diagnostic criterion for the disease. To objectively characterize mast cell gated event distributions, we performed cluster analysis using FLOCK, a computational approach to identify cell subsets in multidimensional flow cytometry data in an unbiased, automated fashion. FLOCK identified discrete mast cell populations in most cases of SM (56/75 [75%]) but only a minority of non-SM cases (17/124 [14%]). FLOCK-identified mast cell populations accounted for 2.46% of total cells on average in SM cases and 0.09% of total cells on average in non-SM cases (P < .0001) and were predictive of SM, with a sensitivity of 75%, a specificity of 86%, a positive predictive value of 76%, and a negative predictive value of 85%. FLOCK analysis provides useful diagnostic information for evaluating patients with suspected SM, and may be useful for the analysis of other hematopoietic neoplasms. Copyright© by the American Society for Clinical Pathology.

Flat plate vs. concentrator solar photovoltaic cells - A manufacturing cost analysis

NASA Technical Reports Server (NTRS)

Granon, L. A.; Coleman, M. G.

1980-01-01

The choice of which photovoltaic system (flat plate or concentrator) to use for utilizing solar cells to generate electricity depends mainly on the cost. A detailed, comparative manufacturing cost analysis of the two types of systems is presented. Several common assumptions, i.e., cell thickness, interest rate, power rate, factory production life, polysilicon cost, and direct labor rate are utilized in this analysis. Process sequences, cost variables, and sensitivity analyses have been studied, and results of the latter show that the most important parameters which determine manufacturing costs are concentration ratio, manufacturing volume, and cell efficiency. The total cost per watt of the flat plate solar cell is $1.45, and that of the concentrator solar cell is $1.85, the higher cost being due to the increased process complexity and material costs.

Proton Exchange Membrane Fuel Cells for Electrical Power Generation On-Board Commercial Airplanes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pratt, Joesph W.; Klebanoff, Leonard E.; Munoz-Ramos, Karina

2011-05-01

Deployed on a commercial airplane, proton exchange membrane fuel cells may offer emissions reductions, thermal efficiency gains, and enable locating the power near the point of use. This work seeks to understand whether on-board fuel cell systems are technically feasible, and, if so, if they offer a performance advantage for the airplane as a whole. Through hardware analysis and thermodynamic and electrical simulation, we found that while adding a fuel cell system using today’s technology for the PEM fuel cell and hydrogen storage is technically feasible, it will not likely give the airplane a performance benefit. However, when we re-didmore » the analysis using DOE-target technology for the PEM fuel cell and hydrogen storage, we found that the fuel cell system would provide a performance benefit to the airplane (i.e., it can save the airplane some fuel), depending on the way it is configured.« less

Proton exchange membrane fuel cells for electrical power generation on-board commercial airplanes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Curgus, Dita Brigitte; Munoz-Ramos, Karina; Pratt, Joseph William

2011-05-01

Deployed on a commercial airplane, proton exchange membrane fuel cells may offer emissions reductions, thermal efficiency gains, and enable locating the power near the point of use. This work seeks to understand whether on-board fuel cell systems are technically feasible, and, if so, if they offer a performance advantage for the airplane as a whole. Through hardware analysis and thermodynamic and electrical simulation, we found that while adding a fuel cell system using today's technology for the PEM fuel cell and hydrogen storage is technically feasible, it will not likely give the airplane a performance benefit. However, when we re-didmore » the analysis using DOE-target technology for the PEM fuel cell and hydrogen storage, we found that the fuel cell system would provide a performance benefit to the airplane (i.e., it can save the airplane some fuel), depending on the way it is configured.« less

Systemic Inflammation in Progressive Multiple Sclerosis Involves Follicular T-Helper, Th17- and Activated B-Cells and Correlates with Progression

PubMed Central

Christensen, Jeppe Romme; Börnsen, Lars; Ratzer, Rikke; Piehl, Fredrik; Khademi, Mohsen; Olsson, Tomas; Sørensen, Per Soelberg; Sellebjerg, Finn

2013-01-01

Pathology studies of progressive multiple sclerosis (MS) indicate a major role of inflammation including Th17-cells and meningeal inflammation with ectopic lymphoid follicles, B-cells and plasma cells, the latter indicating a possible role of the newly identified subset of follicular T-helper (TFH) cells. Although previous studies reported increased systemic inflammation in progressive MS it remains unclear whether systemic inflammation contributes to disease progression and intrathecal inflammation. This study aimed to investigate systemic inflammation in progressive MS and its relationship with disease progression, using flow cytometry and gene expression analysis of CD4+ and CD8+T-cells, B-cells, monocytes and dendritic cells. Furthermore, gene expression of cerebrospinal fluid cells was studied. Flow cytometry studies revealed increased frequencies of ICOS+TFH-cells in peripheral blood from relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients. All MS subtypes had decreased frequencies of Th1 TFH-cells, while primary progressive (PPMS) MS patients had increased frequency of Th17 TFH-cells. The Th17-subset, interleukin-23-receptor+CD4+T-cells, was significantly increased in PPMS and SPMS. In the analysis of B-cells, we found a significant increase of plasmablasts and DC-SIGN+ and CD83+B-cells in SPMS. ICOS+TFH-cells and DC-SIGN+B-cells correlated with disease progression in SPMS patients. Gene expression analysis of peripheral blood cell subsets substantiated the flow cytometry findings by demonstrating increased expression of IL21, IL21R and ICOS in CD4+T-cells in progressive MS. Cerebrospinal fluid cells from RRMS and progressive MS (pooled SPMS and PPMS patients) had increased expression of TFH-cell and plasmablast markers. In conclusion, this study is the first to demonstrate the potential involvement of activated TFH-cells in MS. The increased frequencies of Th17-cells, activated TFH- and B-cells parallel findings from pathology studies which, along with the correlation between activated TFH- and B-cells and disease progression, suggest a pathogenic role of systemic inflammation in progressive MS. These observations may have implications for the treatment of progressive MS. PMID:23469245

Design and performance of a new continuous-flow sample-introduction system for flame infrared-emission spectrometry: Applications in process analysis, flow injection analysis, and ion-exchange high-performance liquid chromatography.

PubMed

Lam, C K; Zhang, Y; Busch, M A; Busch, K W

1993-06-01

A new sample introduction system for the analysis of continuously flowing liquid streams by flame infrared-emission (FIRE) spectrometry has been developed. The system uses a specially designed purge cell to strip dissolved CO(2) from solution into a hydrogen gas stream that serves as the fuel for a hydrogen/air flame. Vibrationally excited CO(2) molecules present in the flame are monitored with a simple infrared filter (4.4 mum) photometer. The new system can be used to introduce analytes as a continuous liquid stream (process analysis mode) or on a discrete basis by sample injection (flow injection analysis mode). The key to the success of the method is the new purge-cell design. The small internal volume of the cell minimizes problems associated with purge-cell clean-out and produces sharp, reproducible signals. Spent analytical solution is continuously drained from the cell, making cell disconnection and cleaning between samples unnecessary. Under the conditions employed in this study, samples could be analyzed at a maximum rate of approximately 60/h. The new sample introduction system was successfully tested in both a process analysis- and a flow injection analysis mode for the determination of total inorganic carbon in Waco tap water. For the first time, flame infrared-emission spectrometry was successfully extended to non-volatile organic compounds by using chemical pretreatment with peroxydisulfate in the presence of silver ion to convert the analytes into dissolved carbon dioxide, prior to purging and detection by the FIRE radiometer. A test of the peroxydisulfate/Ag(+) reaction using six organic acids and five sugars indicated that all 11 compounds were oxidized to nearly the same extent. Finally, the new sample introduction system was used in conjunction with a simple filter FIRE radiometer as a detection system in ion-exchange high-performance liquid chromatography. Ion-exchange chromatograms are shown for two aqueous mixtures, one containing six organic acids and the second containing six mono-, di-, and trisaccharides.

Analyzing the texture changes in the quantitative phase maps of adipocytes

NASA Astrophysics Data System (ADS)

Roitshtain, Darina; Sharabani-Yosef, Orna; Gefen, Amit; Shaked, Natan T.

2016-03-01

We present a new analysis tool for studying texture changes in the quantitative phase maps of live cells acquired by wide-field interferometry. The sensitivity of wide-field interferometry systems to small changes in refractive index enables visualizing cells and inner cell organelles without the using fluorescent dyes or other cell-invasive approaches, which may affect the measurement and require external labeling. Our label-free texture-analysis tool is based directly on the optical path delay profile of the sample and does not necessitate decoupling refractive index and thickness in the cell quantitative phase profile; thus, relevant parameters can be calculated using a single-frame acquisition. Our experimental system includes low-coherence wide-field interferometer, combined with simultaneous florescence microscopy system for validation. We used this system and analysis tool for studying lipid droplets formation in adipocytes. The latter demonstration is relevant for various cellular functions such as lipid metabolism, protein storage and degradation to viral replication. These processes are functionally linked to several physiological and pathological conditions, including obesity and metabolic diseases. Quantification of these biological phenomena based on the texture changes in the cell phase map has a potential as a new cellular diagnosis tool.

The use of multi criteria analysis to compare the operating scenarios of the hybrid generation system of wind turbines, photovoltaic modules and a fuel cell

NASA Astrophysics Data System (ADS)

Ceran, Bartosz

2017-11-01

The paper presents the results of the use of multi-criteria analysis to compare hybrid power generation system collaboration scenarios (HSW) consisting of wind turbines, solar panels and energy storage electrolyzer - PEM type fuel cell with electricity system. The following scenarios were examined: the base S-I-hybrid system powers the off-grid mode receiver, S-II, S-III, S-IV scenarios-electricity system covers 25%, 50%, 75% of energy demand by the recipient. The effect of weights of the above-mentioned criteria on the final result of the multi-criteria analysis was examined.

High temperature flow-through device for rapid solubilization and analysis

DOEpatents

West, Jason A. A. [Castro Valley, CA; Hukari, Kyle W [San Ramon, CA; Patel, Kamlesh D [Dublin, CA; Peterson, Kenneth A [Albuquerque, NM; Renzi, Ronald F [Tracy, CA

2009-09-22

Devices and methods for thermally lysing of biological material, for example vegetative bacterial cells and bacterial spores, are provided. Hot solution methods for solubilizing bacterial spores are described. Systems for direct analysis are disclosed including thermal lysers coupled to sample preparation stations. Integrated systems capable of performing sample lysis, labeling and protein fingerprint analysis of biological material, for example, vegetative bacterial cells, bacterial spores and viruses are provided.

High temperature flow-through device for rapid solubilization and analysis

DOEpatents

West, Jason A. A.; Hukari, Kyle W.; Patel, Kamlesh D.; Peterson, Kenneth A.; Renzi, Ronald F.

2013-04-23

Devices and methods for thermally lysing of biological material, for example vegetative bacterial cells and bacterial spores, are provided. Hot solution methods for solubilizing bacterial spores are described. Systems for direct analysis are disclosed including thermal lysers coupled to sample preparation stations. Integrated systems capable of performing sample lysis, labeling and protein fingerprint analysis of biological material, for example, vegetative bacterial cells, bacterial spores and viruses are provided.

Analysis and optimization of solid oxide fuel cell-based auxiliary power units using a generic zero-dimensional fuel cell model

NASA Astrophysics Data System (ADS)

Göll, S.; Samsun, R. C.; Peters, R.

Fuel-cell-based auxiliary power units can help to reduce fuel consumption and emissions in transportation. For this application, the combination of solid oxide fuel cells (SOFCs) with upstream fuel processing by autothermal reforming (ATR) is seen as a highly favorable configuration. Notwithstanding the necessity to improve each single component, an optimized architecture of the fuel cell system as a whole must be achieved. To enable model-based analyses, a system-level approach is proposed in which the fuel cell system is modeled as a multi-stage thermo-chemical process using the "flowsheeting" environment PRO/II™. Therein, the SOFC stack and the ATR are characterized entirely by corresponding thermodynamic processes together with global performance parameters. The developed model is then used to achieve an optimal system layout by comparing different system architectures. A system with anode and cathode off-gas recycling was identified to have the highest electric system efficiency. Taking this system as a basis, the potential for further performance enhancement was evaluated by varying four parameters characterizing different system components. Using methods from the design and analysis of experiments, the effects of these parameters and of their interactions were quantified, leading to an overall optimized system with encouraging performance data.

Effect of power system technology and mission requirements on high altitude long endurance aircraft

NASA Technical Reports Server (NTRS)

Colozza, Anthony J.

1994-01-01

An analysis was performed to determine how various power system components and mission requirements affect the sizing of a solar powered long endurance aircraft. The aircraft power system consists of photovoltaic cells and a regenerative fuel cell. Various characteristics of these components, such as PV cell type, PV cell mass, PV cell efficiency, fuel cell efficiency, and fuel cell specific mass, were varied to determine what effect they had on the aircraft sizing for a given mission. Mission parameters, such as time of year, flight altitude, flight latitude, and payload mass and power, were also altered to determine how mission constraints affect the aircraft sizing. An aircraft analysis method which determines the aircraft configuration, aspect ratio, wing area, and total mass, for maximum endurance or minimum required power based on the stated power system and mission parameters is presented. The results indicate that, for the power system, the greatest benefit can be gained by increasing the fuel cell specific energy. Mission requirements also substantially affect the aircraft size. By limiting the time of year the aircraft is required to fly at high northern or southern latitudes, a significant reduction in aircraft size or increase in payload capacity can be achieved.

Tissue matrix arrays for high throughput screening and systems analysis of cell function

PubMed Central

Beachley, Vince Z.; Wolf, Matthew T.; Sadtler, Kaitlyn; Manda, Srikanth S.; Jacobs, Heather; Blatchley, Michael; Bader, Joel S.; Pandey, Akhilesh; Pardoll, Drew; Elisseeff, Jennifer H.

2015-01-01

Cell and protein arrays have demonstrated remarkable utility in the high-throughput evaluation of biological responses; however, they lack the complexity of native tissue and organs. Here, we describe tissue extracellular matrix (ECM) arrays for screening biological outputs and systems analysis. We spotted processed tissue ECM particles as two-dimensional arrays or incorporated them with cells to generate three-dimensional cell-matrix microtissue arrays. We then investigated the response of human stem, cancer, and immune cells to tissue ECM arrays originating from 11 different tissues, and validated the 2D and 3D arrays as representative of the in vivo microenvironment through quantitative analysis of tissue-specific cellular responses, including matrix production, adhesion and proliferation, and morphological changes following culture. The biological outputs correlated with tissue proteomics, and network analysis identified several proteins linked to cell function. Our methodology enables broad screening of ECMs to connect tissue-specific composition with biological activity, providing a new resource for biomaterials research and translation. PMID:26480475

Electro-triggering and electrochemical monitoring of dopamine exocytosis from a single cell by using ultrathin electrodes based on Au nanowires

NASA Astrophysics Data System (ADS)

Kang, Mijeong; Yoo, Seung Min; Gwak, Raekeun; Eom, Gayoung; Kim, Jihwan; Lee, Sang Yup; Kim, Bongsoo

2015-12-01

A sophisticated set of an Au nanowire (NW) stimulator-Au NW detector system is developed for electrical cell stimulation and electrochemical analysis of subsequent exocytosis with very high spatial resolution. Dopamine release from a rat pheochromocytoma cell is more stimulated by a more negative voltage pulse. This system could help to improve the therapeutic efficacy of electrotherapies by providing valuable information on their healing mechanism.A sophisticated set of an Au nanowire (NW) stimulator-Au NW detector system is developed for electrical cell stimulation and electrochemical analysis of subsequent exocytosis with very high spatial resolution. Dopamine release from a rat pheochromocytoma cell is more stimulated by a more negative voltage pulse. This system could help to improve the therapeutic efficacy of electrotherapies by providing valuable information on their healing mechanism. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06021d

Analysis and performance assessment of a new solar-based multigeneration system integrated with ammonia fuel cell and solid oxide fuel cell-gas turbine combined cycle

NASA Astrophysics Data System (ADS)

Siddiqui, Osamah; Dincer, Ibrahim

2017-12-01

In the present study, a new solar-based multigeneration system integrated with an ammonia fuel cell and solid oxide fuel cell-gas turbine combined cycle to produce electricity, hydrogen, cooling and hot water is developed for analysis and performance assessment. In this regard, thermodynamic analyses and modeling through both energy and exergy approaches are employed to assess and evaluate the overall system performance. Various parametric studies are conducted to study the effects of varying system parameters and operating conditions on the energy and exergy efficiencies. The results of this study show that the overall multigeneration system energy efficiency is obtained as 39.1% while the overall system exergy efficiency is calculated as 38.7%, respectively. The performance of this multigeneration system results in an increase of 19.3% in energy efficiency as compared to single generation system. Furthermore, the exergy efficiency of the multigeneration system is 17.8% higher than the single generation system. Moreover, both energy and exergy efficiencies of the solid oxide fuel cell-gas turbine combined cycle are determined as 68.5% and 55.9% respectively.

Automated cell counts on CSF samples: A multicenter performance evaluation of the GloCyte system.

PubMed

Hod, E A; Brugnara, C; Pilichowska, M; Sandhaus, L M; Luu, H S; Forest, S K; Netterwald, J C; Reynafarje, G M; Kratz, A

2018-02-01

Automated cell counters have replaced manual enumeration of cells in blood and most body fluids. However, due to the unreliability of automated methods at very low cell counts, most laboratories continue to perform labor-intensive manual counts on many or all cerebrospinal fluid (CSF) samples. This multicenter clinical trial investigated if the GloCyte System (Advanced Instruments, Norwood, MA), a recently FDA-approved automated cell counter, which concentrates and enumerates red blood cells (RBCs) and total nucleated cells (TNCs), is sufficiently accurate and precise at very low cell counts to replace all manual CSF counts. The GloCyte System concentrates CSF and stains RBCs with fluorochrome-labeled antibodies and TNCs with nucleic acid dyes. RBCs and TNCs are then counted by digital image analysis. Residual adult and pediatric CSF samples obtained for clinical analysis at five different medical centers were used for the study. Cell counts were performed by the manual hemocytometer method and with the GloCyte System following the same protocol at all sites. The limits of the blank, detection, and quantitation, as well as precision and accuracy of the GloCyte, were determined. The GloCyte detected as few as 1 TNC/μL and 1 RBC/μL, and reliably counted as low as 3 TNCs/μL and 2 RBCs/μL. The total coefficient of variation was less than 20%. Comparison with cell counts obtained with a hemocytometer showed good correlation (>97%) between the GloCyte and the hemocytometer, including at very low cell counts. The GloCyte instrument is a precise, accurate, and stable system to obtain red cell and nucleated cell counts in CSF samples. It allows for the automated enumeration of even very low cell numbers, which is crucial for CSF analysis. These results suggest that GloCyte is an acceptable alternative to the manual method for all CSF samples, including those with normal cell counts. © 2017 John Wiley & Sons Ltd.

Genome Editing of Erythroid Cell Culture Model Systems.

PubMed

Yik, Jinfen J; Crossley, Merlin; Quinlan, Kate G R

2018-01-01

Genome editing to introduce specific mutations or to knock out genes in model cell systems has become an efficient platform for research in the fields of molecular biology, genetics, and cell biology. With recent rapid improvements in genome editing techniques, bench-top manipulation of the genome in cell culture has become progressively easier. The application of this knowledge to erythroid cell culture systems now allows the rapid analysis of the downstream effects of virtually any engineered gene disruption or modification in cell systems. Here, we describe a CRISPR/Cas9-based approach to making genomic modifications in erythroid lineage cells which we have successfully used in both murine (MEL) and human (K562) erythroleukaemia immortalized cell lines.

Single-Cell Protein Analysis

PubMed Central

Wu, Meiye; Singh, Anup K

2012-01-01

Heterogeneity of cellular systems has been widely recognized but only recently have tools become available that allow probing of genes and proteins in single cells to understand it. While the advancement in single cell genomic analysis has been greatly aided by the power of amplification techniques (e.g., PCR), analysis of proteins in single cells has proven to be more challenging. However, recent advances in multi-parameter flow cytometry, microfluidics and other techniques have made it possible to measure wide variety of proteins in single cells. In this review, we highlight key recent developments in analysis of proteins in a single cell, and discuss their significance in biological research. PMID:22189001

In vivo RNAi in the Drosophila Follicular Epithelium: Analysis of Stem Cell Maintenance, Proliferation, and Differentiation.

PubMed

Riechmann, Veit

2017-01-01

In vivo RNAi in Drosophila facilitates simple and rapid analysis of gene functions in a cell- or tissue-specific manner. The versatility of the UAS-GAL4 system allows to control exactly where and when during development the function of a gene is depleted. The epithelium of the ovary is a particularly good model to study in a living animal how stem cells are maintained and how their descendants proliferate and differentiate. Here I provide basic information about the publicly available reagents for in vivo RNAi, and I describe how the oogenesis system can be applied to analyze stem cells and epithelial development at a histological level. Moreover, I give helpful hints to optimize the use of the UAS-GAL4 system for RNAi induction in the follicular epithelium. Finally, I provide detailed step-by-step protocols for ovary dissection, antibody stainings, and ovary mounting for microscopic analysis.

Label-Free Optofluidic Nanobiosensor Enables Real-Time Analysis of Single-Cell Cytokine Secretion.

PubMed

Li, Xiaokang; Soler, Maria; Szydzik, Crispin; Khoshmanesh, Khashayar; Schmidt, Julien; Coukos, George; Mitchell, Arnan; Altug, Hatice

2018-06-01

Single-cell analysis of cytokine secretion is essential to understand the heterogeneity of cellular functionalities and develop novel therapies for multiple diseases. Unraveling the dynamic secretion process at single-cell resolution reveals the real-time functional status of individual cells. Fluorescent and colorimetric-based methodologies require tedious molecular labeling that brings inevitable interferences with cell integrity and compromises the temporal resolution. An innovative label-free optofluidic nanoplasmonic biosensor is introduced for single-cell analysis in real time. The nanobiosensor incorporates a novel design of a multifunctional microfluidic system with small volume microchamber and regulation channels for reliable monitoring of cytokine secretion from individual cells for hours. Different interleukin-2 secretion profiles are detected and distinguished from single lymphoma cells. The sensor configuration combined with optical spectroscopic imaging further allows us to determine the spatial single-cell secretion fingerprints in real time. This new biosensor system is anticipated to be a powerful tool to characterize single-cell signaling for basic and clinical research. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evidence of Dynamically Dysregulated Gene Expression Pathways in Hyperresponsive B Cells from African American Lupus Patients

PubMed Central

Dozmorov, Igor; Dominguez, Nicolas; Sestak, Andrea L.; Robertson, Julie M.; Harley, John B.; James, Judith A.; Guthridge, Joel M.

2013-01-01

Recent application of gene expression profiling to the immune system has shown a great potential for characterization of complex regulatory processes. It is becoming increasingly important to characterize functional systems through multigene interactions to provide valuable insights into differences between healthy controls and autoimmune patients. Here we apply an original systematic approach to the analysis of changes in regulatory gene interconnections between in Epstein-Barr virus transformed hyperresponsive B cells from SLE patients and normal control B cells. Both traditional analysis of differential gene expression and analysis of the dynamics of gene expression variations were performed in combination to establish model networks of functional gene expression. This Pathway Dysregulation Analysis identified known transcription factors and transcriptional regulators activated uniquely in stimulated B cells from SLE patients. PMID:23977035

Dynamic modeling and evaluation of solid oxide fuel cell - combined heat and power system operating strategies

NASA Astrophysics Data System (ADS)

Nanaeda, Kimihiro; Mueller, Fabian; Brouwer, Jacob; Samuelsen, Scott

Operating strategies of solid oxide fuel cell (SOFC) combined heat and power (CHP) systems are developed and evaluated from a utility, and end-user perspective using a fully integrated SOFC-CHP system dynamic model that resolves the physical states, thermal integration and overall efficiency of the system. The model can be modified for any SOFC-CHP system, but the present analysis is applied to a hotel in southern California based on measured electric and heating loads. Analysis indicates that combined heat and power systems can be operated to benefit both the end-users and the utility, providing more efficient electric generation as well as grid ancillary services, namely dispatchable urban power. Design and operating strategies considered in the paper include optimal sizing of the fuel cell, thermal energy storage to dispatch heat, and operating the fuel cell to provide flexible grid power. Analysis results indicate that with a 13.1% average increase in price-of-electricity (POE), the system can provide the grid with a 50% operating range of dispatchable urban power at an overall thermal efficiency of 80%. This grid-support operating mode increases the operational flexibility of the SOFC-CHP system, which may make the technology an important utility asset for accommodating the increased penetration of intermittent renewable power.

Systems Biology Analysis of Heterocellular Signaling.

PubMed

Tape, Christopher J

2016-08-01

Tissues comprise multiple heterotypic cell types (e.g., epithelial, mesenchymal, and immune cells). Communication between heterotypic cell types is essential for biological cohesion and is frequently dysregulated in disease. Despite the importance of heterocellular communication, most systems biology techniques do not report cell-specific signaling data from mixtures of cells. As a result, our existing perspective of cellular behavior under-represents the influence of heterocellular signaling. Recent technical advances now permit the resolution of systems-level cell-specific signaling data. This review discusses how new physical, spatial, and isotopic resolving methods are facilitating unique systems biology studies of heterocellular communication. Copyright © 2016 Elsevier Ltd. All rights reserved.

Systems biology: A tool for charting the antiviral landscape.

PubMed

Bowen, James R; Ferris, Martin T; Suthar, Mehul S

2016-06-15

The host antiviral programs that are initiated following viral infection form a dynamic and complex web of responses that we have collectively termed as "the antiviral landscape". Conventional approaches to studying antiviral responses have primarily used reductionist systems to assess the function of a single or a limited subset of molecules. Systems biology is a holistic approach that considers the entire system as a whole, rather than individual components or molecules. Systems biology based approaches facilitate an unbiased and comprehensive analysis of the antiviral landscape, while allowing for the discovery of emergent properties that are missed by conventional approaches. The antiviral landscape can be viewed as a hierarchy of complexity, beginning at the whole organism level and progressing downward to isolated tissues, populations of cells, and single cells. In this review, we will discuss how systems biology has been applied to better understand the antiviral landscape at each of these layers. At the organismal level, the Collaborative Cross is an invaluable genetic resource for assessing how genetic diversity influences the antiviral response. Whole tissue and isolated bulk cell transcriptomics serves as a critical tool for the comprehensive analysis of antiviral responses at both the tissue and cellular levels of complexity. Finally, new techniques in single cell analysis are emerging tools that will revolutionize our understanding of how individual cells within a bulk infected cell population contribute to the overall antiviral landscape. Copyright © 2016 Elsevier B.V. All rights reserved.

Thermal analysis of a multi-layer microchannel heat sink for cooling concentrator photovoltaic (CPV) cells

NASA Astrophysics Data System (ADS)

Siyabi, Idris Al; Shanks, Katie; Mallick, Tapas; Sundaram, Senthilarasu

2017-09-01

Concentrator Photovoltaic (CPV) technology is increasingly being considered as an alternative option for solar electricity generation. However, increasing the light concentration ratio could decrease the system output power due to the increase in the temperature of the cells. The performance of a multi-layer microchannel heat sink configuration was evaluated using numerical analysis. In this analysis, three dimensional incompressible laminar steady flow model was solved numerically. An electrical and thermal solar cell model was coupled for solar cell temperature and efficiency calculations. Thermal resistance, solar cell temperature and pumping power were used for the system efficiency evaluation. An increase in the number of microchannel layers exhibited the best overall performance in terms of the thermal resistance, solar cell temperature uniformity and pressure drop. The channel height and width has no effect on the solar cell maximum temperature. However, increasing channel height leads to a reduction in the pressure drop and hence less fluid pumping power.

Performance of mid infrared spectroscopy in skin cancer cell type identification

NASA Astrophysics Data System (ADS)

Kastl, Lena; Kemper, Björn; Lloyd, Gavin R.; Nallala, Jayakrupakar; Stone, Nick; Naranjo, Valery; Penaranda, Francisco; Schnekenburger, Jürgen

2017-02-01

Marker free optical spectroscopy is a powerful tool for the rapid inspection of pathologically suspicious skin lesions and the non-invasive detection of early skin tumors. This goal can be reached by the combination of signal localization and the spectroscopical detection of chemical cell signatures. We here present the development and application of mid infrared spectroscopy (midIR) for the analysis of skin tumor cell types and three dimensional tissue phantoms towards the application of midIR spectroscopy for fast and reliable skin diagnostics. We developed standardized in vitro skin systems with increasing complexity, from single skin cell types as fibroblasts, keratinocytes and melanoma cells, to mixtures of these and finally three dimensional skin cancer phantoms. The cell systems were characterized with different systems in the midIR range up to 12 μm. The analysis of the spectra by novel data processing algorithms demonstrated the clear separation of all cell types, especially melanoma cells. Special attention and algorithm training was required for closely related mesenchymal cell types as dedifferentiated melanoma cells and fibroblasts. Proof of concept experiments with mixtures of in vivo fluorescence labelled skin cell types allowed the test of the new algorithms performance for the identification of specific cell types. The intense training of the software systems with various samples resulted in a increased sensitivity and specificity of the combined midIR and software system. These data highlight the potential of midIR spectroscopy as sensitive and specific future optical biopsy technology.

Automatic microscopy for mitotic cell location.

NASA Technical Reports Server (NTRS)

Herron, J.; Ranshaw, R.; Castle, J.; Wald, N.

1972-01-01

Advances are reported in the development of an automatic microscope with which to locate hematologic or other cells in mitosis for subsequent chromosome analysis. The system under development is designed to perform the functions of: slide scanning to locate metaphase cells; conversion of images of selected cells into binary form; and on-line computer analysis of the digitized image for significant cytogenetic data. Cell detection criteria are evaluated using a test sample of 100 mitotic cells and 100 artifacts.

Ultraviolet spectroscopic breath analysis using hollow-optical fiber as gas cell

NASA Astrophysics Data System (ADS)

Iwata, T.; Katagiri, T.; Matsuura, Y.

2017-02-01

For breath analysis on ultraviolet absorption spectroscopy, an analysis system using a hollow optical fiber as gas cell is developed. The hollow optical fiber functions as a long path and extremely small volume gas cell. Firstly, the measurement sensitivity of the system is evaluated by using NO gas as a gas sample. The result shows that NO gas with 50 ppb concentration is measured by using a system with a laser-driven, high intensity light source and a 3-meter long, aluminum-coated hollow optical fiber. Then an absorption spectrum of breath sample is measured in the wavelength region of around 200-300 nm and from the spectrum, it is found that the main absorbing components in breath were H2O, isoprene, and O3 converted from O2 by radiation of ultraviolet light. Then the concentration of isoprene in breath is estimated by using multiple linear regression analysis.

Advanced continuous cultivation methods for systems microbiology.

PubMed

Adamberg, Kaarel; Valgepea, Kaspar; Vilu, Raivo

2015-09-01

Increasing the throughput of systems biology-based experimental characterization of in silico-designed strains has great potential for accelerating the development of cell factories. For this, analysis of metabolism in the steady state is essential as only this enables the unequivocal definition of the physiological state of cells, which is needed for the complete description and in silico reconstruction of their phenotypes. In this review, we show that for a systems microbiology approach, high-resolution characterization of metabolism in the steady state--growth space analysis (GSA)--can be achieved by using advanced continuous cultivation methods termed changestats. In changestats, an environmental parameter is continuously changed at a constant rate within one experiment whilst maintaining cells in the physiological steady state similar to chemostats. This increases the resolution and throughput of GSA compared with chemostats, and, moreover, enables following of the dynamics of metabolism and detection of metabolic switch-points and optimal growth conditions. We also describe the concept, challenge and necessary criteria of the systematic analysis of steady-state metabolism. Finally, we propose that such systematic characterization of the steady-state growth space of cells using changestats has value not only for fundamental studies of metabolism, but also for systems biology-based metabolic engineering of cell factories.

Quantitative refractive index distribution of single cell by combining phase-shifting interferometry and AFM imaging.

PubMed

Zhang, Qinnan; Zhong, Liyun; Tang, Ping; Yuan, Yingjie; Liu, Shengde; Tian, Jindong; Lu, Xiaoxu

2017-05-31

Cell refractive index, an intrinsic optical parameter, is closely correlated with the intracellular mass and concentration. By combining optical phase-shifting interferometry (PSI) and atomic force microscope (AFM) imaging, we constructed a label free, non-invasive and quantitative refractive index of single cell measurement system, in which the accurate phase map of single cell was retrieved with PSI technique and the cell morphology with nanoscale resolution was achieved with AFM imaging. Based on the proposed AFM/PSI system, we achieved quantitative refractive index distributions of single red blood cell and Jurkat cell, respectively. Further, the quantitative change of refractive index distribution during Daunorubicin (DNR)-induced Jurkat cell apoptosis was presented, and then the content changes of intracellular biochemical components were achieved. Importantly, these results were consistent with Raman spectral analysis, indicating that the proposed PSI/AFM based refractive index system is likely to become a useful tool for intracellular biochemical components analysis measurement, and this will facilitate its application for revealing cell structure and pathological state from a new perspective.

Three-dimensional slice cultures from murine fetal gut for investigations of the enteric nervous system.

PubMed

Metzger, Marco; Bareiss, Petra M; Nikolov, Ivan; Skutella, Thomas; Just, Lothar

2007-01-01

Three-dimensional intestinal cultures offer new possibilities for the examination of growth potential, analysis of time specific gene expression, and spatial cellular arrangement of enteric nervous system in an organotypical environment. We present an easy to produce in vitro model of the enteric nervous system for analysis and manipulation of cellular differentiation processes. Slice cultures of murine fetal colon were cultured on membrane inserts for up to 2 weeks without loss of autonomous contractility. After slice preparation, cultured tissue reorganized within the first days in vitro. Afterward, the culture possessed more than 35 cell layers, including high prismatic epithelial cells, smooth muscle cells, glial cells, and neurons analyzed by immunohistochemistry. The contraction frequency of intestinal slice culture could be modulated by the neurotransmitter serotonin and the sodium channel blocker tetrodotoxin. Coculture experiments with cultured neurospheres isolated from enhanced green fluorescent protein (eGFP) transgenic mice demonstrated that differentiating eGFP-positive neurons were integrated into the intestinal tissue culture. This slice culture model of enteric nervous system proved to be useful for studying cell-cell interactions, cellular signaling, and cell differentiation processes in a three-dimensional cell arrangement.

Feasibility analysis of a hydrogen backup power system for Russian telecom market

NASA Astrophysics Data System (ADS)

Borzenko, V. I.; Dunikov, D. O.

2017-11-01

We performed feasibility analysis of 10 kW hydrogen backup power system (H2BS) consisting of a water electrolyzer, a metal hydride hydrogen storage and a fuel cell. Capital investments in H2BS are mostly determined by the costs of the PEM electrolyzer, the fuel cell and solid state hydrogen storage materials, for single unit or small series manufacture the cost of AB5-type intermetallic compound can reach 50% of total system cost. Today the capital investments in H2BS are 3 times higher than in conventional lead-acid system of the same capacity. Wide distribution of fuel cell hydrogen vehicles, development of hydrogen infrastructure, and mass production of hydrogen power systems will for sure lower capital investments in fuel cell backup power. Operational expenditures for H2BS is only 15% from the expenditures for lead acid systems, and after 4-5 years of exploitation the total cost of ownership will become lower than for batteries.

Investigation of the photovoltaic cell/ thermoelectric element hybrid system performance

NASA Astrophysics Data System (ADS)

Cotfas, D. T.; Cotfas, P. A.; Machidon, O. M.; Ciobanu, D.

2016-06-01

The PV/TEG hybrid system, consisting of the photovoltaic cells and thermoelectric element, is presented in the paper. The dependence of the PV/TEG hybrid system parameters on the illumination levels and the temperature is analysed. The maxim power values of the photovoltaic cell, of the thermoelectric element and of the PV/TEG system are calculated and a comparison between them is presented and analysed. An economic analysis is also presented.

Monitoring Cellular Events in Living Mast Cells Stimulated with an Extremely Small Amount of Fluid on a Microchip

NASA Astrophysics Data System (ADS)

Munaka, Tatsuya; Abe, Hirohisa; Kanai, Masaki; Sakamoto, Takashi; Nakanishi, Hiroaki; Yamaoka, Tetsuji; Shoji, Shuichi; Murakami, Akira

2006-07-01

We successfully developed a measurement system for real-time analysis of cellular function using a newly designed microchip. This microchip was equipped with a micro cell incubation chamber (240 nl) and was stimulated by a very small amount of stimuli (as small as 24 nl). Using the microchip system, cultivation of mast cells was successfully carried out. Monitoring of the cellular events after stimulation with an extremely small amount of fluid on a microchip was performed. This system could be applicable for various types of cellular analysis including real-time monitoring of cellular response by stimulation.

Electric terminal performance and characterization of solid oxide fuel cells and systems

NASA Astrophysics Data System (ADS)

Lindahl, Peter Allan

Solid Oxide Fuel Cells (SOFCs) are electrochemical devices which can effect efficient, clean, and quiet conversion of chemical to electrical energy. In contrast to conventional electricity generation systems which feature multiple discrete energy conversion processes, SOFCs are direct energy conversion devices. That is, they feature a fully integrated chemical to electrical energy conversion process where the electric load demanded of the cell intrinsically drives the electrochemical reactions and associated processes internal to the cell. As a result, the cell's electric terminals provide a path for interaction between load side electric demand and the conversion side processes. The implication of this is twofold. First, the magnitude and dynamic characteristics of the electric load demanded of the cell can directly impact the long-term efficacy of the cell's chemical to electrical energy conversion. Second, the electric terminal response to dynamic loads can be exploited for monitoring the cell's conversion side processes and used in diagnostic analysis and degradation-mitigating control schemes. This dissertation presents a multi-tier investigation into this electric terminal based performance characterization of SOFCs through the development of novel test systems, analysis techniques and control schemes. First, a reference-based simulation system is introduced. This system scales up the electric terminal performance of a prototype SOFC system, e.g. a single fuel cell, to that of a full power-level stack. This allows realistic stack/load interaction studies while maintaining explicit ability for post-test analysis of the prototype system. Next, a time-domain least squares fitting method for electrochemical impedance spectroscopy (EIS) is developed for reduced-time monitoring of the electrochemical and physicochemical mechanics of the fuel cell through its electric terminals. The utility of the reference-based simulator and the EIS technique are demonstrated through their combined use in the performance testing of a hybrid-source power management (HSPM) system designed to allow in-situ EIS monitoring of a stack under dynamic loading conditions. The results from the latter study suggest that an HSPM controller allows an opportunity for in-situ electric terminal monitoring and control-based mitigation of SOFC degradation. As such, an exploration of control-based SOFC degradation mitigation is presented and ideas for further work are suggested.

Displacement Damage Effects in Solar Cells: Mining Damage From the Microelectronics and Photonics Test Bed Space Experiment

NASA Technical Reports Server (NTRS)

Hardage, Donna (Technical Monitor); Walters, R. J.; Morton, T. L.; Messenger, S. R.

2004-01-01

The objective is to develop an improved space solar cell radiation response analysis capability and to produce a computer modeling tool which implements the analysis. This was accomplished through analysis of solar cell flight data taken on the Microelectronics and Photonics Test Bed experiment. This effort specifically addresses issues related to rapid technological change in the area of solar cells for space applications in order to enhance system performance, decrease risk, and reduce cost for future missions.

A versatile localization system for microscopic multiparametric analysis of cells.

PubMed

Thaw, H H; Rundquist, I; Johansson, U; Svensson, I; Collins, V P

1983-03-01

A new, simple and relatively inexpensive electronic digital position readout (DPRO) system which can be applied to the rapid localization and recovery of microscopic material is described. It is based upon a commercially available digital position readout system which is routinely utilized by industry for small machine tools and measuring equipment. This has been mounted onto the stage of various microscopic instrumentation to provide X and Y coordinates relative to an arbitrary reference point. The integration of small computers interfaced to scanning interferometric, microdensitometric and fluorescence microscopes were used to demonstrate the reliability, versatility and ease of application of this system to problems of multiparametric measurements and analysis of cultured cells. The system may be expanded and applied to clinical material to obtain automatized, multiparametric measurements of cells in haematology and clinical cytology.

Roles of miRNAs in microcystin-LR-induced Sertoli cell toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Yuan; Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing, Jiangsu 210093; Wang, Hui

2015-08-15

Microcystin (MC)-LR, a cyclic heptapeptide, is a potent reproductive system toxin. To understand the molecular mechanisms of MC-induced reproductive system cytotoxicity, we evaluated global changes of miRNA and mRNA expression in mouse Sertoli cells following MC-LR treatment. Our results revealed that the exposure to MC-LR resulted in an altered miRNA expression profile that might be responsible for the modulation of mRNA expression. Bio-functional analysis indicated that the altered genes were involved in specific cellular processes, including cell death and proliferation. Target gene analysis suggested that junction injury in Sertoli cells exposed to MC-LR might be mediated by miRNAs through themore » regulation of the Sertoli cell-Sertoli cell pathway. Collectively, these findings may enhance our understanding on the modes of action of MC-LR on mouse Sertoli cells as well as the molecular mechanisms underlying the toxicity of MC-LR on the male reproductive system. - Highlights: • miRNAs were altered in Sertoli cells exposed to MC-LR. • Alerted genes were involved in different cell functions including the cell morphology. • MC-LR adversely affected Sertoli cell junction formation through the regulating miRNAs.« less

Automated microbeam observation environment for biological analysis-Custom portable environmental control applied to a vertical microbeam system.

PubMed

England, Matthew J; Bigelow, Alan W; Merchant, Michael J; Velliou, Eirini; Welch, David; Brenner, David J; Kirkby, Karen J

2017-02-01

Vertical Microbeams (VMB) are used to irradiate individual cells with low MeV energy ions. The irradiation of cells using VMBs requires cells to be removed from an incubator; this can cause physiological changes to cells because of the lower CO 2 concentration, temperature and relative humidity outside of the incubator. Consequently, for experiments where cells require irradiation and observation for extended time periods, it is important to provide a controlled environment. The highly customised nature of the microscopes used on VMB systems means that there are no commercially available environmentally controlled microscope systems for VMB systems. The Automated Microbeam Observation Environment for Biological Analysis (AMOEBA) is a highly flexible modular environmental control system used to create incubator conditions on the end of a VMB. The AMOEBA takes advantage of the recent "maker" movement to create an open source control system that can be easily configured by the user to fit their control needs even beyond VMB applications. When applied to the task of controlling cell medium temperature, CO 2 concentration and relative humidity on VMBs it creates a stable environment that allows cells to multiply on the end of a VMB over a period of 36 h, providing a low-cost (costing less than $2700 to build), customisable alternative to commercial time-lapse microscopy systems. AMOEBA adds the potential of VMBs to explore the long-term effects of radiation on single cells opening up new research areas for VMBs.

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

NASA Astrophysics Data System (ADS)

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

2015-03-01

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

A novel microfluidic platform for size and deformability based separation and the subsequent molecular characterization of viable circulating tumor cells.

PubMed

Hvichia, G E; Parveen, Z; Wagner, C; Janning, M; Quidde, J; Stein, A; Müller, V; Loges, S; Neves, R P L; Stoecklein, N H; Wikman, H; Riethdorf, S; Pantel, K; Gorges, T M

2016-06-15

Circulating tumor cells (CTCs) were introduced as biomarkers more than 10 years ago, but capture of viable CTCs at high purity from peripheral blood of cancer patients is still a major technical challenge. Here, we report a novel microfluidic platform designed for marker independent capture of CTCs. The Parsortix™ cell separation system provides size and deformability-based enrichment with automated staining for cell identification, and subsequent recovery (harvesting) of cells from the device. Using the Parsortix™ system, average cell capture inside the device ranged between 42% and 70%. Subsequent harvest of cells from the device ranged between 54% and 69% of cells captured. Most importantly, 99% of the isolated tumor cells were viable after processing in spiking experiments as well as after harvesting from patient samples and still functional for downstream molecular analysis as demonstrated by mRNA characterization and array-based comparative genomic hybridization. Analyzing clinical blood samples from metastatic (n = 20) and nonmetastatic (n = 6) cancer patients in parallel with CellSearch(®) system, we found that there was no statistically significant difference between the quantitative behavior of the two systems in this set of twenty six paired separations. In conclusion, the epitope independent Parsortix™ system enables the isolation of viable CTCs at a very high purity. Using this system, viable tumor cells are easily accessible and ready for molecular and functional analysis. The system's ability for enumeration and molecular characterization of EpCAM-negative CTCs will help to broaden research into the mechanisms of cancer as well as facilitating the use of CTCs as "liquid biopsies." © 2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

Effect of amino acid supplementation on titer and glycosylation distribution in hybridoma cell cultures-Systems biology-based interpretation using genome-scale metabolic flux balance model and multivariate data analysis.

PubMed

Reimonn, Thomas M; Park, Seo-Young; Agarabi, Cyrus D; Brorson, Kurt A; Yoon, Seongkyu

2016-09-01

Genome-scale flux balance analysis (FBA) is a powerful systems biology tool to characterize intracellular reaction fluxes during cell cultures. FBA estimates intracellular reaction rates by optimizing an objective function, subject to the constraints of a metabolic model and media uptake/excretion rates. A dynamic extension to FBA, dynamic flux balance analysis (DFBA), can calculate intracellular reaction fluxes as they change during cell cultures. In a previous study by Read et al. (2013), a series of informed amino acid supplementation experiments were performed on twelve parallel murine hybridoma cell cultures, and this data was leveraged for further analysis (Read et al., Biotechnol Prog. 2013;29:745-753). In order to understand the effects of media changes on the model murine hybridoma cell line, a systems biology approach is applied in the current study. Dynamic flux balance analysis was performed using a genome-scale mouse metabolic model, and multivariate data analysis was used for interpretation. The calculated reaction fluxes were examined using partial least squares and partial least squares discriminant analysis. The results indicate media supplementation increases product yield because it raises nutrient levels extending the growth phase, and the increased cell density allows for greater culture performance. At the same time, the directed supplementation does not change the overall metabolism of the cells. This supports the conclusion that product quality, as measured by glycoform assays, remains unchanged because the metabolism remains in a similar state. Additionally, the DFBA shows that metabolic state varies more at the beginning of the culture but less by the middle of the growth phase, possibly due to stress on the cells during inoculation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1163-1173, 2016. © 2016 American Institute of Chemical Engineers.

Raman tweezers in microfluidic systems for analysis and sorting of living cells

NASA Astrophysics Data System (ADS)

Pilát, Zdeněk.; Ježek, Jan; Kaňka, Jan; Zemánek, Pavel

2014-12-01

We have devised an analytical and sorting system combining optical trapping with Raman spectroscopy in microfluidic environment, dedicated to identification and sorting of biological objects, such as living cells of various unicellular organisms. Our main goal was to create a robust and universal platform for non-destructive and non-contact sorting of micro-objects based on their Raman spectral properties. This approach allowed us to collect spectra containing information about the chemical composition of the objects, such as the presence and composition of pigments, lipids, proteins, or nucleic acids, avoiding artificial chemical probes such as fluorescent markers. The non-destructive nature of this optical analysis and manipulation allowed us to separate individual living cells of our interest in a sterile environment and provided the possibility to cultivate the selected cells for further experiments. We used a mixture of polystyrene micro-particles and algal cells to test and demonstrate the function of our analytical and sorting system. The devised system could find its use in many medical, biotechnological, and biological applications.

Thermodynamic analysis of Direct Urea Solid Oxide Fuel Cell in combined heat and power applications

NASA Astrophysics Data System (ADS)

Abraham, F.; Dincer, I.

2015-12-01

This paper presents a comprehensive steady state modelling and thermodynamic analysis of Direct Urea Solid Oxide Fuel Cell integrated with Gas Turbine power cycle (DU-SOFC/GT). The use of urea as direct fuel mitigates public health and safety risks associated with the use of hydrogen and ammonia. The integration scheme in this study covers both oxygen ion-conducting solid oxide fuel cells (SOFC-O) and hydrogen proton-conducting solid oxide fuel cells (SOFC-H). Parametric case studies are carried out to investigate the effects of design and operating parameters on the overall performance of the system. The results reveal that the fuel cell exhibited the highest level of exergy destruction among other system components. Furthermore, the SOFC-O based system offers better overall performance than that with the SOFC-H option mainly due to the detrimental reverse water-gas shift reaction at the SOFC anode as well as the unique configuration of the system.

Raman tweezers in microfluidic systems for analysis and sorting of living cells

NASA Astrophysics Data System (ADS)

Pilát, Zdenëk; Ježek, Jan; Kaňka, Jan; Zemánek, Pavel

2014-03-01

We have devised an analytical and sorting system combining optical trapping with Raman spectroscopy in microfluidic environment in order to identify and sort biological objects, such as living cells of various prokaryotic and eukaryotic organisms. Our main objective was to create a robust and universal platform for non-contact sorting of microobjects based on their Raman spectral properties. This approach allowed us to collect information about the chemical composition of the objects, such as the presence and composition of lipids, proteins, or nucleic acids without using artificial chemical probes such as fluorescent markers. The non-destructive and non-contact nature of this optical analysis and manipulation allowed us to separate individual living cells of our interest in a sterile environment and provided the possibility to cultivate the selected cells for further experiments. We used differently treated cells of algae to test and demonstrate the function of our analytical and sorting system. The devised system could find its use in many medical, biotechnological, and biological applications.

A CLIPS expert system for clinical flow cytometry data analysis

NASA Technical Reports Server (NTRS)

Salzman, G. C.; Duque, R. E.; Braylan, R. C.; Stewart, C. C.

1990-01-01

An expert system is being developed using CLIPS to assist clinicians in the analysis of multivariate flow cytometry data from cancer patients. Cluster analysis is used to find subpopulations representing various cell types in multiple datasets each consisting of four to five measurements on each of 5000 cells. CLIPS facts are derived from results of the clustering. CLIPS rules are based on the expertise of Drs. Stewart, Duque, and Braylan. The rules incorporate certainty factors based on case histories.

Innovative Tools and Technology for Analysis of Single Cells and Cell-Cell Interaction.

PubMed

Konry, Tania; Sarkar, Saheli; Sabhachandani, Pooja; Cohen, Noa

2016-07-11

Heterogeneity in single-cell responses and intercellular interactions results from complex regulation of cell-intrinsic and environmental factors. Single-cell analysis allows not only detection of individual cellular characteristics but also correlation of genetic content with phenotypic traits in the same cell. Technological advances in micro- and nanofabrication have benefited single-cell analysis by allowing precise control of the localized microenvironment, cell manipulation, and sensitive detection capabilities. Additionally, microscale techniques permit rapid, high-throughput, multiparametric screening that has become essential for -omics research. This review highlights innovative applications of microscale platforms in genetic, proteomic, and metabolic detection in single cells; cell sorting strategies; and heterotypic cell-cell interaction. We discuss key design aspects of single-cell localization and isolation in microfluidic systems, dynamic and endpoint analyses, and approaches that integrate highly multiplexed detection of various intracellular species.

A thermodynamic approach for selecting operating conditions in the design of reversible solid oxide cell energy systems

NASA Astrophysics Data System (ADS)

Wendel, Christopher H.; Kazempoor, Pejman; Braun, Robert J.

2016-01-01

Reversible solid oxide cell (ReSOC) systems are being increasingly considered for electrical energy storage, although much work remains before they can be realized, including cell materials development and system design optimization. These systems store electricity by generating a synthetic fuel in electrolysis mode and subsequently recover electricity by electrochemically oxidizing the stored fuel in fuel cell mode. System thermal management is improved by promoting methane synthesis internal to the ReSOC stack. Within this strategy, the cell-stack operating conditions are highly impactful on system performance and optimizing these parameters to suit both operating modes is critical to achieving high roundtrip efficiency. Preliminary analysis shows the thermoneutral voltage to be a useful parameter for analyzing ReSOC systems and the focus of this study is to quantitatively examine how it is affected by ReSOC operating conditions. The results reveal that the thermoneutral voltage is generally reduced by increased pressure, and reductions in temperature, fuel utilization, and hydrogen-to-carbon ratio. Based on the thermodynamic analysis, many different combinations of these operating conditions are expected to promote efficient energy storage. Pressurized systems can achieve high efficiency at higher temperature and fuel utilization, while non-pressurized systems may require lower stack temperature and suffer from reduced energy density.

Cell module and fuel conditioner development

NASA Technical Reports Server (NTRS)

Feret, J. M.

1981-01-01

A phosphoric acid fuel cell (PAFC) stack design having a 10 kW power rating for operation at higher than atmospheric pressure based on the existing Mark II design configuration is described. Functional analysis, trade studies and thermodynamic cycle analysis for requirements definition and system operating parameter selection purposes were performed. Fuel cell materials and components, and performance testing and evaluation of the repeating electrode components were characterized. The state of the art manufacturing technology for all fuel cell components and the fabrication of short stacks of various sites were established. A 10 kW PAFC stack design for higher pressure operation utilizing the top down systems engineering aproach was developed.

A Transcriptomic Analysis of Xylan Mutants Does Not Support the Existence of a Secondary Cell Wall Integrity System in Arabidopsis

PubMed Central

Faria-Blanc, Nuno; Mortimer, Jenny C.; Dupree, Paul

2018-01-01

Yeast have long been known to possess a cell wall integrity (CWI) system, and recently an analogous system has been described for the primary walls of plants (PCWI) that leads to changes in plant growth and cell wall composition. A similar system has been proposed to exist for secondary cell walls (SCWI). However, there is little data to support this. Here, we analyzed the stem transcriptome of a set of cell wall biosynthetic mutants in order to investigate whether cell wall damage, in this case caused by aberrant xylan synthesis, activates a signaling cascade or changes in cell wall synthesis gene expression. Our data revealed remarkably few changes to the transcriptome. We hypothesize that this is because cells undergoing secondary cell wall thickening have entered a committed programme leading to cell death, and therefore a SCWI system would have limited impact. The absence of transcriptomic responses to secondary cell wall alterations may facilitate engineering of the secondary cell wall of plants. PMID:29636762

A Transcriptomic Analysis of Xylan Mutants Does Not Support the Existence of a Secondary Cell Wall Integrity System in Arabidopsis.

PubMed

Faria-Blanc, Nuno; Mortimer, Jenny C; Dupree, Paul

2018-01-01

Yeast have long been known to possess a cell wall integrity (CWI) system, and recently an analogous system has been described for the primary walls of plants (PCWI) that leads to changes in plant growth and cell wall composition. A similar system has been proposed to exist for secondary cell walls (SCWI). However, there is little data to support this. Here, we analyzed the stem transcriptome of a set of cell wall biosynthetic mutants in order to investigate whether cell wall damage, in this case caused by aberrant xylan synthesis, activates a signaling cascade or changes in cell wall synthesis gene expression. Our data revealed remarkably few changes to the transcriptome. We hypothesize that this is because cells undergoing secondary cell wall thickening have entered a committed programme leading to cell death, and therefore a SCWI system would have limited impact. The absence of transcriptomic responses to secondary cell wall alterations may facilitate engineering of the secondary cell wall of plants.

A gradient method for the quantitative analysis of cell movement and tissue flow and its application to the analysis of multicellular Dictyostelium development.

PubMed

Siegert, F; Weijer, C J; Nomura, A; Miike, H

1994-01-01

We describe the application of a novel image processing method, which allows quantitative analysis of cell and tissue movement in a series of digitized video images. The result is a vector velocity field showing average direction and velocity of movement for every pixel in the frame. We apply this method to the analysis of cell movement during different stages of the Dictyostelium developmental cycle. We analysed time-lapse video recordings of cell movement in single cells, mounds and slugs. The program can correctly assess the speed and direction of movement of either unlabelled or labelled cells in a time series of video images depending on the illumination conditions. Our analysis of cell movement during multicellular development shows that the entire morphogenesis of Dictyostelium is characterized by rotational cell movement. The analysis of cell and tissue movement by the velocity field method should be applicable to the analysis of morphogenetic processes in other systems such as gastrulation and neurulation in vertebrate embryos.

Bragg-cell receiver study

NASA Technical Reports Server (NTRS)

Wilson, Lonnie A.

1987-01-01

Bragg-cell receivers are employed in specialized Electronic Warfare (EW) applications for the measurement of frequency. Bragg-cell receiver characteristics are fully characterized for simple RF emitter signals. This receiver is early in its development cycle when compared to the IFM receiver. Functional mathematical models are derived and presented in this report for the Bragg-cell receiver. Theoretical analysis is presented and digital computer signal processing results are presented for the Bragg-cell receiver. Probability density function analysis are performed for output frequency. Probability density function distributions are observed to depart from assumed distributions for wideband and complex RF signals. This analysis is significant for high resolution and fine grain EW Bragg-cell receiver systems.

Microscopic optical path length difference and polarization measurement system for cell analysis

NASA Astrophysics Data System (ADS)

Satake, H.; Ikeda, K.; Kowa, H.; Hoshiba, T.; Watanabe, E.

2018-03-01

In recent years, noninvasive, nonstaining, and nondestructive quantitative cell measurement techniques have become increasingly important in the medical field. These cell measurement techniques enable the quantitative analysis of living cells, and are therefore applied to various cell identification processes, such as those determining the passage number limit during cell culturing in regenerative medicine. To enable cell measurement, we developed a quantitative microscopic phase imaging system based on a Mach-Zehnder interferometer that measures the optical path length difference distribution without phase unwrapping using optical phase locking. The applicability of our phase imaging system was demonstrated by successful identification of breast cancer cells amongst normal cells. However, the cell identification method using this phase imaging system exhibited a false identification rate of approximately 7%. In this study, we implemented a polarimetric imaging system by introducing a polarimetric module to one arm of the Mach-Zehnder interferometer of our conventional phase imaging system. This module was comprised of a quarter wave plate and a rotational polarizer on the illumination side of the sample, and a linear polarizer on the optical detector side. In addition, we developed correction methods for the measurement errors of the optical path length and birefringence phase differences that arose through the influence of elements other than cells, such as the Petri dish. As the Petri dish holding the fluid specimens was transparent, it did not affect the amplitude information; however, the optical path length and birefringence phase differences were affected. Therefore, we proposed correction of the optical path length and birefringence phase for the influence of elements other than cells, as a prerequisite for obtaining highly precise phase and polarimetric images.

Spectrally And Temporally Resolved Low-Light Level Video Microscopy

NASA Astrophysics Data System (ADS)

Wampler, John E.; Furukawa, Ruth; Fechheimer, Marcus

1989-12-01

The IDG law-light video microscope system was designed to aid studies of localization of subcellular luminescence sources and stimulus/response coupling in single living cells using luminescent probes. Much of the motivation for design of this instrument system came from the pioneering efforts of Dr. Reynolds (Reynolds, Q. Rev. Biophys. 5, 295-347; Reynolds and Taylor, Bioscience 30, 586-592) who showed the value of intensified video camera systems for detection and localizion of fluorescence and bioluminescence signals from biological tissues. Our instrument system has essentially two roles, 1) localization and quantitation of very weak bioluminescence signals and 2) quantitation of intracellular environmental characteristics such as pH and calcium ion concentrations using fluorescent and bioluminescent probes. The instrument system exhibits over one million fold operating range allowing visualization and enhancement of quantum limited images with quantum limited response, spectral analysis of fluorescence signals, and transmitted light imaging. The computer control of the system implements rapid switching between light regimes, spatially resolved spectral scanning, and digital data processing for spectral shape analysis and for detailed analysis of the statistical distribution of single cell measurements. The system design and software algorithms used by the system are summarized. These design criteria are illustrated with examples taken from studies of bioluminescence, applications of bioluminescence to study developmental processes and gene expression in single living cells, and applications of fluorescent probes to study stimulus/response coupling in living cells.

Label-Free Raman Microspectral Analysis for Comparison of Cellular Uptake and Distribution between Non-Targeted and EGFR-Targeted Biodegradable Polymeric Nanoparticles

PubMed Central

Chernenko, Tatyana; Buyukozturk, Fulden; Miljkovic, Milos; Carrier, Rebecca; Diem, Max; Amiji, Mansoor

2013-01-01

Active targeted delivery of nanoparticle-encapsulated agents to tumor cells in vivo is expected to enhance therapeutic effect with significantly less non-specific toxicity. Active targeting is based on surface modification of nanoparticles with ligands that bind with extracellular targets and enhance payload delivery in the cells. In this study, we have used label-free Raman micro-spectral analysis and kinetic modeling to study cellular interactions and intracellular delivery of C6-ceramide using a non-targeted and an epidermal growth factor receptor (EGFR) targeted biodegradable polymeric nano-delivery systems, in EGFR-expressing human ovarian adenocarcinoma (SKOV3) cells. The results show that EGFR peptide-modified nanoparticles were rapidly internalized in SKOV3 cells leading to significant intracellular accumulation as compared to non-specific uptake by the non-targeted nanoparticles. Raman micro-spectral analysis enables visualization and quantification of the carrier system, drug-load, and responses of the biological systems interrogated, without exogenous staining and labeling procedures. PMID:24298430

A simplified solar cell array modelling program

NASA Technical Reports Server (NTRS)

Hughes, R. D.

1982-01-01

As part of the energy conversion/self sufficiency efforts of DSN engineering, it was necessary to have a simplified computer model of a solar photovoltaic (PV) system. This article describes the analysis and simplifications employed in the development of a PV cell array computer model. The analysis of the incident solar radiation, steady state cell temperature and the current-voltage characteristics of a cell array are discussed. A sample cell array was modelled and the results are presented.

Noninvasive Real-Time Assessment of Cell Viability in a Three-Dimensional Tissue.

PubMed

Mahfouzi, Seyed Hossein; Amoabediny, Ghassem; Doryab, Ali; Safiabadi-Tali, Seyed Hamid; Ghanei, Mostafa

2018-04-01

Maintaining cell viability within 3D tissue engineering scaffolds is an essential step toward a functional tissue or organ. Assessment of cell viability in 3D scaffolds is necessary to control and optimize tissue culture process. Monitoring systems based on respiration activity of cells (e.g., oxygen consumption) have been used in various cell cultures. In this research, an online monitoring system based on respiration activity was developed to monitor cell viability within acellular lung scaffolds. First, acellular lung scaffolds were recellularized with human umbilical cord vein endothelial cells, and then, cell viability was monitored during a 5-day period. The real-time monitoring system generated a cell growth profile representing invaluable information on cell viability and proliferative states during the culture period. The cell growth profile obtained by the monitoring system was consistent with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and glucose consumption measurement. This system provided a means for noninvasive, real-time, and repetitive investigation of cell viability. Also, we showed the applicability of this monitoring system by introducing shaking as an operating parameter in a long-term culture.

Rapid cell-free forward engineering of novel genetic ring oscillators

PubMed Central

Niederholtmeyer, Henrike; Sun, Zachary Z; Hori, Yutaka; Yeung, Enoch; Verpoorte, Amanda; Murray, Richard M; Maerkl, Sebastian J

2015-01-01

While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the ‘repressilator’, a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hr. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior. DOI: http://dx.doi.org/10.7554/eLife.09771.001 PMID:26430766

The new numerology of immunity mediated by virus-specific CD8(+) T cells.

PubMed

Doherty, P C

1998-08-01

Our understanding of virus-specific CD8(+) T cell responses is currently being revolutionized by peptide-based assay systems that allow flow cytometric analysis of effector and memory cytotoxic T lymphocyte populations. These techniques are, for the first time, putting the analysis of T-cell-mediated immunity on a quantitative basis.

Modeling integrated photovoltaic–electrochemical devices using steady-state equivalent circuits

PubMed Central

Winkler, Mark T.; Cox, Casandra R.; Nocera, Daniel G.; Buonassisi, Tonio

2013-01-01

We describe a framework for efficiently coupling the power output of a series-connected string of single-band-gap solar cells to an electrochemical process that produces storable fuels. We identify the fundamental efficiency limitations that arise from using solar cells with a single band gap, an arrangement that describes the use of currently economic solar cell technologies such as Si or CdTe. Steady-state equivalent circuit analysis permits modeling of practical systems. For the water-splitting reaction, modeling defines parameters that enable a solar-to-fuels efficiency exceeding 18% using laboratory GaAs cells and 16% using all earth-abundant components, including commercial Si solar cells and Co- or Ni-based oxygen evolving catalysts. Circuit analysis also provides a predictive tool: given the performance of the separate photovoltaic and electrochemical systems, the behavior of the coupled photovoltaic–electrochemical system can be anticipated. This predictive utility is demonstrated in the case of water oxidation at the surface of a Si solar cell, using a Co–borate catalyst.

Comparison of Whole-Cell SELEX Methods for the Identification of Staphylococcus Aureus-Specific DNA Aptamers

PubMed Central

Moon, Jihea; Kim, Giyoung; Park, Saet Byeol; Lim, Jongguk; Mo, Changyeun

2015-01-01

Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria. PMID:25884791

Comparison of whole-cell SELEX methods for the identification of Staphylococcus aureus-specific DNA aptamers.

PubMed

Moon, Jihea; Kim, Giyoung; Park, Saet Byeol; Lim, Jongguk; Mo, Changyeun

2015-04-15

Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria.

Integrated CZE-ESI-MS/MS system with an immobilized trypsin microreactor for online digestion and analysis of picogram amounts of RAW 264.7 cell lysate

PubMed Central

Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J.

2013-01-01

A capillary zone electrophoresis (CZE) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) system was integrated with an immobilized trypsin microreactor. The system was evaluated and then applied for online digestion and analysis of picogram loadings of RAW 264.7 cell lysate. Protein samples were dissolved in a buffer containing 50% (v/v) acetonitrile (ACN), and then directly loaded into the capillary for digestion, followed by CZE separation and MS/MS identification. The organic solvent (50% (v/v) ACN) assisted the immobilized trypsin digestion and simplified the protein sample preparation protocol. Neither protein reduction nor alkylation steps were employed, which minimized sample loss and contamination. The integrated CZE-ESI-MS/MS system generated confident identification of bovine serum albumin (BSA) with 19% sequence coverage and 14 peptide IDs when 20 fmole was loaded. When only 1 fmole BSA was injected, one BSA peptide was consistently detected. For the analysis of a standard protein mixture, the integrated system produced efficient protein digestion and confident identification for proteins with different molecular weights and isoelectric points when low fmole amount was loaded for each protein. We further applied the system for triplicate analysis of a RAW 264.7 cell lysate; 2 ± 1 and 7 ± 2 protein groups were confidently identified from only 300 pg and 3 ng loadings, respectively. The 300 pg sample loading corresponds to the protein content of three RAW 264.7 cells. In addition to high sensitivity analysis, the integrated CZE-ESI-MS/MS system produces good reproducibility in terms of peptide and protein IDs, peptide migration time, and peptide intensity. PMID:23510126

A system architecture for online data interpretation and reduction in fluorescence microscopy

NASA Astrophysics Data System (ADS)

Röder, Thorsten; Geisbauer, Matthias; Chen, Yang; Knoll, Alois; Uhl, Rainer

2010-01-01

In this paper we present a high-throughput sample screening system that enables real-time data analysis and reduction for live cell analysis using fluorescence microscopy. We propose a novel system architecture capable of analyzing a large amount of samples during the experiment and thus greatly minimizing the post-analysis phase that is the common practice today. By utilizing data reduction algorithms, relevant information of the target cells is extracted from the online collected data stream, and then used to adjust the experiment parameters in real-time, allowing the system to dynamically react on changing sample properties and to control the microscope setup accordingly. The proposed system consists of an integrated DSP-FPGA hybrid solution to ensure the required real-time constraints, to execute efficiently the underlying computer vision algorithms and to close the perception-action loop. We demonstrate our approach by addressing the selective imaging of cells with a particular combination of markers. With this novel closed-loop system the amount of superfluous collected data is minimized, while at the same time the information entropy increases.

Natural Resource Information System, design analysis

NASA Technical Reports Server (NTRS)

1972-01-01

The computer-based system stores, processes, and displays map data relating to natural resources. The system was designed on the basis of requirements established in a user survey and an analysis of decision flow. The design analysis effort is described, and the rationale behind major design decisions, including map processing, cell vs. polygon, choice of classification systems, mapping accuracy, system hardware, and software language is summarized.

Imaging cell picker: A morphology-based automated cell separation system on a photodegradable hydrogel culture platform.

PubMed

Shibuta, Mayu; Tamura, Masato; Kanie, Kei; Yanagisawa, Masumi; Matsui, Hirofumi; Satoh, Taku; Takagi, Toshiyuki; Kanamori, Toshiyuki; Sugiura, Shinji; Kato, Ryuji

2018-06-09

Cellular morphology on and in a scaffold composed of extracellular matrix generally represents the cellular phenotype. Therefore, morphology-based cell separation should be interesting method that is applicable to cell separation without staining surface markers in contrast to conventional cell separation methods (e.g., fluorescence activated cell sorting and magnetic activated cell sorting). In our previous study, we have proposed a cloning technology using a photodegradable gelatin hydrogel to separate the individual cells on and in hydrogels. To further expand the applicability of this photodegradable hydrogel culture platform, we here report an image-based cell separation system imaging cell picker for the morphology-based cell separation on a photodegradable hydrogel. We have developed the platform which enables the automated workflow of image acquisition, image processing and morphology analysis, and collection of a target cells. We have shown the performance of the morphology-based cell separation through the optimization of the critical parameters that determine the system's performance, such as (i) culture conditions, (ii) imaging conditions, and (iii) the image analysis scheme, to actually clone the cells of interest. Furthermore, we demonstrated the morphology-based cloning performance of cancer cells in the mixture of cells by automated hydrogel degradation by light irradiation and pipetting. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Fostering synergy between cell biology and systems biology.

PubMed

Eddy, James A; Funk, Cory C; Price, Nathan D

2015-08-01

In the shared pursuit of elucidating detailed mechanisms of cell function, systems biology presents a natural complement to ongoing efforts in cell biology. Systems biology aims to characterize biological systems through integrated and quantitative modeling of cellular information. The process of model building and analysis provides value through synthesizing and cataloging information about cells and molecules, predicting mechanisms and identifying generalizable themes, generating hypotheses and guiding experimental design, and highlighting knowledge gaps and refining understanding. In turn, incorporating domain expertise and experimental data is crucial for building towards whole cell models. An iterative cycle of interaction between cell and systems biologists advances the goals of both fields and establishes a framework for mechanistic understanding of the genome-to-phenome relationship. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Visualization and Image Analysis of Yeast Cells.

PubMed

Bagley, Steve

2016-01-01

When converting real-life data via visualization to numbers and then onto statistics the whole system needs to be considered so that conversion from the analogue to the digital is accurate and repeatable. Here we describe the points to consider when approaching yeast cell analysis visualization, processing, and analysis of a population by screening techniques.

CellStress - open source image analysis program for single-cell analysis

NASA Astrophysics Data System (ADS)

Smedh, Maria; Beck, Caroline; Sott, Kristin; Goksör, Mattias

2010-08-01

This work describes our image-analysis software, CellStress, which has been developed in Matlab and is issued under a GPL license. CellStress was developed in order to analyze migration of fluorescent proteins inside single cells during changing environmental conditions. CellStress can also be used to score information regarding protein aggregation in single cells over time, which is especially useful when monitoring cell signaling pathways involved in e.g. Alzheimer's or Huntington's disease. Parallel single-cell analysis of large numbers of cells is an important part of the research conducted in systems biology and quantitative biology in order to mathematically describe cellular processes. To quantify properties for single cells, large amounts of data acquired during extended time periods are needed. Manual analyses of such data involve huge efforts and could also include a bias, which complicates the use and comparison of data for further simulations or modeling. Therefore, it is necessary to have an automated and unbiased image analysis procedure, which is the aim of CellStress. CellStress utilizes cell contours detected by CellStat (developed at Fraunhofer-Chalmers Centre), which identifies cell boundaries using bright field images, and thus reduces the fluorescent labeling needed.

VIC: A Computer Analysis of Verbal Interaction Category Systems.

ERIC Educational Resources Information Center

Kline, John A.; And Others

VIC is a computer program for the analysis of verbal interaction category systems, especially the Flanders interaction analysis system. The observer codes verbal behavior on coding sheets for later machine scoring. A matrix is produced by the program showing the number and percentages of times that a particular cell describes classroom behavior.…

Final Report: Mass Production Cost Estimation of Direct H2 PEM Fuel Cell Systems for Transportation Applications (2012-2016)

DOE Office of Scientific and Technical Information (OSTI.GOV)

James, Brian David; Huya-Kouadio, Jennie Moton; Houchins, Cassidy

This report summarizes project activities for Strategic Analysis, Inc. (SA) Contract Number DE-EE0005236 to the U.S. Department of Energy titled “Transportation Fuel Cell System Cost Assessment”. The project defined and projected the mass production costs of direct hydrogen Proton Exchange Membrane fuel cell power systems for light-duty vehicles (automobiles) and 40-foot transit buses. In each year of the five-year contract, the fuel cell power system designs and cost projections were updated to reflect technology advances. System schematics, design assumptions, manufacturing assumptions, and cost results are presented.

A comprehensive complex systems approach to the study and analysis of mammalian cell cycle control system in the presence of DNA damage stress.

PubMed

Abroudi, Ali; Samarasinghe, Sandhya; Kulasiri, Don

2017-09-21

Not many models of mammalian cell cycle system exist due to its complexity. Some models are too complex and hard to understand, while some others are too simple and not comprehensive enough. Moreover, some essential aspects, such as the response of G1-S and G2-M checkpoints to DNA damage as well as the growth factor signalling, have not been investigated from a systems point of view in current mammalian cell cycle models. To address these issues, we bring a holistic perspective to cell cycle by mathematically modelling it as a complex system consisting of important sub-systems that interact with each other. This retains the functionality of the system and provides a clearer interpretation to the processes within it while reducing the complexity in comprehending these processes. To achieve this, we first update a published ODE mathematical model of cell cycle with current knowledge. Then the part of the mathematical model relevant to each sub-system is shown separately in conjunction with a diagram of the sub-system as part of this representation. The model sub-systems are Growth Factor, DNA damage, G1-S, and G2-M checkpoint signalling. To further simplify the model and better explore the function of sub-systems, they are further divided into modules. Here we also add important new modules of: chk-related rapid cell cycle arrest, p53 modules expanded to seamlessly integrate with the rapid arrest module, Tyrosine phosphatase modules that activate Cyc_Cdk complexes and play a crucial role in rapid and delay arrest at both G1-S and G2-M, Tyrosine Kinase module that is important for inactivating nuclear transport of CycB_cdk1 through Wee1 to resist M phase entry, Plk1-Related module that is crucial in activating Tyrosine phosphatases and inactivating Tyrosine kinase, and APC-Related module to show steps in CycB degradation. This multi-level systems approach incorporating all known aspects of cell cycle allowed us to (i) study, through dynamic simulation of an ODE model, comprehensive details of cell cycle dynamics under normal and DNA damage conditions revealing the role and value of the added new modules and elements, (ii) assess, through a global sensitivity analysis, the most influential sub-systems, modules and parameters on system response, such as G1-S and G2-M transitions, and (iii) probe deeply into the relationship between DNA damage and cell cycle progression and test the biological evidence that G1-S is relatively inefficient in arresting damaged cells compared to G2-M checkpoint. To perform sensitivity analysis, Self-Organizing Map with Correlation Coefficient Analysis (SOMCCA) is developed which shows that Growth Factor and G1-S Checkpoint sub-systems and 13 parameters in the modules within them are crucial for G1-S and G2-M transitions. To study the relative efficiency of DNA damage checkpoints, a Checkpoint Efficiency Evaluator (CEE) is developed based on perturbation studies and statistical Type II error. Accordingly, cell cycle is about 96% efficient in arresting damaged cells with G2-M checkpoint being more efficient than G1-S. Further, both checkpoint systems are near perfect (98.6%) in passing healthy cells. Thus this study has shown the efficacy of the proposed systems approach to gain a better understanding of different aspects of mammalian cell cycle system separately and as an integrated system that will also be useful in investigating targeted therapy in future cancer treatments. Copyright © 2017 Elsevier Ltd. All rights reserved.

A fully automated cell segmentation and morphometric parameter system for quantifying corneal endothelial cell morphology.

PubMed

Al-Fahdawi, Shumoos; Qahwaji, Rami; Al-Waisy, Alaa S; Ipson, Stanley; Ferdousi, Maryam; Malik, Rayaz A; Brahma, Arun

2018-07-01

Corneal endothelial cell abnormalities may be associated with a number of corneal and systemic diseases. Damage to the endothelial cells can significantly affect corneal transparency by altering hydration of the corneal stroma, which can lead to irreversible endothelial cell pathology requiring corneal transplantation. To date, quantitative analysis of endothelial cell abnormalities has been manually performed by ophthalmologists using time consuming and highly subjective semi-automatic tools, which require an operator interaction. We developed and applied a fully-automated and real-time system, termed the Corneal Endothelium Analysis System (CEAS) for the segmentation and computation of endothelial cells in images of the human cornea obtained by in vivo corneal confocal microscopy. First, a Fast Fourier Transform (FFT) Band-pass filter is applied to reduce noise and enhance the image quality to make the cells more visible. Secondly, endothelial cell boundaries are detected using watershed transformations and Voronoi tessellations to accurately quantify the morphological parameters of the human corneal endothelial cells. The performance of the automated segmentation system was tested against manually traced ground-truth images based on a database consisting of 40 corneal confocal endothelial cell images in terms of segmentation accuracy and obtained clinical features. In addition, the robustness and efficiency of the proposed CEAS system were compared with manually obtained cell densities using a separate database of 40 images from controls (n = 11), obese subjects (n = 16) and patients with diabetes (n = 13). The Pearson correlation coefficient between automated and manual endothelial cell densities is 0.9 (p < 0.0001) and a Bland-Altman plot shows that 95% of the data are between the 2SD agreement lines. We demonstrate the effectiveness and robustness of the CEAS system, and the possibility of utilizing it in a real world clinical setting to enable rapid diagnosis and for patient follow-up, with an execution time of only 6 seconds per image. Copyright © 2018 Elsevier B.V. All rights reserved.

Modelling Spread of Oncolytic Viruses in Heterogeneous Cell Populations

NASA Astrophysics Data System (ADS)

Ellis, Michael; Dobrovolny, Hana

2014-03-01

One of the most promising areas in current cancer research and treatment is the use of viruses to attack cancer cells. A number of oncolytic viruses have been identified to date that possess the ability to destroy or neutralize cancer cells while inflicting minimal damage upon healthy cells. Formulation of predictive models that correctly describe the evolution of infected tumor systems is critical to the successful application of oncolytic virus therapy. A number of different models have been proposed for analysis of the oncolytic virus-infected tumor system, with approaches ranging from traditional coupled differential equations such as the Lotka-Volterra predator-prey models, to contemporary modeling frameworks based on neural networks and cellular automata. Existing models are focused on tumor cells and the effects of virus infection, and offer the potential for improvement by including effects upon normal cells. We have recently extended the traditional framework to a 2-cell model addressing the full cellular system including tumor cells, normal cells, and the impacts of viral infection upon both populations. Analysis of the new framework reveals complex interaction between the populations and potential inability to simultaneously eliminate the virus and tumor populations.

Determination of morphological parameters of biological cells by analysis of scattered-light distributions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burger, D.E.

1979-11-01

The extraction of morphological parameters from biological cells by analysis of light-scatter patterns is described. A light-scattering measurement system has been designed and constructed that allows one to visually examine and photographically record biological cells or cell models and measure the light-scatter pattern of an individual cell or cell model. Using a laser or conventional illumination, the imaging system consists of a modified microscope with a 35 mm camera attached to record the cell image or light-scatter pattern. Models of biological cells were fabricated. The dynamic range and angular distributions of light scattered from these models was compared to calculatedmore » distributions. Spectrum analysis techniques applied on the light-scatter data give the sought after morphological cell parameters. These results compared favorably to shape parameters of the fabricated cell models confirming the mathematical model procedure. For nucleated biological material, correct nuclear and cell eccentricity as well as the nuclear and cytoplasmic diameters were determined. A method for comparing the flow equivalent of nuclear and cytoplasmic size to the actual dimensions is shown. This light-scattering experiment provides baseline information for automated cytology. In its present application, it involves correlating average size as measured in flow cytology to the actual dimensions determined from this technique. (ERB)« less

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

NASA Astrophysics Data System (ADS)

Zimny, Philip; Juncker, David; Reisner, Walter

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

Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics.

PubMed

Spaethling, Jennifer M; Na, Young-Ji; Lee, Jaehee; Ulyanova, Alexandra V; Baltuch, Gordon H; Bell, Thomas J; Brem, Steven; Chen, H Isaac; Dueck, Hannah; Fisher, Stephen A; Garcia, Marcela P; Khaladkar, Mugdha; Kung, David K; Lucas, Timothy H; O'Rourke, Donald M; Stefanik, Derek; Wang, Jinhui; Wolf, John A; Bartfai, Tamas; Grady, M Sean; Sul, Jai-Yoon; Kim, Junhyong; Eberwine, James H

2017-01-17

Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single-cell analysis of live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells, permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes, including hundreds of cell-type-enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type- and patient-specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. Because these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Rapid detection of bacterial contamination in cell or tissue cultures based on Raman spectroscopy

NASA Astrophysics Data System (ADS)

Bolwien, Carsten; Sulz, Gerd; Becker, Sebastian; Thielecke, Hagen; Mertsching, Heike; Koch, Steffen

2008-02-01

Monitoring the sterility of cell or tissue cultures is an essential task, particularly in the fields of regenerative medicine and tissue engineering when implanting cells into the human body. We present a system based on a commercially available microscope equipped with a microfluidic cell that prepares the particles found in the solution for analysis, a Raman-spectrometer attachment optimized for non-destructive, rapid recording of Raman spectra, and a data acquisition and analysis tool for identification of the particles. In contrast to conventional sterility testing in which samples are incubated over weeks, our system is able to analyze milliliters of supernatant or cell suspension within hours by filtering relevant particles and placing them on a Raman-friendly substrate in the microfluidic cell. Identification of critical particles via microscopic imaging and subsequent image analysis is carried out before micro-Raman analysis of those particles is then carried out with an excitation wavelength of 785 nm. The potential of this setup is demonstrated by results of artificial contamination of samples with a pool of bacteria, fungi, and spores: single-channel spectra of the critical particles are automatically baseline-corrected without using background data and classified via hierarchical cluster analysis, showing great promise for accurate and rapid detection and identification of contaminants.

Systems modelling methodology for the analysis of apoptosis signal transduction and cell death decisions.

PubMed

Rehm, Markus; Prehn, Jochen H M

2013-06-01

Systems biology and systems medicine, i.e. the application of systems biology in a clinical context, is becoming of increasing importance in biology, drug discovery and health care. Systems biology incorporates knowledge and methods that are applied in mathematics, physics and engineering, but may not be part of classical training in biology. We here provide an introduction to basic concepts and methods relevant to the construction and application of systems models for apoptosis research. We present the key methods relevant to the representation of biochemical processes in signal transduction models, with a particular reference to apoptotic processes. We demonstrate how such models enable a quantitative and temporal analysis of changes in molecular entities in response to an apoptosis-inducing stimulus, and provide information on cell survival and cell death decisions. We introduce methods for analyzing the spatial propagation of cell death signals, and discuss the concepts of sensitivity analyses that enable a prediction of network responses to disturbances of single or multiple parameters. Copyright © 2013 Elsevier Inc. All rights reserved.

The long-time dynamics of two hydrodynamically-coupled swimming cells.

PubMed

Michelin, Sébastien; Lauga, Eric

2010-05-01

Swimming microorganisms such as bacteria or spermatozoa are typically found in dense suspensions, and exhibit collective modes of locomotion qualitatively different from that displayed by isolated cells. In the dilute limit where fluid-mediated interactions can be treated rigorously, the long-time hydrodynamics of a collection of cells result from interactions with many other cells, and as such typically eludes an analytical approach. Here, we consider the only case where such problem can be treated rigorously analytically, namely when the cells have spatially confined trajectories, such as the spermatozoa of some marine invertebrates. We consider two spherical cells swimming, when isolated, with arbitrary circular trajectories, and derive the long-time kinematics of their relative locomotion. We show that in the dilute limit where the cells are much further away than their size, and the size of their circular motion, a separation of time scale occurs between a fast (intrinsic) swimming time, and a slow time where hydrodynamic interactions lead to change in the relative position and orientation of the swimmers. We perform a multiple-scale analysis and derive the effective dynamical system--of dimension two--describing the long-time behavior of the pair of cells. We show that the system displays one type of equilibrium, and two types of rotational equilibrium, all of which are found to be unstable. A detailed mathematical analysis of the dynamical systems further allows us to show that only two cell-cell behaviors are possible in the limit of t-->infinity, either the cells are attracted to each other (possibly monotonically), or they are repelled (possibly monotonically as well), which we confirm with numerical computations. Our analysis shows therefore that, even in the dilute limit, hydrodynamic interactions lead to new modes of cell-cell locomotion.

Contribution to solving the energy crisis - Simulating the prospects for low cost energy through silicon solar cells

NASA Technical Reports Server (NTRS)

Kran, A.

1978-01-01

PECAN (Photovoltaic Energy Conversion Analysis) is a highly interactive decision analysis and support system. It simulates the prospects for widespread use of solar cells for the generation of electrical power. PECAN consists of a set of integrated APL functions for evaluating the potential of terrestrial photovoltaics. Specifically, the system is a deterministic simulator, which translates present and future manufacturing technology into economic and financial terms, using the production unit concept. It guides solar cell development in three areas: tactical decision making, strategic planning, and the formulation of alternative options.

The glia of the adult Drosophila nervous system

PubMed Central

Kremer, Malte C.; Jung, Christophe; Batelli, Sara; Rubin, Gerald M.

2017-01-01

Glia play crucial roles in the development and homeostasis of the nervous system. While the GLIA in the Drosophila embryo have been well characterized, their study in the adult nervous system has been limited. Here, we present a detailed description of the glia in the adult nervous system, based on the analysis of some 500 glial drivers we identified within a collection of synthetic GAL4 lines. We find that glia make up ∼10% of the cells in the nervous system and envelop all compartments of neurons (soma, dendrites, axons) as well as the nervous system as a whole. Our morphological analysis suggests a set of simple rules governing the morphogenesis of glia and their interactions with other cells. All glial subtypes minimize contact with their glial neighbors but maximize their contact with neurons and adapt their macromorphology and micromorphology to the neuronal entities they envelop. Finally, glial cells show no obvious spatial organization or registration with neuronal entities. Our detailed description of all glial subtypes and their regional specializations, together with the powerful genetic toolkit we provide, will facilitate the functional analysis of glia in the mature nervous system. GLIA 2017 GLIA 2017;65:606–638 PMID:28133822

Illuminating Cell Biology

NASA Technical Reports Server (NTRS)

2002-01-01

NASA's Ames Research Center awarded Ciencia, Inc., a Small Business Innovation Research contract to develop the Cell Fluorescence Analysis System (CFAS) to address the size, mass, and power constraints of using fluorescence spectroscopy in the International Space Station's Life Science Research Facility. The system will play an important role in studying biological specimen's long-term adaptation to microgravity. Commercial applications for the technology include diverse markets such as food safety, in situ environmental monitoring, online process analysis, genomics and DNA chips, and non-invasive diagnostics. Ciencia has already sold the system to the private sector for biosensor applications.

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

PubMed

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-04-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis.

Printing 2-Dimentional Droplet Array for Single-Cell Reverse Transcription Quantitative PCR Assay with a Microfluidic Robot

PubMed Central

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-01-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis. PMID:25828383

Detection of Her2-overexpressing cancer cells using keyhole shaped chamber array employing a magnetic droplet-handling system.

PubMed

Okochi, Mina; Koike, Shinji; Tanaka, Masayoshi; Honda, Hiroyuki

2017-07-15

An on-chip gene expression analysis compartmentalized in droplets was developed for detection of cancer cells at a single-cell level. The chip consists of a keyhole-shaped reaction chamber with hydrophobic modification employing a magnetic bead-droplet-handling system with a gate for bead separation. Using three kinds of water-based droplets in oil, a droplet with sample cells, a lysis buffer with magnetic beads, and RT-PCR buffer, parallel magnetic manipulation and fusion of droplets were performed using a magnet-handling device containing small external magnet patterns in an array. The actuation with the magnet offers a simple system for droplet manipulation that allows separation and fusion of droplets containing magnetic beads. After reverse transcription and amplification by thermal cycling, fluorescence was obtained for detection of overexpressing genes. For clinical detection of gastric cancer cells in peritoneal washing, the Her2-overexpressing gastric cancer cells spiked within normal cells was detected by gene expression analysis of droplets containing an average of 2.5 cells. Our developed droplet-based cancer detection system manipulated by external magnetic force without pumps or valves offers a simple and flexible set-up for transcriptional detection of cancer cells, and will be greatly advantageous for less-invasive clinical diagnosis and prognostic prediction. Copyright © 2016 Elsevier B.V. All rights reserved.

Low-cost motility tracking system (LOCOMOTIS) for time-lapse microscopy applications and cell visualisation.

PubMed

Lynch, Adam E; Triajianto, Junian; Routledge, Edwin

2014-01-01

Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81 ± 0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17 ± 0.004 (MDA-MB-231 breast cancer cells), 1.24 ± 0.006 (SC5 mouse Sertoli cells) and 2.21 ± 0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers.

Low-Cost Motility Tracking System (LOCOMOTIS) for Time-Lapse Microscopy Applications and Cell Visualisation

PubMed Central

Lynch, Adam E.; Triajianto, Junian; Routledge, Edwin

2014-01-01

Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81±0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17±0.004 (MDA-MB-231 breast cancer cells), 1.24±0.006 (SC5 mouse Sertoli cells) and 2.21±0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers. PMID:25121722

Antigen S1, encoded by the MIC1 gene, is characterized as an epitope of human CD59, enabling measurement of mutagen-induced intragenic deletions in the AL cell system

NASA Technical Reports Server (NTRS)

Wilson, A. B.; Seilly, D.; Willers, C.; Vannais, D. B.; McGraw, M.; Waldren, C. A.; Hei, T. K.; Davies, A.; Chatterjee, A. (Principal Investigator)

1999-01-01

S1 cell membrane antigen is encoded by the MIC1 gene on human chromosome 11. This antigen has been widely used as a marker for studies in gene mapping or in analysis of mutagen-induced gene deletions/mutations, which utilized the human-hamster hybrid cell-line, AL-J1, carrying human chromosome 11. Evidence is presented here which identifies S1 as an epitope of CD59, a cell membrane complement inhibiting protein. E7.1 monoclonal antibody, specific for the S1 determinant, was found to react strongly with membrane CD59 in Western blotting, and to bind to purified, urinary form of CD59 in ELISAs. Cell membrane expression of S1 on various cell lines always correlated with that of CD59 when examined by immunofluorescent staining. In addition, E7.1 antibody inhibited the complement regulatory function of CD59. Identification of S1 protein as CD59 has increased the scope of the AL cell system by enabling analysis of intragenic mutations, and multiplex PCR analysis of mutated cells is described, showing variable loss of CD59 exons.

Fuel Cell Backup Power System for Grid Service and Micro-Grid in Telecommunication Applications: Preprint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Zhiwen; Eichman, Joshua D; Kurtz, Jennifer M

This paper presents the feasibility and economics of using fuel cell backup power systems in telecommunication cell towers to provide grid services (e.g., ancillary services, demand response). The fuel cells are able to provide power for the cell tower during emergency conditions. This study evaluates the strategic integration of clean, efficient, and reliable fuel cell systems with the grid for improved economic benefits. The backup systems have potential as enhanced capability through information exchanges with the power grid to add value as grid services that depend on location and time. The economic analysis has been focused on the potential revenuemore » for distributed telecommunications fuel cell backup units to provide value-added power supply. This paper shows case studies on current fuel cell backup power locations and regional grid service programs. The grid service benefits and system configurations for different operation modes provide opportunities for expanding backup fuel cell applications responsive to grid needs.« less

Automated Microfluidic Filtration and Immunocytochemistry Detection System for Capture and Enumeration of Circulating Tumor Cells and Other Rare Cell Populations in Blood.

PubMed

Pugia, Michael; Magbanua, Mark Jesus M; Park, John W

2017-01-01

Isolation by size using a filter membrane offers an antigen-independent method for capturing rare cells present in blood of cancer patients. Multiple cell types, including circulating tumor cells (CTCs), captured on the filter membrane can be simultaneously identified via immunocytochemistry (ICC) analysis of specific cellular biomarkers. Here, we describe an automated microfluidic filtration method combined with a liquid handling system for sequential ICC assays to detect and enumerate non-hematologic rare cells in blood.

Passive hybridization of a photovoltaic module with lithium-ion battery cells: A model-based analysis

NASA Astrophysics Data System (ADS)

Joos, Stella; Weißhar, Björn; Bessler, Wolfgang G.

2017-04-01

Standard photovoltaic battery systems based on AC or DC architectures require power electronics and controllers, including inverters, MPP tracker, and battery charger. Here we investigate an alternative system design based on the parallel connection of a photovoltaic module with battery cells without any intermediate voltage conversion. This approach, for which we use the term passive hybridization, is based on matching the solar cell's and battery cell's respective current/voltage behavior. A battery with flat discharge characteristics can allow to pin the solar cell to its maximum power point (MPP) independently of the external power consumption. At the same time, upon battery full charge, voltage increase will drive the solar cell towards zero current and therefore self-prevent battery overcharge. We present a modeling and simulation analysis of passively hybridizing a 5 kWp PV system with a 5 kWh LFP/graphite lithium-ion battery. Dynamic simulations with 1-min time resolution are carried out for three exemplary summer and winter days using historic weather data and a synthetic single-family household consumer profile. The results demonstrate the feasibility of the system. The passive hybrid allows for high self-sufficiencies of 84.6% in summer and 25.3% in winter, which are only slightly lower than those of a standard system.

Chemical imaging of molecular changes in a hydrated single cell by dynamic secondary ion mass spectrometry and super-resolution microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hua, Xin; Szymanski, Craig; Wang, Zhaoying

2016-01-01

Chemical imaging of single cells is important in capturing biological dynamics. Single cell correlative imaging is realized between structured illumination microscopy (SIM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) using System for Analysis at the Liquid Vacuum Interface (SALVI), a multimodal microreactor. SIM characterized cells and guided subsequent ToF-SIMS analysis. Dynamic ToF-SIMS provided time- and space-resolved cell molecular mapping. Lipid fragments were identified in the hydrated cell membrane. Principal component analysis was used to elucidate chemical component differences among mouse lung cells that uptake zinc oxide nanoparticles. Our results provided submicron chemical spatial mapping for investigations of cell dynamics atmore » the molecular level.« less

Phosphoric acid fuel cell power plant system performance model and computer program

NASA Technical Reports Server (NTRS)

Alkasab, K. A.; Lu, C. Y.

1984-01-01

A FORTRAN computer program was developed for analyzing the performance of phosphoric acid fuel cell power plant systems. Energy mass and electrochemical analysis in the reformer, the shaft converters, the heat exchangers, and the fuel cell stack were combined to develop a mathematical model for the power plant for both atmospheric and pressurized conditions, and for several commercial fuels.

Interdisciplinary Team Science in Cell Biology.

PubMed

Horwitz, Rick

2016-11-01

The cell is complex. With its multitude of components, spatial-temporal character, and gene expression diversity, it is challenging to comprehend the cell as an integrated system and to develop models that predict its behaviors. I suggest an approach to address this issue, involving system level data analysis, large scale team science, and philanthropy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Low-temperature fuel cell systems for commercial airplane auxiliary power.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Curgus, Dita Brigitte; Pratt, Joseph William; Akhil, Abbas Ali

2010-11-01

This presentation briefly describes the ongoing study of fuel cell systems on-board a commercial airplane. Sandia's current project is focused on Proton Exchange Membrane (PEM) fuel cells applied to specific on-board electrical power needs. They are trying to understand how having a fuel cell on an airplane would affect overall performance. The fuel required to accomplish a mission is used to quantify the performance. Our analysis shows the differences between the base airplane and the airplane with the fuel cell. There are many ways of designing a system, depending on what you do with the waste heat. A system thatmore » requires ram air cooling has a large mass penalty due to increased drag. The bottom-line impact can be expressed as additional fuel required to complete the mission. Early results suggest PEM fuel cells can be used on airplanes with manageable performance impact if heat is rejected properly. For PEMs on aircraft, we are continuing to perform: (1) thermodynamic analysis (investigate configurations); (2) integrated electrical design (with dynamic modeling of the micro grid); (3) hardware assessment (performance, weight, and volume); and (4) galley and peaker application.« less

Single-cell systems level analysis of human Toll-Like-Receptor activation defines a chemokine signature in Systemic Lupus Erythematosus

PubMed Central

O'Gorman, William E.; Hsieh, Elena W.Y.; Savig, Erica S.; Gherardini, Pier Federico; Hernandez, Joseph D.; Hansmann, Leo; Balboni, Imelda M.; Utz, Paul J.; Bendall, Sean C.; Fantl, Wendy J.; Lewis, David B.; Nolan, Garry P.; Davis, Mark M.

2015-01-01

Background Activation of Toll-Like Receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in Systemic Lupus Erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has previously not been described. Objective To characterize TLR activation across multiple immune cell subsets and individuals, with the goal of establishing a reference framework against which to compare pathological processes. Methods Peripheral whole blood samples were stimulated with TLR ligands, and analyzed by mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied seventeen healthy volunteer donors and eight newly diagnosed untreated SLE patients. Results Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of NK and T cells selectively induced NF-κB in response to TLR2 ligands. CD14hi monocytes exhibited the most polyfunctional cytokine expression patterns, with over 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared amongst a group of healthy donors, with minimal intra- and inter- individual variability. Furthermore, autoimmune disease altered baseline cytokine production, as newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity. Conclusion Mass cytometry analysis defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in inflammatory disease, such as SLE. PMID:26037552

Analysis of hybrid interface cooling system using air ventilation and nanofluid

NASA Astrophysics Data System (ADS)

Rani, M. F. H.; Razlan, Z. M.; Bakar, S. A.; Desa, H.; Wan, W. K.; Ibrahim, I.; Kamarrudin, N. S.; Bin-Abdun, Nazih A.

2017-09-01

The hybrid interface cooling system needs to be designed for maintaining the electric vehicle's battery cell temperature at 25°C. The hybrid interface cooling system is a combination of two individual systems, where the primary cooling system (R-134a) and the secondary cooling system (CuO + Water) will be used to absorb the heat generated by the battery cells. The ventilation system is designed using air as the medium to transfer the heat from the batteries to the refrigeration system (R-134a). Research will focus on determining the suitable compressor displacement, the heat exchanger volume and the expansion valve resistance value. The analysis for the secondary cooling system is focused on the cooling coil where low temperature nanofluid is passing through each interval of the battery cells. For analysing purposes, the thermal properties of the mixture of 50 grams, Copper (II) Oxide and the base fluid have been determined. The hybrid interface cooling system are able to achieve 57.82% increments in term of rate of heat transfer as compared to the individual refrigeration system.

Potential of mid IR spectroscopy in the rapid label free identification of skin malignancies

NASA Astrophysics Data System (ADS)

Kastl, Lena; Kemper, Björn; Lloyd, Gavin R.; Nallala, Jayakrupakar; Stone, Nick; Naranjo, Valery; Penaranda, Francisco; Schnekenburger, Jürgen

2016-03-01

The rapid inspection of suspicious skin lesions for pathological cell types is the objective of optical point of care diagnostics technologies. A marker free fast diagnosis of skin malignancies would overcome the limitations of the current gold standard surgical biopsy. The time consuming and costly biopsy procedure requires the inspection of each sample by a trained pathologist, which limits the analysis of potentially malignant lesions. Optical technologies like RAMAN or infrared spectroscopy, which provide both, localization and chemical information, can be used to differentiate malignant from healthy tissue by the analysis of multi cell structures and cell type specific spectra. We here report the application of midIR spectroscopy towards fast and reliable skin diagnostics. Within the European research project MINERVA we developed standardized in vitro skin systems with increasing complexity, from single skin cell types as fibroblasts, keratinocytes and melanoma cells, to mixtures of these and finally three dimensional human skin equivalents. The standards were characterized in the established midIR range and also with newly developed systems for fast imaging up to 12 μm. The analysis of the spectra by novel data processing algorithms demonstrated the clear separation of all cell types, especially the tumor cells. The signals from single cell layers were sufficient for cell type differentiation. We have compared different midIR systems and found all of them suitable for specific cell type identification. Our data demonstrate the potential of midIR spectroscopy for fast image acquisition and an improved data processing as sensitive and specific optical biopsy technology.

Protein Expression Profile of Rat Type Two Alveolar Epithelial Cells During Hyperoxic Stress and Recovery

NASA Astrophysics Data System (ADS)

Bhargava, Maneesh

Rationale: In rodent model systems, the sequential changes in lung morphology resulting from hyperoxic injury are well characterized, and are similar to changes in human acute respiratory distress syndrome (ARDS). In the injured lung, alveolar type two (AT2) epithelial cells play a critical role restoring the normal alveolar structure. Thus characterizing the changes in AT2 cells will provide insights into the mechanisms underpinning the recovery from lung injury. Methods: We applied an unbiased systems level proteomics approach to elucidate molecular mechanisms contributing to lung repair in a rat hyperoxic lung injury model. AT2 cells were isolated from rat lungs at predetermined intervals during hyperoxic injury and recovery. Protein expression profiles were determined by using iTRAQRTM with tandem mass spectrometry. Results: Of 959 distinct proteins identified, 183 significantly changed in abundance during the injury-recovery cycle. Gene Ontology enrichment analysis identified cell cycle, cell differentiation, cell metabolism, ion homeostasis, programmed cell death, ubiquitination, and cell migration to be significantly enriched by these proteins. Gene Set Enrichment Analysis of data acquired during lung repair revealed differential expression of gene sets that control multicellular organismal development, systems development, organ development, and chemical homeostasis. More detailed analysis identified activity in two regulatory pathways, JNK and miR 374. A Short Time-series Expression Miner (STEM) algorithm identified protein clusters with coherent changes during injury and repair. Conclusion: Coherent changes occur in the AT2 cell proteome in response to hyperoxic stress. These findings offer guidance regarding the specific molecular mechanisms governing repair of the injured lung.

Fuzzy-based failure mode and effect analysis (FMEA) of a hybrid molten carbonate fuel cell (MCFC) and gas turbine system for marine propulsion

NASA Astrophysics Data System (ADS)

Ahn, Junkeon; Noh, Yeelyong; Park, Sung Ho; Choi, Byung Il; Chang, Daejun

2017-10-01

This study proposes a fuzzy-based FMEA (failure mode and effect analysis) for a hybrid molten carbonate fuel cell and gas turbine system for liquefied hydrogen tankers. An FMEA-based regulatory framework is adopted to analyze the non-conventional propulsion system and to understand the risk picture of the system. Since the participants of the FMEA rely on their subjective and qualitative experiences, the conventional FMEA used for identifying failures that affect system performance inevitably involves inherent uncertainties. A fuzzy-based FMEA is introduced to express such uncertainties appropriately and to provide flexible access to a risk picture for a new system using fuzzy modeling. The hybrid system has 35 components and has 70 potential failure modes, respectively. Significant failure modes occur in the fuel cell stack and rotary machine. The fuzzy risk priority number is used to validate the crisp risk priority number in the FMEA.

Fuzzy energy management for hybrid fuel cell/battery systems for more electric aircraft

NASA Astrophysics Data System (ADS)

Corcau, Jenica-Ileana; Dinca, Liviu; Grigorie, Teodor Lucian; Tudosie, Alexandru-Nicolae

2017-06-01

In this paper is presented the simulation and analysis of a Fuzzy Energy Management for Hybrid Fuel cell/Battery Systems used for More Electric Aircraft. The fuel cell hybrid system contains of fuel cell, lithium-ion batteries along with associated dc to dc boost converters. In this configuration the battery has a dc to dc converter, because it is an active in the system. The energy management scheme includes the rule based fuzzy logic strategy. This scheme has a faster response to load change and is more robust to measurement imprecisions. Simulation will be provided using Matlab/Simulink based models. Simulation results are given to show the overall system performance.

Proton exchange membrane fuel cell system diagnosis based on the signed directed graph method

NASA Astrophysics Data System (ADS)

Hua, Jianfeng; Lu, Languang; Ouyang, Minggao; Li, Jianqiu; Xu, Liangfei

The fuel-cell powered bus is becoming the favored choice for electric vehicles because of its extended driving range, zero emissions, and high energy conversion efficiency when compared with battery-operated electric vehicles. In China, a demonstration program for the fuel cell bus fleet operated at the Beijing Olympics in 2008 and the Shanghai Expo in 2010. It is necessary to develop comprehensive proton exchange membrane fuel cell (PEMFC) diagnostic tools to increase the reliability of these systems. It is especially critical for fuel-cell city buses serving large numbers of passengers using public transportation. This paper presents a diagnostic analysis and implementation study based on the signed directed graph (SDG) method for the fuel-cell system. This diagnostic system was successfully implemented in the fuel-cell bus fleet at the Shanghai Expo in 2010.

Stromal vascular fraction isolated from lipo-aspirates using an automated processing system: bench and bed analysis.

PubMed

Doi, Kentaro; Tanaka, Shinsuke; Iida, Hideo; Eto, Hitomi; Kato, Harunosuke; Aoi, Noriyuki; Kuno, Shinichiro; Hirohi, Toshitsugu; Yoshimura, Kotaro

2013-11-01

The heterogeneous stromal vascular fraction (SVF), containing adipose-derived stem/progenitor cells (ASCs), can be easily isolated through enzymatic digestion of aspirated adipose tissue. In clinical settings, however, strict control of technical procedures according to standard operating procedures and validation of cell-processing conditions are required. Therefore, we evaluated the efficiency and reliability of an automated system for SVF isolation from adipose tissue. SVF cells, freshly isolated using the automated procedure, showed comparable number and viability to those from manual isolation. Flow cytometric analysis confirmed an SVF cell composition profile similar to that after manual isolation. In addition, the ASC yield after 1 week in culture was also not significantly different between the two groups. Our clinical study, in which SVF cells isolated with the automated system were transplanted with aspirated fat tissue for soft tissue augmentation/reconstruction in 42 patients, showed satisfactory outcomes with no serious side-effects. Taken together, our results suggested that the automated isolation system is as reliable a method as manual isolation and may also be useful in clinical settings. Automated isolation is expected to enable cell-based clinical trials in small facilities with an aseptic room, without the necessity of a good manufacturing practice-level cell processing area. Copyright © 2012 John Wiley & Sons, Ltd.

IEEE Photovoltaic Specialists Conference, 20th, Las Vegas, NV, Sept. 26-30, 1988, Conference Record. Volumes 1 & 2

NASA Astrophysics Data System (ADS)

Various papers on photovoltaics are presented. The general topics considered include: amorphous materials and cells; amorphous silicon-based solar cells and modules; amorphous silicon-based materials and processes; amorphous materials characterization; amorphous silicon; high-efficiency single crystal solar cells; multijunction and heterojunction cells; high-efficiency III-V cells; modeling and characterization of high-efficiency cells; LIPS flight experience; space mission requirements and technology; advanced space solar cell technology; space environmental effects and modeling; space solar cell and array technology; terrestrial systems and array technology; terrestrial utility and stand-alone applications and testing; terrestrial concentrator and storage technology; terrestrial stand-alone systems applications; terrestrial systems test and evaluation; terrestrial flatplate and concentrator technology; use of polycrystalline materials; polycrystalline II-VI compound solar cells; analysis of and fabrication procedures for compound solar cells.

Design and Analysis of Single-Cell Sequencing Experiments.

PubMed

Grün, Dominic; van Oudenaarden, Alexander

2015-11-05

Recent advances in single-cell sequencing hold great potential for exploring biological systems with unprecedented resolution. Sequencing the genome of individual cells can reveal somatic mutations and allows the investigation of clonal dynamics. Single-cell transcriptome sequencing can elucidate the cell type composition of a sample. However, single-cell sequencing comes with major technical challenges and yields complex data output. In this Primer, we provide an overview of available methods and discuss experimental design and single-cell data analysis. We hope that these guidelines will enable a growing number of researchers to leverage the power of single-cell sequencing. Copyright © 2015 Elsevier Inc. All rights reserved.

A topological coordinate system for the diamond cubic grid.

PubMed

Čomić, Lidija; Nagy, Benedek

2016-09-01

Topological coordinate systems are used to address all cells of abstract cell complexes. In this paper, a topological coordinate system for cells in the diamond cubic grid is presented and some of its properties are detailed. Four dependent coordinates are used to address the voxels (triakis truncated tetrahedra), their faces (hexagons and triangles), their edges and the points at their corners. Boundary and co-boundary relations, as well as adjacency relations between the cells, can easily be captured by the coordinate values. Thus, this coordinate system is apt for implementation in various applications, such as visualizations, morphological and topological operations and shape analysis.

Proteome-wide analysis and CXCL4 as a biomarker in systemic sclerosis.

PubMed

van Bon, Lenny; Affandi, Alsya J; Broen, Jasper; Christmann, Romy B; Marijnissen, Renoud J; Stawski, Lukasz; Farina, Giuseppina A; Stifano, Giuseppina; Mathes, Allison L; Cossu, Marta; York, Michael; Collins, Cindy; Wenink, Mark; Huijbens, Richard; Hesselstrand, Roger; Saxne, Tore; DiMarzio, Mike; Wuttge, Dirk; Agarwal, Sandeep K; Reveille, John D; Assassi, Shervin; Mayes, Maureen; Deng, Yanhui; Drenth, Joost P H; de Graaf, Jacqueline; den Heijer, Martin; Kallenberg, Cees G M; Bijl, Marc; Loof, Arnoud; van den Berg, Wim B; Joosten, Leo A B; Smith, Vanessa; de Keyser, Filip; Scorza, Rafaella; Lunardi, Claudio; van Riel, Piet L C M; Vonk, Madelon; van Heerde, Waander; Meller, Stephan; Homey, Bernhard; Beretta, Lorenzo; Roest, Mark; Trojanowska, Maria; Lafyatis, Robert; Radstake, Timothy R D J

2014-01-30

Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon. We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo. Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis. Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.).

Proteome-wide Analysis and CXCL4 as a Biomarker in Systemic Sclerosis

PubMed Central

van Bon, L.; Affandi, A.J.; Broen, J.; Christmann, R.B.; Marijnissen, R.J.; Stawski, L.; Farina, G.A.; Stifano, G.; Mathes, A.L.; Cossu, M.; York, M.; Collins, C.; Wenink, M.; Huijbens, R.; Hesselstrand, R.; Saxne, T.; DiMarzio, M.; Wuttge, D.; Agarwal, S.K.; Reveille, J.D.; Assassi, S.; Mayes, M.; Deng, Y.; Drenth, J.P.H.; de Graaf, J.; den Heijer, M.; Kallenberg, C.G.M.; Bijl, M.; Loof, A.; van den Berg, W.B.; Joosten, L.A.B.; Smith, V.; de Keyser, F.; Scorza, R.; Lunardi, C.; van Riel, P.L.C.M.; Vonk, M.; van Heerde, W.; Meller, S.; Homey, B.; Beretta, L.; Roest, M.; Trojanowska, M.; Lafyatis, R.; Radstake, T.R.D.J.

2014-01-01

Background Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon. Methods We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo. Results Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 downregulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis. Conclusions Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.) PMID:24350901

An assessment of alternative fuel cell designs for residential and commercial cogeneration

NASA Technical Reports Server (NTRS)

Wakefield, R. A.

1980-01-01

A comparative assessment of three fuel cell systems for application in different buildings and geographic locations is presented. The study was performed at the NASA Lewis Center and comprised the fuel cell design, performance in different conditions, and the economic parameters. Applications in multifamily housing, stores and hospitals were considered, with a load of 10kW-1 MW. Designs were traced through system sizing, simulation/evaluation, and reliability analysis, and a computer simulation based on a fourth-order representation of a generalized system was performed. The cells were all phosphoric acid type cells, and were found to be incompatible with gas/electric systems and more favorable economically than the gas/electric systems in hospital uses. The methodology used provided an optimized energy-use pattern and minimized back-up system turn-on.

Cell-free protein synthesis: applications in proteomics and biotechnology.

PubMed

He, Mingyue

2008-01-01

Protein production is one of the key steps in biotechnology and functional proteomics. Expression of proteins in heterologous hosts (such as in E. coli) is generally lengthy and costly. Cell-free protein synthesis is thus emerging as an attractive alternative. In addition to the simplicity and speed for protein production, cell-free expression allows generation of functional proteins that are difficult to produce by in vivo systems. Recent exploitation of cell-free systems enables novel development of technologies for rapid discovery of proteins with desirable properties from very large libraries. This article reviews the recent development in cell-free systems and their application in the large scale protein analysis.

New Frontiers and Challenges for Single-Cell Electrochemical Analysis.

PubMed

Zhang, Jingjing; Zhou, Junyu; Pan, Rongrong; Jiang, Dechen; Burgess, James D; Chen, Hong-Yuan

2018-02-23

Previous measurements of cell populations might obscure many important cellular differences, and new strategies for single-cell analyses are urgently needed to re-examine these fundamental biological principles for better diagnosis and treatment of diseases. Electrochemistry is a robust technique for the analysis of single living cells that has the advantages of minor interruption of cellular activity and provides the capability of high spatiotemporal resolution. The achievements of the past 30 years have revealed significant information about the exocytotic events of single cells to elucidate the mechanisms of cellular activity. Currently, the rapid developments of micro/nanofabrication and optoelectronic technologies drive the development of multifunctional electrodes and novel electrochemical approaches with higher resolution for single cells. In this Perspective, three new frontiers in this field, namely, electrochemical microscopy, intracellular analysis, and single-cell analysis in a biological system (i.e., neocortex and retina), are reviewed. The unique features and remaining challenges of these techniques are discussed.

Fast Metabolic Response to Drug Intervention through Analysis on a Miniaturized, Highly Integrated Molecular Imaging System

PubMed Central

Wang, Jun; Hwang, Kiwook; Braas, Daniel; Dooraghi, Alex; Nathanson, David; Campbell, Dean O.; Gu, Yuchao; Sandberg, Troy; Mischel, Paul; Radu, Caius; Chatziioannou, Arion F.; Phelps, Michael E.; Christofk, Heather; Heath, James R.

2014-01-01

We report on a radiopharmaceutical imaging platform designed to capture the kinetics of cellular responses to drugs. Methods A portable in vitro molecular imaging system, comprised of a microchip and a beta-particle imaging camera, permits routine cell-based radioassays on small number of either suspension or adherent cells. We investigate the response kinetics of model lymphoma and glioblastoma cancer cell lines to [18F]fluorodeoxyglucose ([18F]FDG) uptake following drug exposure. Those responses are correlated with kinetic changes in the cell cycle, or with changes in receptor-tyrosine kinase signaling. Results The platform enables radioassays directly on multiple cell types, and yields results comparable to conventional approaches, but uses smaller sample sizes, permits a higher level of quantitation, and doesn’t require cell lysis. Conclusion The kinetic analysis enabled by the platform provides a rapid (~1 hour) drug screening assay. PMID:23978446

Analysis of a crossed Bragg cell acousto-optical spectrometer for SETI

NASA Technical Reports Server (NTRS)

Gulkis, S.

1989-01-01

The search for radio signals from extraterrestrial intelligent beings (SETI) requires the use of large instantaneous bandwidth (500 MHz) and high resolution (20 Hz) spectrometers. Digital systems with a high degree of modularity can be used to provide this capability, and this method has been widely discussed. Another technique for meeting the SETI requirement is to use a crossed Bragg cell spectrometer as described by Psaltis and Casasent. This technique makes use of the Folded Spectrum concept, introduced by Thomas. The Folded Spectrum is a 2-D Fourier Transform of a raster scanned 1-D signal. It is directly related to the long 1-D spectrum of the original signal and is ideally suited for optical signal processing. The folded spectrum technique has received little attention to date, primarily because early systems made use of photographic film which are unsuitable for the real time data analysis and voluminous data requirements of SETI. An analysis of the crossed Bragg cell spectrometer is presented as a method to achieve the spectral processing requirements for SETI. Systematic noise contributions unique to the Bragg cell system will be discussed.

Analysis of a crossed Bragg cell acousto-optical spectrometer for SETI.

PubMed

Gulkis, S

1989-01-01

The search for radio signals from extraterrestrial intelligent beings (SETI) requires the use of large instantaneous bandwidth (500 MHz) and high resolution (20 Hz) spectrometers. Digital systems with a high degree of modularity can be used to provide this capability, and this method has been widely discussed. Another technique for meeting the SETI requirement is to use a crossed Bragg cell spectrometer as described by Psaltis and Casasent. This technique makes use of the Folded Spectrum concept, introduced by Thomas. The Folded Spectrum is a 2-D Fourier Transform of a raster scanned 1-D signal. It is directly related to the long 1-D spectrum of the original signal and is ideally suited for optical signal processing. The folded spectrum technique has received little attention to date, primarily because early systems made use of photographic film which are unsuitable for the real time data analysis and voluminous data requirements of SETI. An analysis of the crossed Bragg cell spectrometer is presented as a method to achieve the spectral processing requirements for SETI. Systematic noise contributions unique to the Bragg cell system will be discussed.

Analysis of a crossed Bragg cell acousto-optical spectrometer for SETI

NASA Astrophysics Data System (ADS)

Gulkis, Samuel

The search for radio signals from extraterrestrial intelligent beings (SETI) requires the use of large instantaneous bandwidth (500 MHz) and high resolution (20 Hz) spectrometers. Digital systems with a high degree of modularity can be used to provide this capability, and this method has been widely discussed. Another technique for meeting the SETI requirement is to use a crossed Bragg cell spectrometer as described by Psaltis and Casasent. This technique makes use of the Folded Spectrum concept, introduced by Thomas. The Folded Spectrum is a 2-D Fourier Transform of a raster scanned 1-D signal. It is directly related to the long 1-D spectrum of the original signal and is ideally suited for optical signal processing. The folded spectrum technique has received little attention to date, primarily because early systems made use of photographic film which are unsuitable for the real time data analysis and voluminous data requirements of SETI. An analysis of the crossed Bragg cell spectrometer is presented as a method to achieve the spectral processing requirements for SETI. Systematic noise contributions unique to the Bragg cell system will be discussed.

Optical analysis and thermal management of 2-cell strings linear concentrating photovoltaic system

NASA Astrophysics Data System (ADS)

Reddy, K. S.; Kamnapure, Nikhilesh R.

2015-09-01

This paper presents the optical and thermal analyses for a linear concentrating photovoltaic/thermal collector under different operating conditions. Linear concentrating photovoltaic system (CPV) consists of a highly reflective mirror, a receiver and semi-dual axis tracking mechanism. The CPV receiver embodies two strings of triple-junction cells (100 cells in each string) adhered to a mild steel circular tube mounted at the focal length of trough. This system provides 560 W of electricity and 1580 W of heat which needs to be dissipated by active cooling. The Al2O3/Water nanofluid is used as heat transfer fluid (HTF) flowing through circular receiver for CPV cells cooling. Optical analysis of linear CPV system with 3.35 m2 aperture and geometric concentration ratio (CR) of 35 is carried out using Advanced System Analysis Program (ASAP) an optical simulation tool. Non-uniform intensity distribution model of solar disk is used to model the sun in ASAP. The impact of random errors including slope error (σslope), tracking error (σtrack) and apparent change in sun's width (σsun) on optical performance of collector is shown. The result from the optical simulations shows the optical efficiency (ηo) of 88.32% for 2-cell string CPV concentrator. Thermal analysis of CPV receiver is carried out with conjugate heat transfer modeling in ANSYS FLUENT-14. Numerical simulations of Al2O3/Water nanofluid turbulent forced convection are performed for various parameters such as nanoparticle volume fraction (φ), Reynolds number (Re). The addition of the nanoparticle in water enhances the heat transfer in the ranges of 3.28% - 35.6% for φ = 1% - 6%. Numerical results are compared with literature data which shows the reasonable agreement.

New tools for the analysis of glial cell biology in Drosophila.

PubMed

Awasaki, Takeshi; Lee, Tzumin

2011-09-01

Because of its genetic, molecular, and behavioral tractability, Drosophila has emerged as a powerful model system for studying molecular and cellular mechanisms underlying the development and function of nervous systems. The Drosophila nervous system has fewer neurons and exhibits a lower glia:neuron ratio than is seen in vertebrate nervous systems. Despite the simplicity of the Drosophila nervous system, glial organization in flies is as sophisticated as it is in vertebrates. Furthermore, fly glial cells play vital roles in neural development and behavior. In addition, powerful genetic tools are continuously being created to explore cell function in vivo. In taking advantage of these features, the fly nervous system serves as an excellent model system to study general aspects of glial cell development and function in vivo. In this article, we review and discuss advanced genetic tools that are potentially useful for understanding glial cell biology in Drosophila. Copyright © 2011 Wiley-Liss, Inc.

Feasibility Study of Seawater Electrolysis for Photovoltaic/Fuel Cell Hybrid Power System for the Coastal Areas in Thailand

NASA Astrophysics Data System (ADS)

Srisiriwat, A.; Pirom, W.

2017-10-01

Solar photovoltaic cell and fuel cell are the practicable options to realize as a possible hybrid power system because the power of the sun cannot be utilized at night or cloudy days but hydrogen has been found as an ideal energy carrier for being transportable, storable, and converting energy though fuel cell. Hydrogen storage is chosen for its ability to obtain a clean energy option. Electrolysis, which is the simplest process to produce hydrogen, can be powered by the dc voltage from the photovoltaic cell instead of using the battery as power supply. This paper concentrates on a feasibility study of seawater electrolysis using photovoltaic power integrated fuel cell system for the coastal cities in Thailand. The proposed system composed of photovoltaic arrays, seawater electrolyzer and fuel cell is presented when the 10-kW of fuel cell electrical power is considered. The feasibility study of hydrogen production and energy analysis of this proposed system is also evaluated.

Rapid analysis of NSAIDs binding to β-cyclodextrin using the simultaneous measurement of absorption and circular dichroism with a novel multi-cell low-volume device.

PubMed

Aboel Dahab, Ali; El-Hag, Dhia

2012-10-01

One of the relatively recent and most widely used approaches to reduce side effects associated with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) is the complexation of NSAIDs with Cyclodextrins (CyD). So far, CyD interaction with drugs is not well understood. There have been many reports along these lines; however, rarely do these studies exploit the full potential of optical techniques. The purpose of this work is to produce a versatile, compact, low-volume, routine apparatus for the simultaneous measurements of absorbance and circular dichroism (CD) which allows for the concurrent use of three different pathlengths for binding studies of NSAIDs/CyD as a function of pH. A new rotating multi-cell holder which holds four cells was designed and manufactured. The work was achieved using an effective novel method for binding titration employing four separate flow cells connected in series in a flow system involving a titration flask and a pump. The pK(a), binding constants, stoichiometry and structural co-conformations of NSAIDs/β-CyD complexes were elucidated and determined with accuracy. The system proved to be efficient and the analysis time was reduced to less than or equal to one fourth of total analysis time used in one-cell systems, with possible automation for high-throughput analysis.

Systems cell biology

PubMed Central

Mast, Fred D.; Ratushny, Alexander V.

2014-01-01

Systems cell biology melds high-throughput experimentation with quantitative analysis and modeling to understand many critical processes that contribute to cellular organization and dynamics. Recently, there have been several advances in technology and in the application of modeling approaches that enable the exploration of the dynamic properties of cells. Merging technology and computation offers an opportunity to objectively address unsolved cellular mechanisms, and has revealed emergent properties and helped to gain a more comprehensive and fundamental understanding of cell biology. PMID:25225336

Damage Thresholds for Exposure to NIR and Blue Lasers in an In Vitro RPE Cell System

DTIC Science & Technology

2006-07-01

damage , and to identify antioxidants capable of protecting these cells from laser-in- duced cell death. MATERIALS AND METHODS The human RPE cell...melanosomes in blue laser-induced damage in vitro, which confirms the view that melanin plays an important role in photochemical damage mechanisms in...community has only a validating role in the animal ED50 damage threshold data used by safety committees. Systems of in vitro analysis must be

Cell module and fuel conditioner development

NASA Technical Reports Server (NTRS)

Feret, J. M.

1982-01-01

The efforts performed to develop a phosphoric acid fuel cell (PAFC) stack design having a 10 kW power rating for operation at higher than atmospheric pressure based on the existing Mark II design configuration are described. The work involves: (1) Performance of pertinent functional analysis, trade studies and thermodynamic cycle analysis for requirements definition and system operating parameter selection purposes, (2) characterization of fuel cell materials and components, and performance testing and evaluation of the repeating electrode components, (3) establishment of the state-of-the-art manufacturing technology for all fuel cell components at Westinghouse and the fabrication of short stacks of various sites, and (4) development of a 10 kW PAFC stack design for higher pressure operation utilizing the top down systems engineering approach.

Current reprogramming systems in regenerative medicine: from somatic cells to induced pluripotent stem cells.

PubMed

Hu, Chenxia; Li, Lanjuan

2016-01-01

Induced pluripotent stem cells (iPSCs) paved the way for research fields including cell therapy, drug screening, disease modeling and the mechanism of embryonic development. Although iPSC technology has been improved by various delivery systems, direct transduction and small molecule regulation, low reprogramming efficiency and genomic modification steps still inhibit its clinical use. Improvements in current vectors and the exploration of novel vectors are required to balance efficiency and genomic modification for reprogramming. Herein, we set out a comprehensive analysis of current reprogramming systems for the generation of iPSCs from somatic cells. By clarifying advantages and disadvantages of the current reprogramming systems, we are striding toward an effective route to generate clinical grade iPSCs.

Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL) Reveals the Sequential Differentiation of Sieve Element-Like Cells.

PubMed

Kondo, Yuki; Nurani, Alif Meem; Saito, Chieko; Ichihashi, Yasunori; Saito, Masato; Yamazaki, Kyoko; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Fukuda, Hiroo

2016-06-01

Cell differentiation is a complex process involving multiple steps, from initial cell fate specification to final differentiation. Procambial/cambial cells, which act as vascular stem cells, differentiate into both xylem and phloem cells during vascular development. Recent studies have identified regulatory cascades for xylem differentiation. However, the molecular mechanism underlying phloem differentiation is largely unexplored due to technical challenges. Here, we established an ectopic induction system for phloem differentiation named Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL). Our results verified similarities between VISUAL-induced Arabidopsis thaliana phloem cells and in vivo sieve elements. We performed network analysis using transcriptome data with VISUAL to dissect the processes underlying phloem differentiation, eventually identifying a factor involved in the regulation of the master transcription factor gene APL Thus, our culture system opens up new avenues not only for genetic studies of phloem differentiation, but also for future investigations of multidirectional differentiation from vascular stem cells. © 2016 American Society of Plant Biologists. All rights reserved.

Cost projections for Redox Energy storage systems

NASA Technical Reports Server (NTRS)

Michaels, K.; Hall, G.

1980-01-01

A preliminary design and system cost analysis was performed for the redox energy storage system. A conceptual design and cost estimate was prepared for each of two energy applications: (1) electric utility 100-MWh requirement (10-MW for ten hours) for energy storage for utility load leveling application, and (2) a 500-kWh requirement (10-kW for 50 hours) for use with a variety of residential or commercial applications, including stand alone solar photovoltaic systems. The conceptual designs were based on cell performance levels, system design parameters, and special material costs. These data were combined with estimated thermodynamic and hydraulic analysis to provide preliminary system designs. Results indicate that the redox cell stack to be amenable to mass production techniques with a relatively low material cost.

Development of a Monitoring Method for Nonlabeled Human Pluripotent Stem Cell Growth by Time-Lapse Image Analysis.

PubMed

Suga, Mika; Kii, Hiroaki; Niikura, Keiichi; Kiyota, Yasujiro; Furue, Miho K

2015-07-01

: Cell growth is an important criterion for determining healthy cell conditions. When somatic cells or cancer cells are dissociated into single cells for passaging, the cell numbers can be counted at each passage, providing information on cell growth as an indicator of the health conditions of these cells. In the case of human pluripotent stem cells (hPSCs), because the cells are usually dissociated into cell clumps of ∼50-100 cells for passaging, cell counting is time-consuming. In the present study, using a time-lapse imaging system, we developed a method to determine the growth of hPSCs from nonlabeled live cell phase-contrast images without damaging these cells. Next, the hPSC colony areas and number of nuclei were determined and used to derive equations to calculate the cell number in hPSC colonies, which were assessed on time-lapse images acquired using a culture observation system. The relationships between the colony areas and nuclei numbers were linear, although the equation coefficients were dependent on the cell line used, colony size, colony morphology, and culture conditions. When the culture conditions became improper, the change in cell growth conditions could be detected by analysis of the phase-contrast images. This method provided real-time information on colony growth and cell growth rates without using treatments that can damage cells and could be useful for basic research on hPSCs and cell processing for hPSC-based therapy. This is the first study to use a noninvasive method using images to systemically determine the growth of human pluripotent stem cells (hPSCs) without damaging or wasting cells. This method would be useful for quality control during cell culture of clinical hPSCs. ©AlphaMed Press.

Neuron analysis of visual perception

NASA Technical Reports Server (NTRS)

Chow, K. L.

1980-01-01

The receptive fields of single cells in the visual system of cat and squirrel monkey were studied investigating the vestibular input affecting the cells, and the cell's responses during visual discrimination learning process. The receptive field characteristics of the rabbit visual system, its normal development, its abnormal development following visual deprivation, and on the structural and functional re-organization of the visual system following neo-natal and prenatal surgery were also studied. The results of each individual part of each investigation are detailed.

Real-Time Analysis of Isoprene in Breath by Using Ultraviolet-Absorption Spectroscopy with a Hollow Optical Fiber Gas Cell

PubMed Central

Iwata, Takuro; Katagiri, Takashi; Matsuura, Yuji

2016-01-01

A breath analysis system based on ultraviolet-absorption spectroscopy was developed by using a hollow optical fiber as a gas cell for real-time monitoring of isoprene in breath. The hollow optical fiber functions as an ultra-small-volume gas cell with a long path. The measurement sensitivity of the system was evaluated by using nitric-oxide gas as a gas sample. The evaluation result showed that the developed system, using a laser-driven, high-intensity light source and a 3-m-long, aluminum-coated hollow optical fiber, could successfully measure nitric-oxide gas with a 50 ppb concentration. An absorption spectrum of a breath sample in the wavelength region of around 200–300 nm was measured, and the measured spectrum revealed the main absorbing components in breath as water vapor, isoprene, and ozone converted from oxygen by radiation of ultraviolet light. The concentration of isoprene in breath was estimated by multiple linear regression. The regression analysis results showed that the proposed analysis system enables real-time monitoring of isoprene during the exhaling of breath. Accordingly, it is suitable for measuring the circadian variation of isoprene. PMID:27929387

Real-Time Analysis of Isoprene in Breath by Using Ultraviolet-Absorption Spectroscopy with a Hollow Optical Fiber Gas Cell.

PubMed

Iwata, Takuro; Katagiri, Takashi; Matsuura, Yuji

2016-12-05

A breath analysis system based on ultraviolet-absorption spectroscopy was developed by using a hollow optical fiber as a gas cell for real-time monitoring of isoprene in breath. The hollow optical fiber functions as an ultra-small-volume gas cell with a long path. The measurement sensitivity of the system was evaluated by using nitric-oxide gas as a gas sample. The evaluation result showed that the developed system, using a laser-driven, high-intensity light source and a 3-m-long, aluminum-coated hollow optical fiber, could successfully measure nitric-oxide gas with a 50 ppb concentration. An absorption spectrum of a breath sample in the wavelength region of around 200-300 nm was measured, and the measured spectrum revealed the main absorbing components in breath as water vapor, isoprene, and ozone converted from oxygen by radiation of ultraviolet light. The concentration of isoprene in breath was estimated by multiple linear regression. The regression analysis results showed that the proposed analysis system enables real-time monitoring of isoprene during the exhaling of breath. Accordingly, it is suitable for measuring the circadian variation of isoprene.

Prognostic value of circulating tumor cells detected with the CellSearch System in patients with gastric cancer: evidence from a meta-analysis.

PubMed

Yang, Chaogang; Zou, Kun; Yuan, Zewei; Guo, Tangxi; Xiong, Bin

2018-01-01

Circulating tumor cells (CTCs) have been proposed as a marker for predicting the prognosis of cancer. However, the prognostic value of CTCs detected with the CellSearch System in patients with gastric cancer (GC) remains controversial. We performed a meta-analysis of available studies to investigate this topic. Two authors systematically searched the studies independently in PubMed, Science Citation Index, Cochrane Database, Embase, and the references in relevant studies (up to September 2017) using keywords. Our meta-analysis was performed in Stata software, version 12.0 (2011; Stata Corp, College Station, TX, USA), with the risk ratio (RR), hazard ratio (HR), and 95% CI as the effect measures. Subgroup analyses and meta-regression were also conducted. Seven studies (including eight sets of data) containing 579 patients with GC from four countries were included in this meta-analysis. The pooled results showed CTC-positive status detected by the CellSearch System was significantly associated with poor overall survival (HR =2.09, 95% CI [1.71, 2.55], P <0.001, I 2 =31.5%) and progression-free survival (HR =2.11, 95% CI [1.25, 3.57], P =0.005, I 2 =75.6%) of patients with GC, regardless of sampling time. The disease control rate of CTC-positive group was lower than that of CTC-negative group for both baseline and intra-therapy, although no statistical difference existed at both sampling time points (baseline: 69.5% versus 81.8%, RR=0.79, 95% CI [0.54, 1.16], P =0.23, I 2 =68.0%; intra-therapy: 50.0% versus 85.9%, RR=0.24, 95% CI [0.02, 3.13], P =0.28, I 2 =87.4%). Our meta-analysis demonstrated that CTCs detected with the CellSearch System from the peripheral blood had significant prognostic value and might predict poor response to chemotherapy for patients with GC.

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

PubMed

Zhou, Ting; Matsunami, Hiroaki

2018-05-01

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

Single-cell printer: automated, on demand, and label free.

PubMed

Gross, Andre; Schöndube, Jonas; Niekrawitz, Sonja; Streule, Wolfgang; Riegger, Lutz; Zengerle, Roland; Koltay, Peter

2013-12-01

Within the past years, single-cell analysis has developed into a key topic in cell biology to study cellular functions that are not accessible by investigation of larger cell populations. Engineering approaches aiming to access single cells to extract information about their physiology, phenotype, and genotype at the single-cell level are going manifold ways, meanwhile allowing separation, sorting, culturing, and analysis of individual cells. Based on our earlier research toward inkjet-like printing of single cells, this article presents further characterization results obtained with a fully automated prototype instrument for printing of single living cells in a noncontact inkjet-like manner. The presented technology is based on a transparent microfluidic drop-on-demand dispenser chip coupled with a camera-assisted automatic detection system. Cells inside the chip are detected and classified with this detection system before they are expelled from the nozzle confined in microdroplets, thus enabling a "one cell per droplet" printing mode. To demonstrate the prototype instrument's suitability for biological and biomedical applications, basic experiments such as printing of single-bead and cell arrays as well as deposition and culture of single cells in microwell plates are presented. Printing efficiencies greater than 80% and viability rates about 90% were achieved.

Stem cells in the Drosophila digestive system.

PubMed

Zeng, Xiankun; Chauhan, Chhavi; Hou, Steven X

2013-01-01

Adult stem cells maintain tissue homeostasis by continuously replenishing damaged, aged and dead cells in any organism. Five types of region and organ-specific multipotent adult stem cells have been identified in the Drosophila digestive system: intestinal stem cells (ISCs) in the posterior midgut; hindgut intestinal stem cells (HISCs) at the midgut/hindgut junction; renal and nephric stem cells (RNSCs) in the Malpighian Tubules; type I gastric stem cells (GaSCs) at foregut/midgut junction; and type II gastric stem cells (GSSCs) at the middle of the midgut. Despite the fact that each type of stem cell is unique to a particular organ, they share common molecular markers and some regulatory signaling pathways. Due to the simpler tissue structure, ease of performing genetic analysis, and availability of abundant mutants, Drosophila serves as an elegant and powerful model system to study complex stem cell biology. The recent discoveries, particularly in the Drosophila ISC system, have greatly advanced our understanding of stem cell self-renewal, differentiation, and the role of stem cells play in tissue homeostasis/regeneration and adaptive tissue growth.

Asymptotic behaviors of a cell-to-cell HIV-1 infection model perturbed by white noise

NASA Astrophysics Data System (ADS)

Liu, Qun

2017-02-01

In this paper, we analyze a mathematical model of cell-to-cell HIV-1 infection to CD4+ T cells perturbed by stochastic perturbations. First of all, we investigate that there exists a unique global positive solution of the system for any positive initial value. Then by using Lyapunov analysis methods, we study the asymptotic property of this solution. Moreover, we discuss whether there is a stationary distribution for this system and if it owns the ergodic property. Numerical simulations are presented to illustrate the theoretical results.

High-recovery visual identification and single-cell retrieval of circulating tumor cells for genomic analysis using a dual-technology platform integrated with automated immunofluorescence staining.

PubMed

Campton, Daniel E; Ramirez, Arturo B; Nordberg, Joshua J; Drovetto, Nick; Clein, Alisa C; Varshavskaya, Paulina; Friemel, Barry H; Quarre, Steve; Breman, Amy; Dorschner, Michael; Blau, Sibel; Blau, C Anthony; Sabath, Daniel E; Stilwell, Jackie L; Kaldjian, Eric P

2015-05-06

Circulating tumor cells (CTCs) are malignant cells that have migrated from solid cancers into the blood, where they are typically present in rare numbers. There is great interest in using CTCs to monitor response to therapies, to identify clinically actionable biomarkers, and to provide a non-invasive window on the molecular state of a tumor. Here we characterize the performance of the AccuCyte®--CyteFinder® system, a comprehensive, reproducible and highly sensitive platform for collecting, identifying and retrieving individual CTCs from microscopic slides for molecular analysis after automated immunofluorescence staining for epithelial markers. All experiments employed a density-based cell separation apparatus (AccuCyte) to separate nucleated cells from the blood and transfer them to microscopic slides. After staining, the slides were imaged using a digital scanning microscope (CyteFinder). Precisely counted model CTCs (mCTCs) from four cancer cell lines were spiked into whole blood to determine recovery rates. Individual mCTCs were removed from slides using a single-cell retrieval device (CytePicker™) for whole genome amplification and subsequent analysis by PCR and Sanger sequencing, whole exome sequencing, or array-based comparative genomic hybridization. Clinical CTCs were evaluated in blood samples from patients with different cancers in comparison with the CellSearch® system. AccuCyte--CyteFinder presented high-resolution images that allowed identification of mCTCs by morphologic and phenotypic features. Spike-in mCTC recoveries were between 90 and 91%. More than 80% of single-digit spike-in mCTCs were identified and even a single cell in 7.5 mL could be found. Analysis of single SKBR3 mCTCs identified presence of a known TP53 mutation by both PCR and whole exome sequencing, and confirmed the reported karyotype of this cell line. Patient sample CTC counts matched or exceeded CellSearch CTC counts in a small feasibility cohort. The AccuCyte--CyteFinder system is a comprehensive and sensitive platform for identification and characterization of CTCs that has been applied to the assessment of CTCs in cancer patient samples as well as the isolation of single cells for genomic analysis. It thus enables accurate non-invasive monitoring of CTCs and evolving cancer biology for personalized, molecularly-guided cancer treatment.

Suppression of T24 human bladder cancer cells by ROS from locally delivered hematoporphyrin-containing polyurethane films.

PubMed

Kim, Dohyun; Lee, Mi Hee; Koo, Min-Ah; Kwon, Byeong-Ju; Kim, Min Sung; Seon, Gyeung Mi; Hong, Seung Hee; Park, Jong-Chul

2018-06-13

Systemic injection of a photosensitizer is a general method in photodynamic therapy, but it has complications due to the unintended systemic distribution and remnants of photosensitizers. This study focused on the possibility of suppressing luminal proliferative cells by excessive reactive oxygen species from locally delivered photosensitizer with biocompatible polyurethane, instead of the systemic injection method. We used human bladder cancer cells, hematoporphyrin as the photosensitizer, and polyurethane film as the photosensitizer-delivering container. The light source was a self-made LED (510 nm, 5 mW cm-2) system. The cancer cells were cultured on different doses of hematoporphyrin-containing polyurethane film and irradiated with LED for 15 minutes and 30 minutes each. After irradiating with LED and incubating for 24 hours, cell viability analysis, cell cycle analysis, apoptosis assay, intracellular and extracellular ROS generation study and western blot were performed. The cancer cell suppression effects of different concentrations of the locally delivered hematoporphyrin with PDT were compared. Apoptosis dominant cancer cell suppressions were shown to be hematoporphyrin dose-dependent. However, after irradiation, intracellular ROS amounts were similar in all the groups having different doses of hematoporphyrin, but these values were definitely higher than those in the control group. Excessive extracellular ROS from the intended, locally delivered photosensitizer for photodynamic treatment application had an inhibitory effect on luminal proliferative cancer cells. This method can be another possibility for PDT application on contactable or attachable lesions.

An Analysis for Capital Expenditure Decisions at a Naval Regional Medical Center.

DTIC Science & Technology

1981-12-01

Service Equipment Review Committee 1. Portable defibrilator Computed tomographic scanner and cardioscope 2. ECG cart Automated blood cell counter 3. Gas...system sterilizer Gas system sterilizer 4. Automated blood cell Portable defibrilator and counter cardioscope 5. Computed tomographic ECG cart scanner...dictating and automated typing) systems. e. Filing equipment f. Automatic data processing equipment including data communications equipment. g

The cell monolayer trajectory from the system state point of view.

PubMed

Stys, Dalibor; Vanek, Jan; Nahlik, Tomas; Urban, Jan; Cisar, Petr

2011-10-01

Time-lapse microscopic movies are being increasingly utilized for understanding the derivation of cell states and predicting cell future. Often, fluorescence and other types of labeling are not available or desirable, and cell state-definitions based on observable structures must be used. We present the methodology for cell behavior recognition and prediction based on the short term cell recurrent behavior analysis. This approach has theoretical justification in non-linear dynamics theory. The methodology is based on the general stochastic systems theory which allows us to define the cell states, trajectory and the system itself. We introduce the usage of a novel image content descriptor based on information contribution (gain) by each image point for the cell state characterization as the first step. The linkage between the method and the general system theory is presented as a general frame for cell behavior interpretation. We also discuss extended cell description, system theory and methodology for future development. This methodology may be used for many practical purposes, ranging from advanced, medically relevant, precise cell culture diagnostics to very utilitarian cell recognition in a noisy or uneven image background. In addition, the results are theoretically justified.

Microfabricated systems and assays for studying the cytoskeletal organization, micromechanics, and motility patterns of cancerous cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huda, Sabil; Pilans, Didzis; Makurath, Monika

Cell motions are driven by coordinated actions of the intracellular cytoskeleton – actin, microtubules (MTs) and substrate/focal adhesions (FAs). This coordination is altered in metastatic cancer cells resulting in deregulated and increased cellular motility. Microfabrication tools, including photolithography, micromolding, microcontact printing, wet stamping and microfluidic devices have emerged as a powerful set of experimental tools with which to probe and define the differences in cytoskeleton organization/dynamics and cell motility patterns in non-metastatic and metastatic cancer cells. In this paper, we discuss four categories of microfabricated systems: (i) micropatterned substrates for studying of cell motility sub-processes (for example, MT targeting ofmore » FAs or cell polarization); (ii) systems for studying cell mechanical properties, (iii) systems for probing overall cell motility patterns within challenging geometric confines relevant to metastasis (for example, linear and ratchet geometries), and (iv) microfluidic devices that incorporate co-cultures of multiple cell types and chemical gradients to mimic in vivo intravasation/extravasation steps of metastasis. Finally, together, these systems allow for creating controlled microenvironments that not only mimic complex soft tissues, but are also compatible with live cell high-resolution imaging and quantitative analysis of single cell behavior.« less

Microfabricated systems and assays for studying the cytoskeletal organization, micromechanics, and motility patterns of cancerous cells

DOE PAGES

Huda, Sabil; Pilans, Didzis; Makurath, Monika; ...

2014-08-28

Cell motions are driven by coordinated actions of the intracellular cytoskeleton – actin, microtubules (MTs) and substrate/focal adhesions (FAs). This coordination is altered in metastatic cancer cells resulting in deregulated and increased cellular motility. Microfabrication tools, including photolithography, micromolding, microcontact printing, wet stamping and microfluidic devices have emerged as a powerful set of experimental tools with which to probe and define the differences in cytoskeleton organization/dynamics and cell motility patterns in non-metastatic and metastatic cancer cells. In this paper, we discuss four categories of microfabricated systems: (i) micropatterned substrates for studying of cell motility sub-processes (for example, MT targeting ofmore » FAs or cell polarization); (ii) systems for studying cell mechanical properties, (iii) systems for probing overall cell motility patterns within challenging geometric confines relevant to metastasis (for example, linear and ratchet geometries), and (iv) microfluidic devices that incorporate co-cultures of multiple cell types and chemical gradients to mimic in vivo intravasation/extravasation steps of metastasis. Finally, together, these systems allow for creating controlled microenvironments that not only mimic complex soft tissues, but are also compatible with live cell high-resolution imaging and quantitative analysis of single cell behavior.« less

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

PubMed Central

Chen, Zixi; Chen, Lei; Zhang, Weiwen

2017-01-01

Microbiologists traditionally study population rather than individual cells, as it is generally assumed that the status of individual cells will be similar to that observed in the population. However, the recent studies have shown that the individual behavior of each single cell could be quite different from that of the whole population, suggesting the importance of extending traditional microbiology studies to single-cell level. With recent technological advances, such as flow cytometry, next-generation sequencing (NGS), and microspectroscopy, single-cell microbiology has greatly enhanced the understanding of individuality and heterogeneity of microbes in many biological systems. Notably, the application of multiple ‘omics’ in single-cell analysis has shed light on how individual cells perceive, respond, and adapt to the environment, how heterogeneity arises under external stress and finally determines the fate of the whole population, and how microbes survive under natural conditions. As single-cell analysis involves no axenic cultivation of target microorganism, it has also been demonstrated as a valuable tool for dissecting the microbial ‘dark matter.’ In this review, current state-of-the-art tools and methods for genomic and transcriptomic analysis of microbes at single-cell level were critically summarized, including single-cell isolation methods and experimental strategies of single-cell analysis with NGS. In addition, perspectives on the future trends of technology development in the field of single-cell analysis was also presented. PMID:28979258

Systems cell biology.

PubMed

Mast, Fred D; Ratushny, Alexander V; Aitchison, John D

2014-09-15

Systems cell biology melds high-throughput experimentation with quantitative analysis and modeling to understand many critical processes that contribute to cellular organization and dynamics. Recently, there have been several advances in technology and in the application of modeling approaches that enable the exploration of the dynamic properties of cells. Merging technology and computation offers an opportunity to objectively address unsolved cellular mechanisms, and has revealed emergent properties and helped to gain a more comprehensive and fundamental understanding of cell biology. © 2014 Mast et al.

Develop and test fuel cell powered on-site integrated total energy system

NASA Technical Reports Server (NTRS)

Kaufman, A.; Feigenbaum, H.; Wang, C. L.; Werth, J.; Whelan, J. A.

1983-01-01

Test results are presented for a 24 cell, two sq ft (4kW) stack. This stack is a precursor to a 25kW stack that is a key milestone. Results are discussed in terms of cell performance, electrolyte management, thermal management, and reactant gas manifolding. The results obtained in preliminary testing of a 50kW methanol processing subsystem are discussed. Subcontracting activities involving application analysis for fuel cell on site integrated energy systems are updated.

Microfabricated Electrochemical Cell-Based Biosensors for Analysis of Living Cells In Vitro

PubMed Central

Wang, Jun; Wu, Chengxiong; Hu, Ning; Zhou, Jie; Du, Liping; Wang, Ping

2012-01-01

Cellular biochemical parameters can be used to reveal the physiological and functional information of various cells. Due to demonstrated high accuracy and non-invasiveness, electrochemical detection methods have been used for cell-based investigation. When combined with improved biosensor design and advanced measurement systems, the on-line biochemical analysis of living cells in vitro has been applied for biological mechanism study, drug screening and even environmental monitoring. In recent decades, new types of miniaturized electrochemical biosensor are emerging with the development of microfabrication technology. This review aims to give an overview of the microfabricated electrochemical cell-based biosensors, such as microelectrode arrays (MEA), the electric cell-substrate impedance sensing (ECIS) technique, and the light addressable potentiometric sensor (LAPS). The details in their working principles, measurement systems, and applications in cell monitoring are covered. Driven by the need for high throughput and multi-parameter detection proposed by biomedicine, the development trends of electrochemical cell-based biosensors are also introduced, including newly developed integrated biosensors, and the application of nanotechnology and microfluidic technology. PMID:25585708

Investigation of VOCs associated with different characteristics of breast cancer cells

PubMed Central

Lavra, Luca; Catini, Alexandro; Ulivieri, Alessandra; Capuano, Rosamaria; Baghernajad Salehi, Leila; Sciacchitano, Salvatore; Bartolazzi, Armando; Nardis, Sara; Paolesse, Roberto; Martinelli, Eugenio; Di Natale, Corrado

2015-01-01

The efficacy of breath volatile organic compounds (VOCs) analysis for the screening of patients bearing breast cancer lesions has been demonstrated by using gas chromatography and artificial olfactory systems. On the other hand, in-vitro studies suggest that VOCs detection could also give important indications regarding molecular and tumorigenic characteristics of tumor cells. Aim of this study was to analyze VOCs in the headspace of breast cancer cell lines in order to ascertain the potentiality of VOCs signatures in giving information about these cells and set-up a new sensor system able to detect breast tumor-associated VOCs. We identified by Gas Chromatography-Mass Spectrometry analysis a VOCs signature that discriminates breast cancer cells for: i) transformed condition; ii) cell doubling time (CDT); iii) Estrogen and Progesterone Receptors (ER, PgR) expression, and HER2 overexpression. Moreover, the signals obtained from a temperature modulated metal oxide semiconductor gas sensor can be classified in order to recognize VOCs signatures associated with breast cancer cells, CDT and ER expression. Our results demonstrate that VOCs analysis could give clinically relevant information about proliferative and molecular features of breast cancer cells and pose the basis for the optimization of a low-cost diagnostic device to be used for tumors characterization. PMID:26304457

Gap junction systems in the rat vestibular labyrinth: immunohistochemical and ultrastructural analysis.

PubMed

Kikuchi, T; Adams, J C; Paul, D L; Kimura, R S

1994-09-01

The distribution of gap junctions within the vestibular labyrinth was investigated using immunohistochemistry and transmission electron microscopy. Connexin26-like immunoreactivity was observed among supporting cells in each vestibular sensory epithelium. Reaction product was also present in the transitional epithelium of each vestibular endorgan and in the planum semilunatum of crista ampullaris. No connexin26-like immunoreactivity was observed among thin wall epithelial cells or among vestibular dark cells. In addition, fibrocytes within vestibular connective tissue were positively immunostained. Reaction product was also detected in the melanocyte area just beneath dark cells. Ultrastructural observations indicated that a gap junction network of vestibular supporting cells extends to the transitional epithelium and planum semilunatum and forms an isolated epithelial cell gap junction system in each vestibular endorgan. In contrast, no gap junctions were found among wall epithelial cells or among dark cells. Fibrocytes and melanocytes were coupled by gap junctions and belong to the connective tissue cell gap junction system, which is continuous throughout the vestibular system and the cochlea. The possible functional significance of these gap junction systems is discussed.

Real-time analysis of Drosophila post-embryonic haemocyte behaviour.

PubMed

Sampson, Christopher J; Williams, Michael J

2012-01-01

The larval stage of the model organism Drosophila is frequently used to study host-pathogen interactions. During embryogenesis the cellular arm of the immune response, consisting of macrophage-like cells known as plasmatocytes, is extremely motile and functions to phagocytise pathogens and apoptotic bodies, as well as produce extracellular matrix. The cellular branch of the larval (post-embryonic) innate immune system consists of three cell types--plasmatocytes, crystal cells and lamellocytes--which are involved in the phagocytosis, encapsulation and melanisation of invading pathogens. Post-embryonic haemocyte motility is poorly understood thus further characterisation is required, for the purpose of standardisation. In order to examine post-embryonic haemocyte cytoskeletal dynamics or migration, the most commonly used system is in vitro cell lines. The current study employs an ex vivo system (an adaptation of in vitro cell incubation using primary cells), in which primary larval or pre-pupal haemocytes are isolated for short term analysis, in order to discover various aspects of their behaviour during events requiring cytoskeleton dynamics. The ex vivo method allows for real-time analysis and manipulation of primary post-embryonic haemocytes. This technique was used to characterise, and potentially standardised, larval and pre-pupal haemocyte cytoskeleton dynamics, assayed on different extracellular matrices. Using this method it was determined that, while larval haemocytes are unable to migrate, haemocytes recovered from pre-pupae are capable of migration.

Type conversion of secretomes in a 3D TAM2 and HCC cell co-culture system and functional importance of CXCL2 in HCC

PubMed Central

Lu, Yu; Li, Shan; Ma, Liping; Li, Yan; Zhang, Xiaolian; Peng, Qiliu; Mo, Cuiju; Huang, Li; Qin, Xue; Liu, Yinkun

2016-01-01

Macrophages play important roles in the tumor microenvironment, driving cancer progression and metastasis, particularly in hepatocellular carcinoma (HCC). However, few studies have assessed the exact secretome composition in HCC. In the present study, the impact of different phenotype of macrophages on HCC cells was investigated. Alternatively activated macrophages (M2) were found to significantly increase the proliferation, migration, and invasion abilities of SMMC7721 cells (all P < 0.05). M2 were then co-cultured with SMMC7721 cells to reconstruct the tumor microenvironment. Conditioned medium from 3D single cultures of M2, SMMC7721 cells, and their co-culture system were analyzed using quantitative proteomics via iTRAQ labeling combined with mass spectrometric analysis. Secretome analysis revealed a total of 159 differential secreted proteins in the co-culture system compared to the single culture systems, with 63 being up-regulated (>1.3-fold) and 96 down-regulated (<0.7-fold). CXCL2 was confirmed to have higher expression in the co-culture system and HCC tissues, and was selected for further investigation. Functional effects data suggested that recombinant human CXCL2 significantly enhanced the migration, invasion ability of SMMC7721 cells, and weakened adhesion ability. While CXCL2 neutralization and CXCR2 blockage significantly inhibited the effects of CXCL2 on SMMC7721 cells, indicating that CXCL2 may play pivotal role in HCC metastasis. PMID:27117207

Type conversion of secretomes in a 3D TAM2 and HCC cell co-culture system and functional importance of CXCL2 in HCC.

PubMed

Lu, Yu; Li, Shan; Ma, Liping; Li, Yan; Zhang, Xiaolian; Peng, Qiliu; Mo, Cuiju; Huang, Li; Qin, Xue; Liu, Yinkun

2016-04-27

Macrophages play important roles in the tumor microenvironment, driving cancer progression and metastasis, particularly in hepatocellular carcinoma (HCC). However, few studies have assessed the exact secretome composition in HCC. In the present study, the impact of different phenotype of macrophages on HCC cells was investigated. Alternatively activated macrophages (M2) were found to significantly increase the proliferation, migration, and invasion abilities of SMMC7721 cells (all P < 0.05). M2 were then co-cultured with SMMC7721 cells to reconstruct the tumor microenvironment. Conditioned medium from 3D single cultures of M2, SMMC7721 cells, and their co-culture system were analyzed using quantitative proteomics via iTRAQ labeling combined with mass spectrometric analysis. Secretome analysis revealed a total of 159 differential secreted proteins in the co-culture system compared to the single culture systems, with 63 being up-regulated (>1.3-fold) and 96 down-regulated (<0.7-fold). CXCL2 was confirmed to have higher expression in the co-culture system and HCC tissues, and was selected for further investigation. Functional effects data suggested that recombinant human CXCL2 significantly enhanced the migration, invasion ability of SMMC7721 cells, and weakened adhesion ability. While CXCL2 neutralization and CXCR2 blockage significantly inhibited the effects of CXCL2 on SMMC7721 cells, indicating that CXCL2 may play pivotal role in HCC metastasis.

Development of the automated circulating tumor cell recovery system with microcavity array.

PubMed

Negishi, Ryo; Hosokawa, Masahito; Nakamura, Seita; Kanbara, Hisashige; Kanetomo, Masafumi; Kikuhara, Yoshihito; Tanaka, Tsuyoshi; Matsunaga, Tadashi; Yoshino, Tomoko

2015-05-15

Circulating tumor cells (CTCs) are well recognized as useful biomarker for cancer diagnosis and potential target of drug discovery for metastatic cancer. Efficient and precise recovery of extremely low concentrations of CTCs from blood has been required to increase the detection sensitivity. Here, an automated system equipped with a microcavity array (MCA) was demonstrated for highly efficient and reproducible CTC recovery. The use of MCA allows selective recovery of cancer cells from whole blood on the basis of differences in size between tumor and blood cells. Intra- and inter-assays revealed that the automated system achieved high efficiency and reproducibility equal to the assay manually performed by well-trained operator. Under optimized assay workflow, the automated system allows efficient and precise cell recovery for non-small cell lung cancer cells spiked in whole blood. The automated CTC recovery system will contribute to high-throughput analysis in the further clinical studies on large cohort of cancer patients. Copyright © 2014 Elsevier B.V. All rights reserved.

Interrogating the variation of element masses and distribution patterns in single cells using ICP-MS with a high efficiency cell introduction system.

PubMed

Wang, Hailong; Wang, Meng; Wang, Bing; Zheng, Lingna; Chen, Hanqing; Chai, Zhifang; Feng, Weiyue

2017-02-01

Cellular heterogeneity is an inherent condition of cell populations, which results from stochastic expression of genes, proteins, and metabolites. The heterogeneity of individual cells can dramatically influence cellular decision-making and cell fate. So far, our knowledge about how the variation of endogenous metals and non-metals in individual eukaryotic cells is limited. In this study, ICP-MS equipped with a high efficiency cell introduction system (HECIS) was developed as a method of single-cell ICP-MS (SC-ICP-MS). The method was applied to the single-cell analysis of Mn, Fe, Co, Cu, Zn, P, and S in human cancer cell lines (HeLa and A549) and normal human bronchial epithelial cell line (16HBE). The analysis showed obvious variation of the masses of Cu, Fe, Zn, and P in individual HeLa cells, and variation of Fe, Zn, and P in individual A549 cells. On the basis of the single-cell data, a multimodal distribution of the elements in the cell population was fitted, which showed marked differences among the various cell lines. Importantly, subpopulations of the elements were found in the cell populations, especially in the HeLa cancer cells. This study demonstrates that SC-ICP-MS is able to unravel the extent of variation of endogenous elements in individual cells, which will help to improve our fundamental understanding of cellular biology and reveal novel insights into human biology and medicine. Graphical abstract The variations of masses and distribution patterns of elements Mn, Fe, Co, Cu, Zn, P, and S in single cells were successfully detected by ICP-MS coupled with a high efficiency cell introduction system (HECIS).

Treatment Analysis in a Cancer Stem Cell Context Using a Tumor Growth Model Based on Cellular Automata.

PubMed

Monteagudo, Ángel; Santos, José

2015-01-01

Cancer can be viewed as an emergent behavior in terms of complex system theory and artificial life, Cellular Automata (CA) being the tool most used for studying and characterizing the emergent behavior. Different approaches with CA models were used to model cancer growth. The use of the abstract model of acquired cancer hallmarks permits the direct modeling at cellular level, where a cellular automaton defines the mitotic and apoptotic behavior of cells, and allows for an analysis of different dynamics of the cellular system depending on the presence of the different hallmarks. A CA model based on the presence of hallmarks in the cells, which includes a simulation of the behavior of Cancer Stem Cells (CSC) and their implications for the resultant growth behavior of the multicellular system, was employed. This modeling of cancer growth, in the avascular phase, was employed to analyze the effect of cancer treatments in a cancer stem cell context. The model clearly explains why, after treatment against non-stem cancer cells, the regrowth capability of CSCs generates a faster regrowth of tumor behavior, and also shows that a continuous low-intensity treatment does not favor CSC proliferation and differentiation, thereby allowing an unproblematic control of future tumor regrowth. The analysis performed indicates that, contrary to the current attempts at CSC control, trying to make CSC proliferation more difficult is an important point to consider, especially in the immediate period after a standard treatment for controlling non-stem cancer cell proliferation.

Applications of laser-induced breakdown spectrometry (LIBS) in surface analysis.

PubMed

Vadillo, J M; Palanco, S; Romero, M D; Laserna, J J

1996-07-01

The applicability of laser-induced breakdown spectrometry (LIBS) for surface analysis is presented in terms of its lateral and depth resolution. A pulsed N(2) laser at 337.1 nm (3.65 J/cm(2)) was used to irradiate solar cells employed for photovoltaic energy production. Laser produced plasmas were collected and detected using a charge-coupled device. An experimental device developed in the laboratory permits an exact synchronization of sample positioning using an XY motorized system with laser pulses. Multielement analysis with lateral resolution of up to 30 microm is feasible with the present system. Three-dimensional capabilities of the system are used for studies on the distribution of carbon impurities at the surface of the solar cells.

Bioculture System: Expanding ISS Space Bioscience Capabilities for Fundamental Stem Cell Research and Commercial Applications

NASA Astrophysics Data System (ADS)

Blaber, Elizabeth; Dvorochkin, Natalya; Almeida, Eduardo; Fitzpatrick, Garret; Ellingson, Lance; Mitchell, Sarah; Yang, Anthony; Kosnik, Cristine; Rayl, Nicole; Cannon, Tom; Austin, Edward; Sato, Kevin

With the recent call by the 2011 Decadal Report and the 2010 Space Biosciences Roadmap for the International Space Station (ISS) to be used as a National Laboratory for scientific research, there is now a need for new laboratory instruments on ISS to enable such research to occur. The Bioculture System supports the extended culturing of multiple cell types and microbiological specimens. It consists of a docking station that carries ten independent incubation units or ‘Cassettes’. Each Cassette contains a cooling chamber (5(°) C) for temperature sensitive solutions and samples, or long duration fluids and sample storage, as well as an incubation chamber (ambient up to 42(°) C). Each Cassette houses an independent fluidics system comprised of a biochamber, medical-grade fluid tubing, medium warming module, oxygenation module, fluid pump, and sixteen solenoid valves for automated biochamber injections of sampling. The Bioculture System provides the user with the ability to select the incubation temperature, fluid flow rate and automated biochamber sampling or injection events for each separate Cassette. Furthermore, the ISS crew can access the biochamber, media bag, and accessory bags on-orbit using the Microgravity Science Glovebox. The Bioculture System also permits initiation of cultures, subculturing, injection of compounds, and removal of samples for on-orbit processing using ISS facilities. The Bioculture System therefore provides a unique opportunity for the study of stem cells and other cell types in space. The first validation flight of the Bioculture System will be conducted on SpaceX5, consisting of 8 Cassettes and lasting for 30-37 days. During this flight we plan to culture two different mammalian cell types in bioreactors: a mouse osteocytic-like cell line, and human induced pluripotent stem cell (iPS)-derived cardiomyocytes. Specifically, the osteocytic line will enable the study of a type of cell that has been flown on the Bioculture System’s predecessor, the Cell Culture Module, whilst demonstrating the Bioculture Systems bead-based sub-culturing capabilities, automated sampling and fixation, manual sample removal/storage by ISS crew members, and whole bioreactor fixation. These activities will enable, for the first time, the long-duration culture of a proliferative cell line. Furthermore, these activities will facilitate genetic and proteomic analysis of these cells at several time points to determine cell health throughout the culture period. The long-duration culture of iPS-derived cardiomyocytes will afford us the capability to assess the maturation and formation of a cardiac-like tissue in microgravity conditions. Automated sampling of this culture immediately prior to un-berthing from the ISS will enable genetic analysis of the mature cardiomyocyte tissue, whilst still enabling the return of live cultures for analysis of cardiomyocyte morphology, contractility, and viability in response to spaceflight. This validation flight will demonstrate the new functional capabilities of the Bioculture System and the System will enable, for the first time, the study of the response of stem cells and other cell lineages to long-duration spaceflight exposure, whilst enabling normal cell culturing techniques to be automatically conducted on ISS.

Differential Sox10 Genomic Occupancy in Myelinating Glia

PubMed Central

Lopez-Anido, Camila; Sun, Guannan; Koenning, Matthias; Srinivasan, Rajini; Hung, Holly A.; Emery, Ben; Keles, Sunduz; Svaren, John

2015-01-01

Myelin is formed by specialized myelinating glia: oligodendrocytes and Schwann cells in the central and peripheral nervous systems, respectively. While there are distinct developmental aspects and regulatory pathways in these two cell types, myelination in both systems requires the transcriptional activator Sox10. Sox10 interacts with cell type-specific transcription factors at some loci to induce myelin gene expression, but it is largely unknown how Sox10 transcriptional networks globally compare between oligodendrocytes and Schwann cells. We used in vivo ChIP-Seq analysis of spinal cord and peripheral nerve (sciatic nerve) to identify unique and shared Sox10 binding sites and assess their correlation with active enhancers and transcriptional profiles in oligodendrocytes and Schwann cells. Sox10 binding sites overlap with active enhancers and critical cell type-specific regulators of myelination, such as Olig2 and Myrf in oligodendrocytes, and Egr2/Krox20 in Schwann cells. Sox10 sites also associate with genes critical for myelination in both oligodendrocytes and Schwann cells, and are found within super-enhancers previously defined in brain. In Schwann cells, Sox10 sites contain binding motifs of putative partners in the Sp/Klf, Tead, and nuclear receptor protein families. Specifically, siRNA analysis of nuclear receptors Nr2f1 and Nr2f2 revealed downregulation of myelin genes Mbp and Ndrg1 in primary Schwann cells. Our analysis highlights different mechanisms that establish cell type-specific genomic occupancy of Sox10, which reflects the unique characteristics of oligodendrocyte and Schwann cell differentiation. PMID:25974668

Miniaturized Mass-Spectrometry-Based Analysis System for Fully Automated Examination of Conditioned Cell Culture Media

PubMed Central

Weber, Emanuel; Pinkse, Martijn W. H.; Bener-Aksam, Eda; Vellekoop, Michael J.; Verhaert, Peter D. E. M.

2012-01-01

We present a fully automated setup for performing in-line mass spectrometry (MS) analysis of conditioned media in cell cultures, in particular focusing on the peptides therein. The goal is to assess peptides secreted by cells in different culture conditions. The developed system is compatible with MS as analytical technique, as this is one of the most powerful analysis methods for peptide detection and identification. Proof of concept was achieved using the well-known mating-factor signaling in baker's yeast, Saccharomyces cerevisiae. Our concept system holds 1 mL of cell culture medium and allows maintaining a yeast culture for, at least, 40 hours with continuous supernatant extraction (and medium replenishing). The device's small dimensions result in reduced costs for reagents and open perspectives towards full integration on-chip. Experimental data that can be obtained are time-resolved peptide profiles in a yeast culture, including information about the appearance of mating-factor-related peptides. We emphasize that the system operates without any manual intervention or pipetting steps, which allows for an improved overall sensitivity compared to non-automated alternatives. MS data confirmed previously reported aspects of the physiology of the yeast-mating process. Moreover, matingfactor breakdown products (as well as evidence for a potentially responsible protease) were found. PMID:23091722

Solar-powered unmanned aerial vehicles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reinhardt, K.C.; Lamp, T.R.; Geis, J.W.

1996-12-31

An analysis was performed to determine the impact of various power system components and mission requirements on the size of solar-powered high altitude long endurance (HALE)-type aircraft. The HALE unmanned aerial vehicle (UAV) has good potential for use in many military and civil applications. The primary power system components considered in this study were photovoltaic (PV) modules for power generation and regenerative fuel cells for energy storage. The impact of relevant component performance on UAV size and capability were considered; including PV module efficiency and mass, power electronics efficiency, and fuel cell specific energy. Mission parameters such as time ofmore » year, flight altitude, flight latitude, and payload mass and power were also varied to determine impact on UAV size. The aircraft analysis method used determines the required aircraft wing aspect ratio, wing area, and total mass based on maximum endurance or minimum required power calculations. The results indicate that the capacity of the energy storage system employed, fuel cells in this analysis, greatly impacts aircraft size, whereas the impact of PV module efficiency and mass is much less important. It was concluded that an energy storage specific energy (total system) of 250--500 Whr/kg is required to enable most useful missions, and that PV cells with efficiencies greater than {approximately} 12% are suitable for use.« less

Revisiting the immune microenvironment of diffuse large B-cell lymphoma using a tissue microarray and immunohistochemistry: robust semi-automated analysis reveals CD3 and FoxP3 as potential predictors of response to R-CHOP

PubMed Central

Coutinho, Rita; Clear, Andrew J.; Mazzola, Emanuele; Owen, Andrew; Greaves, Paul; Wilson, Andrew; Matthews, Janet; Lee, Abigail; Alvarez, Rute; da Silva, Maria Gomes; Cabeçadas, José; Neuberg, Donna; Calaminici, Maria; Gribben, John G.

2015-01-01

Gene expression studies have identified the microenvironment as a prognostic player in diffuse large B-cell lymphoma. However, there is a lack of simple immune biomarkers that can be applied in the clinical setting and could be helpful in stratifying patients. Immunohistochemistry has been used for this purpose but the results are inconsistent. We decided to reinvestigate the immune microenvironment and its impact using immunohistochemistry, with two systems of image analysis, in a large set of patients with diffuse large B-cell lymphoma. Diagnostic tissue from 309 patients was arrayed onto tissue microarrays. Results from 161 chemoimmunotherapy-treated patients were used for outcome prediction. Positive cells, percentage stained area and numbers of pixels/area were quantified and results were compared with the purpose of inferring consistency between the two semi-automated systems. Measurement cutpoints were assessed using a recursive partitioning algorithm classifying results according to survival. Kaplan-Meier estimators and Fisher exact tests were evaluated to check for significant differences between measurement classes, and for dependence between pairs of measurements, respectively. Results were validated by multivariate analysis incorporating the International Prognostic Index. The concordance between the two systems of image analysis was surprisingly high, supporting their applicability for immunohistochemistry studies. Patients with a high density of CD3 and FoxP3 by both methods had a better outcome. Automated analysis should be the preferred method for immunohistochemistry studies. Following the use of two methods of semi-automated analysis we suggest that CD3 and FoxP3 play a role in predicting response to chemoimmunotherapy in diffuse large B-cell lymphoma. PMID:25425693

Revisiting the immune microenvironment of diffuse large B-cell lymphoma using a tissue microarray and immunohistochemistry: robust semi-automated analysis reveals CD3 and FoxP3 as potential predictors of response to R-CHOP.

PubMed

Coutinho, Rita; Clear, Andrew J; Mazzola, Emanuele; Owen, Andrew; Greaves, Paul; Wilson, Andrew; Matthews, Janet; Lee, Abigail; Alvarez, Rute; da Silva, Maria Gomes; Cabeçadas, José; Neuberg, Donna; Calaminici, Maria; Gribben, John G

2015-03-01

Gene expression studies have identified the microenvironment as a prognostic player in diffuse large B-cell lymphoma. However, there is a lack of simple immune biomarkers that can be applied in the clinical setting and could be helpful in stratifying patients. Immunohistochemistry has been used for this purpose but the results are inconsistent. We decided to reinvestigate the immune microenvironment and its impact using immunohistochemistry, with two systems of image analysis, in a large set of patients with diffuse large B-cell lymphoma. Diagnostic tissue from 309 patients was arrayed onto tissue microarrays. Results from 161 chemoimmunotherapy-treated patients were used for outcome prediction. Positive cells, percentage stained area and numbers of pixels/area were quantified and results were compared with the purpose of inferring consistency between the two semi-automated systems. Measurement cutpoints were assessed using a recursive partitioning algorithm classifying results according to survival. Kaplan-Meier estimators and Fisher exact tests were evaluated to check for significant differences between measurement classes, and for dependence between pairs of measurements, respectively. Results were validated by multivariate analysis incorporating the International Prognostic Index. The concordance between the two systems of image analysis was surprisingly high, supporting their applicability for immunohistochemistry studies. Patients with a high density of CD3 and FoxP3 by both methods had a better outcome. Automated analysis should be the preferred method for immunohistochemistry studies. Following the use of two methods of semi-automated analysis we suggest that CD3 and FoxP3 play a role in predicting response to chemoimmunotherapy in diffuse large B-cell lymphoma. Copyright© Ferrata Storti Foundation.

Analysis of integrated photovoltaic-thermal systems using solar concentrators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yusoff, M.B.

1983-01-01

An integrated photovoltaic-thermal system using solar concentrators utilizes the solar radiation spectrum in the production of electrical and thermal energy. The electrical conversion efficiency of this system decreases with increasing solar cell temperature. Since a high operating temperature is desirable to maximize the quality of thermal output of the planned integrated system, a proper choice of the operating temperature for the unit cell is of vital importance. The analysis predicts performance characteristics of the unit cell by considering the dependence of the heat generation, the heat absorption and the heat transmission on the material properties of the unit cell structure.more » An analytical model has been developed to describe the heat transport phenomena occurring in the unit cell structure. The range of applicability of the one-dimensional and the two-dimensional models, which have closed-form solutions, has been demonstrated. Parametric and design studies point out the requirements for necessary good electrical and thermal performance. A procedure utilizing functional forms of component characteristics in the form of partial coefficients of the dependent variable has been developed to design and operate the integrated system to have a desirable value of the thermal to electrical output ratio both at design and operating modes.« less

A model-based approach for automated in vitro cell tracking and chemotaxis analyses.

PubMed

Debeir, Olivier; Camby, Isabelle; Kiss, Robert; Van Ham, Philippe; Decaestecker, Christine

2004-07-01

Chemotaxis may be studied in two main ways: 1) counting cells passing through an insert (e.g., using Boyden chambers), and 2) directly observing cell cultures (e.g., using Dunn chambers), both in response to stationary concentration gradients. This article promotes the use of Dunn chambers and in vitro cell-tracking, achieved by video microscopy coupled with automatic image analysis software, in order to extract quantitative and qualitative measurements characterizing the response of cells to a diffusible chemical agent. Previously, we set up a videomicroscopy system coupled with image analysis software that was able to compute cell trajectories from in vitro cell cultures. In the present study, we are introducing a new software increasing the application field of this system to chemotaxis studies. This software is based on an adapted version of the active contour methodology, enabling each cell to be efficiently tracked for hours and resulting in detailed descriptions of individual cell trajectories. The major advantages of this method come from an improved robustness with respect to variability in cell morphologies between different cell lines and dynamical changes in cell shape during cell migration. Moreover, the software includes a very small number of parameters which do not require overly sensitive tuning. Finally, the running time of the software is very short, allowing improved possibilities in acquisition frequency and, consequently, improved descriptions of complex cell trajectories, i.e. trajectories including cell division and cell crossing. We validated this software on several artificial and real cell culture experiments in Dunn chambers also including comparisons with manual (human-controlled) analyses. We developed new software and data analysis tools for automated cell tracking which enable cell chemotaxis to be efficiently analyzed. Copyright 2004 Wiley-Liss, Inc.

Single-cell technologies to study the immune system.

PubMed

Proserpio, Valentina; Mahata, Bidesh

2016-02-01

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

Validation of a single-platform method for hematopoietic CD34+ stem cells enumeration according to accreditation procedure.

PubMed

Massin, Frédéric; Huili, Cai; Decot, Véronique; Stoltz, Jean-François; Bensoussan, Danièle; Latger-Cannard, Véronique

2015-01-01

Stem cells for autologous and allogenic transplantation are obtained from several sources including bone marrow, peripheral blood or cord blood. Accurate enumeration of viable CD34+ hematopoietic stem cells (HSC) is routinely used in clinical settings, especially to monitor progenitor cell mobilization and apheresis. The number of viable CD34+ HSC has also been shown to be the most critical factor in haematopoietic engraftment. The International Society for Cellular Therapy actually recommends the use of single-platform flow cytometry system using 7-AAD as a viability dye. In a way to move routine analysis from a BD FACSCaliburTM instrument to a BD FACSCantoTM II, according to ISO 15189 standard guidelines, we define laboratory performance data of the BDTM Stem Cell Enumeration (SCE) kit on a CE-IVD system including a BD FACSCanto II flow cytometer and the BD FACSCantoTM Clinical Software. InterQCTM software, a real time internet laboratory QC management system developed by VitroTM and distributed by Becton DickinsonTM, was also tested to monitor daily QC data, to define the internal laboratory statistics and to compare them to external laboratories. Precision was evaluated with BDTM Stem Cell Control (high and low) results and the InterQC software, an internet laboratory QC management system by Vitro. This last one drew Levey-Jennings curves and generated numeral statistical parameters allowing detection of potential changes in the system performances as well as interlaboratory comparisons. Repeatability, linearity and lower limits of detection were obtained with routine samples from different origins. Agreement evaluation between BD FACSCanto II system versus BD FACSCalibur system was tested on fresh peripheral blood, freeze-thawed apheresis, fresh bone marrow and fresh cord blood samples. Instrument's measure and staining repeatability clearly evidenced acceptable variability on the different samples tested. Intra- and inter-laboratory CV in CD34+ cell absolute count are consistent and reproducible. Linearity analysis, established between 2 and 329 cells/μl showed a linear relation between expected counts and measured counts (R2=0.97). Linear regression and Bland-Altman representations showed an excellent correlation on samples from different sources between the two systems and allowed the transfer of routine analysis from BD FACSCalibur to BD FACSCanto II. The BD SCE kit provides an accurate measure of the CD34 HSC, and can be used in daily routine to optimize the enumeration of hematopoietic CD34+ stem cells by flow cytometry. Moreover, the InterQC system seems to be a very useful tool for laboratory daily quality monitoring and thus for accreditation.

Single molecule analysis of B cell receptor motion during signaling activation

NASA Astrophysics Data System (ADS)

Rey Suarez, Ivan; Koo, Peter; Zhou, Shu; Wheatley, Brittany; Song, Wenxia; Mochrie, Simon; Upadhyaya, Arpita

B cells are an essential part of the adaptive immune system. They patrol the body for signs of infection in the form of antigen on the surface of antigen presenting cells. B cell receptor (BCR) binding to antigen induces a signaling cascade that leads to B cell activation and spreading. During activation, BCR form signaling microclusters that later coalesce as the cell contracts. We have studied the dynamics of BCRs on activated murine primary B cells using single particle tracking. The tracks are analyzed using perturbation expectation-maximization (pEM), a systems-level analysis, which allows identification of different short-time diffusive states from single molecule tracks. We identified four dominant diffusive states, two of which correspond to BCRs interacting with signaling molecules. For wild-type cells, the number of BCR in signaling states increases as the cell spreads and then decreases during cell contraction. In contrast, cells lacking the actin regulatory protein, N-WASP, are unable to contract and BCRs remain in the signaling states for longer times. These observations indicate that actin cytoskeleton dynamics modulate BCR diffusion and clustering. Our results provide novel information regarding the timescale of interaction between BCR and signaling molecules.

[Metabolic changes in cells under electromagnetic radiation of mobile communication systems].

PubMed

Iakimenko, I L; Sidorik, E P; Tsybulin, A S

2011-01-01

Review is devoted to the analysis of biological effects of microwaves. The results of last years' researches indicated the potential risks of long-term low-level microwaves exposure for human health. The analysis of metabolic changes in living cells under the exposure of microwaves from mobile communication systems indicates that this factor is stressful for cells. Among the reproducible effects of low-level microwave radiation are overexpression of heat shock proteins, an increase of reactive oxygen species level, an increase of intracellular Ca2+, damage of DNA, inhibition of DNA reparation, and induction of apoptosis. Extracellular-signal-regulated kinases ERK and stress-related kinases p38MAPK are involved in metabolic changes. Analysis of current data suggests that the concept of exceptionally thermal mechanism of biological effects of microwaves is not correct. In turn, this raises the question of the need to revaluation of modern electromagnetic standards based on thermal effects of non-ionizing radiation on biological systems.

TLM-Tracker: software for cell segmentation, tracking and lineage analysis in time-lapse microscopy movies.

PubMed

Klein, Johannes; Leupold, Stefan; Biegler, Ilona; Biedendieck, Rebekka; Münch, Richard; Jahn, Dieter

2012-09-01

Time-lapse imaging in combination with fluorescence microscopy techniques enable the investigation of gene regulatory circuits and uncovered phenomena like culture heterogeneity. In this context, computational image processing for the analysis of single cell behaviour plays an increasing role in systems biology and mathematical modelling approaches. Consequently, we developed a software package with graphical user interface for the analysis of single bacterial cell behaviour. A new software called TLM-Tracker allows for the flexible and user-friendly interpretation for the segmentation, tracking and lineage analysis of microbial cells in time-lapse movies. The software package, including manual, tutorial video and examples, is available as Matlab code or executable binaries at http://www.tlmtracker.tu-bs.de.

Steroid Hormone Signaling Is Essential to Regulate Innate Immune Cells and Fight Bacterial Infection in Drosophila

PubMed Central

Regan, Jennifer C.; Brandão, Ana S.; Leitão, Alexandre B.; Mantas Dias, Ângela Raquel; Sucena, Élio; Jacinto, António; Zaidman-Rémy, Anna

2013-01-01

Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells. PMID:24204269

Central Facilities Area Sewage Lagoon Evaluation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giesbrecht, Alan

2015-03-01

The Central Facilities Area (CFA) located in Butte County, Idaho at Idaho National Laboratory (INL) has an existing wastewater system to collect and treat sanitary wastewater and non contact cooling water from the facility. The existing treatment facility consists of three cells: Cell 1 has a surface area of 1.7 acres, Cell 2 has a surface area of 10.3 acres, and Cell 3 has a surface area of 0.5 acres. If flows exceed the evaporative capacity of the cells, wastewater is discharged to a 73.5 acre land application site that utilizes a center pivot irrigation sprinkler system. The purpose ofmore » this current study is to update the analysis and conclusions of the December 2013 study. In this current study, the new seepage rate and influent flow rate data have been used to update the calculations, model, and analysis.« less

High-Content Electrophysiological Analysis of Human Pluripotent Stem Cell-Derived Cardiomyocytes (hPSC-CMs).

PubMed

Kong, Chi-Wing; Geng, Lin; Li, Ronald A

2018-01-01

Considerable interest has been raised to develop human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as a model for drug discovery and cardiotoxicity screening. High-content electrophysiological analysis of currents generated by transmembrane cell surface ion channels has been pursued to complement such emerging applications. Here we describe practical procedures and considerations for accomplishing successful assays of hPSC-CMs using an automated planar patch-clamp system.

Analysis of a Concentrated Solar Thermophotovoltaic System with Thermal Energy Storage

NASA Astrophysics Data System (ADS)

Seyf, Hamid Reza; Henry, Asegun

2017-01-01

We analyzed a high temperature concentrated solar thermophotovoltaic (TPV) system with thermal energy storage (TES), which is enabled by the potential usage of liquid metal as a high temperature heat transfer fluid. The system concept combines the great advantages of TES with the potential for low cost and high performance derived from photovoltaic cells fabricated on reusable substrates, with a high reflectivity back reflector for photon recycling. The TES makes the electricity produced dispatchable, and thus the system studied should be compared to technologies such as concentrated solar power (CSP) with TES (e.g., using a turbine) or PV with electrochemical batteries, instead of direct and intermittent electricity generation from flat plate PV alone. Thus, the addition of TES places the system in a different class than has previously been considered and based on the model results, appears worthy of increased attention. The system level analysis presented identifies important cell level parameters that have the greatest impact on the overall system performance, and as a result can help to set the priorities for future TPV cell development.

Analysis of a Concentrated Solar Thermophotovoltaic System with Thermal Energy Storage

NASA Astrophysics Data System (ADS)

Seyf, Hamid Reza; Henry, Asegun

We analyzed a high temperature concentrated solar thermophotovoltaic (TPV) system with thermal energy storage (TES), which is enabled by the potential usage of liquid metal as a high temperature heat transfer fluid. The system concept combines the great advantages of TES with the potential for low cost and high performance derived from photovoltaic cells fabricated on reusable substrates, with a high reflectivity back reflector for photon recycling. The TES makes the electricity produced dispatchable, and thus the system studied should be compared to technologies such as concentrated solar power (CSP) with TES (e.g., using a turbine) or PV with electrochemical batteries, instead of direct and intermittent electricity generation from flat plate PV alone. Thus, the addition of TES places the system in a different class than has previously been considered and based on the model results, appears worthy of increased attention. The system level analysis presented identifies important cell level parameters that have the greatest impact on the overall system performance, and as a result can help to set the priorities for future TPV cell development.

Hybrid Solid Oxide Fuel Cell/Gas Turbine System Design for High Altitude Long Endurance Aerospace Missions

NASA Technical Reports Server (NTRS)

Himansu, Ananda; Freeh, Joshua E.; Steffen, Christopher J., Jr.; Tornabene, Robert T.; Wang, Xiao-Yen J.

2006-01-01

A system level analysis, inclusive of mass, is carried out for a cryogenic hydrogen fueled hybrid solid oxide fuel cell and bottoming gas turbine (SOFC/GT) power system. The system is designed to provide primary or secondary electrical power for an unmanned aerial vehicle (UAV) over a high altitude, long endurance mission. The net power level and altitude are parametrically varied to examine their effect on total system mass. Some of the more important technology parameters, including turbomachinery efficiencies and the SOFC area specific resistance, are also studied for their effect on total system mass. Finally, two different solid oxide cell designs are compared to show the importance of the individual solid oxide cell design on the overall system. We show that for long mission durations of 10 days or more, the fuel mass savings resulting from the high efficiency of a SOFC/GT system more than offset the larger powerplant mass resulting from the low specific power of the SOFC/GT system. These missions therefore favor high efficiency, low power density systems, characteristics typical of fuel cell systems in general.

xCELLigence system for real-time label-free monitoring of growth and viability of cell lines from hematological malignancies.

PubMed

Martinez-Serra, Jordi; Gutierrez, Antonio; Muñoz-Capó, Saúl; Navarro-Palou, María; Ros, Teresa; Amat, Juan Carlos; Lopez, Bernardo; Marcus, Toni F; Fueyo, Laura; Suquia, Angela G; Gines, Jordi; Rubio, Francisco; Ramos, Rafael; Besalduch, Joan

2014-01-01

The xCELLigence system is a new technological approach that allows the real-time cell analysis of adherent tumor cells. To date, xCELLigence has not been able to monitor the growth or cytotoxicity of nonadherent cells derived from hematological malignancies. The basis of its technology relies on the use of culture plates with gold microelectrodes located in their base. We have adapted the methodology described by others to xCELLigence, based on the pre-coating of the cell culture surface with specific substrates, some of which are known to facilitate cell adhesion in the extracellular matrix. Pre-coating of the culture plates with fibronectin, compared to laminin, collagen, or gelatin, significantly induced the adhesion of most of the leukemia/lymphoma cells assayed (Jurkat, L1236, KMH2, and K562). With a fibronectin substrate, nonadherent cells deposited in a monolayer configuration, and consequently, the cell growth and viability were robustly monitored. We further demonstrate the feasibility of xCELLigence for the real-time monitoring of the cytotoxic properties of several antineoplastic agents. In order to validate this technology, the data obtained through real-time cell analysis was compared with that obtained from using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. This provides an excellent label-free tool for the screening of drug efficacy in nonadherent cells and discriminates optimal time points for further molecular analysis of cellular events associated with treatments, reducing both time and costs.

JSC systems using solid ceramic oxygen electrolyte cells to measure oxygen fugacites in gas-mixing systems

NASA Technical Reports Server (NTRS)

Williams, R. J.; Mullins, O.

1981-01-01

Details are given for the construction and operation of a 101.3 KN/sq meter (1 atmosphere) redox control system. A solid ceramic oxygen electrolyte cell is used to monitor the oxygen fugacity in the furnace. The system consists of a vertical quench gas mixing furnace with heads designed for mounting the electrolyte cell and with facilities for inserting and removing the samples, a simplified version of a gas mixing apparatus, and devices for experiments under controlled rates of change of temperature. A thermogravimetric analysis system employing these techniques of redox control and measurement is also described. The calibration and maintenance of the system are discussed.

Measuring bovine gamma delta T cell function at the site of Mycobacterium bovis infection

USDA-ARS?s Scientific Manuscript database

Bovine gamma delta T cells are amongst the first cells to accumulate at the site of Mycobacterium bovis infection; however, their role in the developing lesion remains unclear. We utilized transcriptomics analysis, in situ hybridization, and a macrophage/gamma delta T cell co-culture system to eluc...

High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells.

PubMed

Steyer, Benjamin; Carlson-Stevermer, Jared; Angenent-Mari, Nicolas; Khalil, Andrew; Harkness, Ty; Saha, Krishanu

2016-04-01

Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24h after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery. CRISPR-Cas9 is a new gene-editing technology for "genome surgery" that is anticipated to treat genetic diseases. This technology uses multiple components of the Cas9 system to cut out disease-causing mutations in the human genome and precisely suture in therapeutic sequences. Biomaterials based delivery strategies could help transition these technologies to the clinic. The design space for materials based delivery strategies is vast and optimization is essential to ensuring the safety and efficacy of these treatments. Therefore, new methods are required to rapidly and systematically screen gene-editing efficacy in human cells. This work utilizes an innovative platform to generate and screen many formulations of synthetic biomaterials and components of the CRISPR-Cas9 system in parallel. On this platform, we watch genome surgery in action using high content image analysis. These capabilities enabled us to identify formulation parameters for Cas9-material complexes that can optimize gene-editing in a specific human cell type. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Automated Analysis of siRNA Screens of Virus Infected Cells Based on Immunofluorescence Microscopy

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

We present an image analysis approach as part of a high-throughput microscopy screening system based on cell arrays for the identification of genes involved in Hepatitis C and Dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in cells, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behavior of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

Rate-limiting step analysis of the microbial desulfurization of dibenzothiophene in a model oil system

PubMed Central

Abin-Fuentes, Andres; Leung, James C.; Mohamed, Magdy El-Said; Wang, Daniel IC; Prather, Kristala LJ

2014-01-01

A mechanistic analysis of the various mass transport and kinetic steps in the microbial desulfurization of dibenzothiophene (DBT) by Rhodococcus erythropolis IGTS8 in a model biphasic (oil-water), small-scale system was performed. The biocatalyst was distributed into three populations, free cells in the aqueous phase, cell aggregates and oil-adhered cells, and the fraction of cells in each population was measured. The power input per volume (P/V) and the impeller tip speed (vtip) were identified as key operating parameters in determining whether the system is mass transport controlled or kinetically controlled. Oil-water DBT mass transport was found to not be limiting under the conditions tested. Experimental results at both the 100 mL and 4L (bioreactor) scales suggest that agitation leading to P/V greater than 10,000 W/ m3 and/or vtip greater than 0.67 m/s is sufficient to overcome the major mass transport limitation in the system, which was the diffusion of DBT within the biocatalyst aggregates. PMID:24284557

Effect of intravenous administration of D-lysergic acid diethylamide on initiation of protein synthesis in a cell-free system derived from brain.

PubMed

Cosgrove, J W; Brown, I R

1984-05-01

An initiating cell-free protein synthesis system derived from brain was utilized to demonstrate that the intravenous injection of D-lysergic acid diethylamide (LSD) to rabbits resulted in a lesion at the initiation stage of brain protein synthesis. Three inhibitors of initiation, edeine, poly(I), and aurintricarboxylic acid were used to demonstrate a reduction in initiation-dependent amino acid incorporation in the brain cell-free system. One hour after LSD injection, there was also a measurable decrease in the formation of 40S and 80S initiation complexes in vitro, using either [35S]methionine or [35S]Met-tRNAf. Analysis of the methionine pool size after LSD administration indicated there was no change in methionine levels. Analysis of the formation of initiation complexes in the brain cell-free protein synthesis system prepared 6 h after LSD administration indicated that there was a return to control levels at this time. The effects of LSD on steps in the initiation process are thus reversible.

Metabolic Compartmentation – A System Level Property of Muscle Cells

PubMed Central

Saks, Valdur; Beraud, Nathalie; Wallimann, Theo

2008-01-01

Problems of quantitative investigation of intracellular diffusion and compartmentation of metabolites are analyzed. Principal controversies in recently published analyses of these problems for the living cells are discussed. It is shown that the formal theoretical analysis of diffusion of metabolites based on Fick's equation and using fixed diffusion coefficients for diluted homogenous aqueous solutions, but applied for biological systems in vivo without any comparison with experimental results, may lead to misleading conclusions, which are contradictory to most biological observations. However, if the same theoretical methods are used for analysis of actual experimental data, the apparent diffusion constants obtained are orders of magnitude lower than those in diluted aqueous solutions. Thus, it can be concluded that local restrictions of diffusion of metabolites in a cell are a system-level properties caused by complex structural organization of the cells, macromolecular crowding, cytoskeletal networks and organization of metabolic pathways into multienzyme complexes and metabolons. This results in microcompartmentation of metabolites, their channeling between enzymes and in modular organization of cellular metabolic networks. The perspectives of further studies of these complex intracellular interactions in the framework of Systems Biology are discussed. PMID:19325782

Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems.

PubMed

Díaz-Triviño, Sara; Long, Yuchen; Scheres, Ben; Blilou, Ikram

2017-01-01

In plant biology, transient expression systems have become valuable approaches used routinely to rapidly study protein expression, subcellular localization, protein-protein interactions, and transcriptional activity prior to in vivo studies. When studying transcriptional regulation, luciferase reporter assays offer a sensitive readout for assaying promoter behavior in response to different regulators or environmental contexts and to confirm and assess the functional relevance of predicted binding sites in target promoters. This chapter aims to provide detailed methods for using luciferase reporter system as a rapid, efficient, and versatile assay to analyze transcriptional regulation of target genes by transcriptional regulators. We describe a series of optimized transient expression systems consisting of Arabidopsis thaliana protoplasts, infiltrated Nicotiana benthamiana leaves, and human HeLa cells to study the transcriptional regulations of two well-characterized transcriptional regulators SCARECROW (SCR) and SHORT-ROOT (SHR) on one of their targets, CYCLIN D6 (CYCD6).Here, we illustrate similarities and differences in outcomes when using different systems. The plant-based systems revealed that the SCR-SHR complex enhances CYCD6 transcription, while analysis in HeLa cells showed that the complex is not sufficient to strongly induce CYCD6 transcription, suggesting that additional, plant-specific regulators are required for full activation. These results highlight the importance of the system and suggest that including heterologous systems, such as HeLa cells, can provide a more comprehensive analysis of a complex gene regulatory network.

Stability of Materials in High Temperature Water Vapor: SOFC Applications

NASA Technical Reports Server (NTRS)

Opila, E. J.; Jacobson, N. S.

2010-01-01

Solid oxide fuel cell material systems require long term stability in environments containing high-temperature water vapor. Many materials in fuel cell systems react with high-temperature water vapor to form volatile hydroxides which can degrade cell performance. In this paper, experimental methods to characterize these volatility reactions including the transpiration technique, thermogravimetric analysis, and high pressure mass spectrometry are reviewed. Experimentally determined data for chromia, silica, and alumina volatility are presented. In addition, data from the literature for the stability of other materials important in fuel cell systems are reviewed. Finally, methods for predicting material recession due to volatilization reactions are described.

The MqsRA Toxin-Antitoxin System from Xylella fastidiosa Plays a Key Role in Bacterial Fitness, Pathogenicity, and Persister Cell Formation.

PubMed

Merfa, Marcus V; Niza, Bárbara; Takita, Marco A; De Souza, Alessandra A

2016-01-01

Through the formation of persister cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of persister cells because they are able to induce cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in persister cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis-CVC) activated by copper stress, and has been described in Escherichia coli as related to the formation of persister cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of persister cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X. fastidiosa and reveal new insights into the physiology of phytopathogen-host interactions.

The MqsRA Toxin-Antitoxin System from Xylella fastidiosa Plays a Key Role in Bacterial Fitness, Pathogenicity, and Persister Cell Formation

PubMed Central

Merfa, Marcus V.; Niza, Bárbara; Takita, Marco A.; De Souza, Alessandra A.

2016-01-01

Through the formation of persister cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of persister cells because they are able to induce cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in persister cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis—CVC) activated by copper stress, and has been described in Escherichia coli as related to the formation of persister cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of persister cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X. fastidiosa and reveal new insights into the physiology of phytopathogen-host interactions. PMID:27375608

Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

PubMed

Radtke, Andrea L; Herbst-Kralovetz, Melissa M

2012-04-03

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties. The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type(1, 7-13). Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

A multi-tissue type genome-scale metabolic network for analysis of whole-body systems physiology

PubMed Central

2011-01-01

Background Genome-scale metabolic reconstructions provide a biologically meaningful mechanistic basis for the genotype-phenotype relationship. The global human metabolic network, termed Recon 1, has recently been reconstructed allowing the systems analysis of human metabolic physiology and pathology. Utilizing high-throughput data, Recon 1 has recently been tailored to different cells and tissues, including the liver, kidney, brain, and alveolar macrophage. These models have shown utility in the study of systems medicine. However, no integrated analysis between human tissues has been done. Results To describe tissue-specific functions, Recon 1 was tailored to describe metabolism in three human cells: adipocytes, hepatocytes, and myocytes. These cell-specific networks were manually curated and validated based on known cellular metabolic functions. To study intercellular interactions, a novel multi-tissue type modeling approach was developed to integrate the metabolic functions for the three cell types, and subsequently used to simulate known integrated metabolic cycles. In addition, the multi-tissue model was used to study diabetes: a pathology with systemic properties. High-throughput data was integrated with the network to determine differential metabolic activity between obese and type II obese gastric bypass patients in a whole-body context. Conclusion The multi-tissue type modeling approach presented provides a platform to study integrated metabolic states. As more cell and tissue-specific models are released, it is critical to develop a framework in which to study their interdependencies. PMID:22041191

Redox flow cell development and demonstration project, calendar year 1977

NASA Technical Reports Server (NTRS)

1979-01-01

Research and development on the redox flow cell conducted from January 1, 1977, to December 31, 1977, are described in this report. The major focus of the effort during 1977 was the key technology issues that directly influence the fundamental feasibility of the overall redox concept. These issues were the development of a suitable ion exchange membrane for the system, the screening and study of candidate redox couples to achieve optimum cell performance, and the carrying out of systems analysis and modeling to develop system performance goals and cost estimates.

Professional Advanced Research and Analysis

NASA Technical Reports Server (NTRS)

Coulman, George A.

1996-01-01

Reported here is a summary of studies examining some problems in an energy conversion system. Regenerative fuel cell systems have been suggested for future manned space missions, but to meet the needed specific power requirements substantial improvements in the state-of-the-art technologies are needed. Similar improvements are needed, with emphasis on cost reduction in addition to higher conversion efficiency, for fuel cell systems that have potential for terrestrial applications. Polymer Electrolyte Membrane (PEM) fuel cells have been identified as promising candidates for development that would lead to the desired cost reduction and increased efficiency.

AI-augmented time stretch microscopy

NASA Astrophysics Data System (ADS)

Mahjoubfar, Ata; Chen, Claire L.; Lin, Jiahao; Jalali, Bahram

2017-02-01

Cell reagents used in biomedical analysis often change behavior of the cells that they are attached to, inhibiting their native signaling. On the other hand, label-free cell analysis techniques have long been viewed as challenging either due to insufficient accuracy by limited features, or because of low throughput as a sacrifice of improved precision. We present a recently developed artificial-intelligence augmented microscope, which builds upon high-throughput time stretch quantitative phase imaging (TS-QPI) and deep learning to perform label-free cell classification with record high-accuracy. Our system captures quantitative optical phase and intensity images simultaneously by frequency multiplexing, extracts multiple biophysical features of the individual cells from these images fused, and feeds these features into a supervised machine learning model for classification. The enhanced performance of our system compared to other label-free assays is demonstrated by classification of white blood T-cells versus colon cancer cells and lipid accumulating algal strains for biofuel production, which is as much as five-fold reduction in inaccuracy. This system obtains the accuracy required in practical applications such as personalized drug development, while the cells remain intact and the throughput is not sacrificed. Here, we introduce a data acquisition scheme based on quadrature phase demodulation that enables interruptionless storage of TS-QPI cell images. Our proof of principle demonstration is capable of saving 40 TB of cell images in about four hours, i.e. pictures of every single cell in 10 mL of a sample.

Peroxisystem: harnessing systems cell biology to study peroxisomes.

PubMed

Schuldiner, Maya; Zalckvar, Einat

2015-04-01

In recent years, high-throughput experimentation with quantitative analysis and modelling of cells, recently dubbed systems cell biology, has been harnessed to study the organisation and dynamics of simple biological systems. Here, we suggest that the peroxisome, a fascinating dynamic organelle, can be used as a good candidate for studying a complete biological system. We discuss several aspects of peroxisomes that can be studied using high-throughput systematic approaches and be integrated into a predictive model. Such approaches can be used in the future to study and understand how a more complex biological system, like a cell and maybe even ultimately a whole organism, works. © 2015 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Kinetic Limitations of Cooperativity-Based Drug Delivery Systems

NASA Astrophysics Data System (ADS)

Licata, Nicholas A.; Tkachenko, Alexei V.

2008-04-01

We study theoretically a novel drug delivery system that utilizes the overexpression of certain proteins in cancerous cells for cell-specific chemotherapy. The system consists of dendrimers conjugated with “keys” (ex: folic acid) which “key-lock” bind to particular cell-membrane proteins (ex: folate receptor). The increased concentration of “locks” on the surface leads to a longer residence time for the dendrimer and greater incorporation into the cell. Cooperative binding of the nanocomplexes leads to an enhancement of cell specificity. However, both our theory and detailed analysis of in vitro experiments indicate that the degree of cooperativity is kinetically limited. We demonstrate that cooperativity and hence the specificity to particular cell type can be increased by making the strength of individual bonds weaker, and suggest a particular implementation of this idea.

Plasma cell quantification in bone marrow by computer-assisted image analysis.

PubMed

Went, P; Mayer, S; Oberholzer, M; Dirnhofer, S

2006-09-01

Minor and major criteria for the diagnosis of multiple meloma according to the definition of the WHO classification include different categories of the bone marrow plasma cell count: a shift from the 10-30% group to the > 30% group equals a shift from a minor to a major criterium, while the < 10% group does not contribute to the diagnosis. Plasma cell fraction in the bone marrow is therefore critical for the classification and optimal clinical management of patients with plasma cell dyscrasias. The aim of this study was (i) to establish a digital image analysis system able to quantify bone marrow plasma cells and (ii) to evaluate two quantification techniques in bone marrow trephines i.e. computer-assisted digital image analysis and conventional light-microscopic evaluation. The results were compared regarding inter-observer variation of the obtained results. Eighty-seven patients, 28 with multiple myeloma, 29 with monoclonal gammopathy of undetermined significance, and 30 with reactive plasmocytosis were included in the study. Plasma cells in H&E- and CD138-stained slides were quantified by two investigators using light-microscopic estimation and computer-assisted digital analysis. The sets of results were correlated with rank correlation coefficients. Patients were categorized according to WHO criteria addressing the plasma cell content of the bone marrow (group 1: 0-10%, group 2: 11-30%, group 3: > 30%), and the results compared by kappa statistics. The degree of agreement in CD138-stained slides was higher for results obtained using the computer-assisted image analysis system compared to light microscopic evaluation (corr.coeff. = 0.782), as was seen in the intra- (corr.coeff. = 0.960) and inter-individual results correlations (corr.coeff. = 0.899). Inter-observer agreement for categorized results (SM/PW: kappa 0.833) was in a high range. Computer-assisted image analysis demonstrated a higher reproducibility of bone marrow plasma cell quantification. This might be of critical importance for diagnosis, clinical management and prognostics when plasma cell numbers are low, which makes exact quantifications difficult.

Analysis system for characterisation of simple, low-cost microfluidic components

NASA Astrophysics Data System (ADS)

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

2014-06-01

There is an inherent trade-off between cost and operational integrity of microfluidic components, especially when intended for use in point-of-care devices. We present an analysis system developed to characterise microfluidic components for performing blood cell counting, enabling the balance between function and cost to be established quantitatively. Microfluidic components for sample and reagent introduction, mixing and dispensing of fluids were investigated. A simple inlet port plugging mechanism is used to introduce and dispense a sample of blood, while a reagent is released into the microfluidic system through compression and bursting of a blister pack. Mixing and dispensing of the sample and reagent are facilitated via air actuation. For these microfluidic components to be implemented successfully, a number of aspects need to be characterised for development of an integrated point-of-care device design. The functional components were measured using a microfluidic component analysis system established in-house. Experiments were carried out to determine: 1. the force and speed requirements for sample inlet port plugging and blister pack compression and release using two linear actuators and load cells for plugging the inlet port, compressing the blister pack, and subsequently measuring the resulting forces exerted, 2. the accuracy and repeatability of total volumes of sample and reagent dispensed, and 3. the degree of mixing and dispensing uniformity of the sample and reagent for cell counting analysis. A programmable syringe pump was used for air actuation to facilitate mixing and dispensing of the sample and reagent. Two high speed cameras formed part of the analysis system and allowed for visualisation of the fluidic operations within the microfluidic device. Additional quantitative measures such as microscopy were also used to assess mixing and dilution accuracy, as well as uniformity of fluid dispensing - all of which are important requirements towards the successful implementation of a blood cell counting system.

Fourier Ptychographic Microscopy for Rapid, High-Resolution Imaging of Circulating Tumor Cells Enriched by Microfiltration.

PubMed

Williams, Anthony; Chung, Jaebum; Yang, Changhuei; Cote, Richard J

2017-01-01

Examining the hematogenous compartment for evidence of metastasis has increased significantly within the oncology research community in recent years, due to the development of technologies aimed at the enrichment of circulating tumor cells (CTCs), the subpopulation of primary tumor cells that gain access to the circulatory system and are responsible for colonization at distant sites. In contrast to other technologies, filtration-based CTC enrichment, which exploits differences in size between larger tumor cells and surrounding smaller, non-tumor blood cells, has the potential to improve CTC characterization through isolation of tumor cell populations with greater molecular heterogeneity. However, microscopic analysis of uneven filtration surfaces containing CTCs is laborious, time-consuming, and inconsistent, preventing widespread use of filtration-based enrichment technologies. Here, integrated with a microfiltration-based CTC and rare cell enrichment device we have previously described, we present a protocol for Fourier Ptychographic Microscopy (FPM), a method that, unlike many automated imaging platforms, produces high-speed, high-resolution images that can be digitally refocused, allowing users to observe objects of interest present on multiple focal planes within the same image frame. The development of a cost-effective and high-throughput CTC analysis system for filtration-based enrichment technologies could have profound clinical implications for improved CTC detection and analysis.

Thermal analysis and management of lithium-titanate batteries

NASA Astrophysics Data System (ADS)

Giuliano, Michael R.; Advani, Suresh G.; Prasad, Ajay K.

2011-08-01

Battery electric vehicles and hybrid electric vehicles demand batteries that can store large amounts of energy in addition to accommodating large charge and discharge currents without compromising battery life. Lithium-titanate batteries have recently become an attractive option for this application. High current thresholds allow these cells to be charged quickly as well as supply the power needed to drive such vehicles. These large currents generate substantial amounts of waste heat due to loss mechanisms arising from the cell's internal chemistry and ohmic resistance. During normal vehicle operation, an active cooling system must be implemented to maintain a safe cell temperature and improve battery performance and life. This paper outlines a method to conduct thermal analysis of lithium-titanate cells under laboratory conditions. Thermochromic liquid crystals were implemented to instantaneously measure the entire surface temperature field of the cell. The resulting temperature measurements were used to evaluate the effectiveness of an active cooling system developed and tested in our laboratory for the thermal management of lithium-titanate cells.

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

PubMed Central

Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

2013-01-01

The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

The Synthesis of Human Placental Lactogen by Ribosomes Derived from Human Placenta

PubMed Central

Boime, Irving; Boguslawski, Sophie

1974-01-01

In a very active cell-free system containing polysomes derived from human placenta and a cell-sap fraction prepared from ascites tumor cells, the synthesis of the hormone human placental lactogen (HPL) was detected. The identification was based on the following: (a) The in vitro synthesized protein labeled with [35S]methionine migrated at the same rate as authentic HPL on sodium dodecyl sulfate-polyacrylamide gels and (b) tryptic fingerprint analysis of the labeled protein yielded peptides having the same mobilities as seen with the same analysis of purified HPL. The amount of HPL synthesized in a cell-free system containing polysomes derived from term placenta was about 10% of the total proteins synthesized and in a comparable system containing first trimester ribosomes the level of synthesis was about 5%. These data suggest the potential for quantitating the HPL mRNA activity as a function of the period of gestation and for isolating the mRNA itself. Images PMID:4524639

Involvement of LAT1 and LAT2 in the high- and low-affinity transport of L-leucine in human retinal pigment epithelial cells (ARPE-19 cells).

PubMed

Yamamoto, Atsushi; Akanuma, Shin-Ichi; Tachikawa, Masanori; Hosoya, Ken-Ichi

2010-05-01

System L, which is encoded by LAT1 and LAT2, is an amino acid transport system that transports neutral amino acids, including several essential amino acids in an Na+-independent manner. Due to its broad substrate selectivity, system L has been proposed to mediate the transport of amino-acid-related drugs across the blood-tissue barriers. We characterized L-leucine transport and its corresponding transporter in a human retinal pigment epithelial cell line (ARPE-19 cells) as an in vitro model of the outer blood-retinal barrier. [3H]L-leucine uptake by ARPE-19 cells took place in an Na+-, Cl(-)-independent and saturable manner with K(m) values of 8.71 and 220 microM. This process was more potently cis-inhibited by substrates of LAT1 than those of LAT2. [3H]L-leucine efflux from ARPE-19 cells was trans-stimulated by substrates of LAT1 and LAT2 through the obligatory exchange mechanism of system L. Although RT-PCR analysis demonstrated that LAT1 and LAT2 mRNA are expressed in ARPE-19 cells, the LAT1 mRNA concentration is 42-fold higher than that of LAT2. Moreover, immunoblot analysis demonstrated that LAT1 is expressed in ARPE-19 cells. In conclusion, although the transport function of LAT1 is greater than that of LAT2, LAT1 and LAT2 are involved in L-leucine transport in ARPE-19 cells.

Propulsion Investigation for Zero and Near-Zero Emissions Aircraft

NASA Technical Reports Server (NTRS)

Snyder, Christopher A.; Berton, Jeffrey J.; Brown, Gerald v.; Dolce, James L.; Dravid, Marayan V.; Eichenberg, Dennis J.; Freeh, Joshua E.; Gallo, Christopher A.; Jones, Scott M.; Kundu, Krishna P.;

Coexpression network analysis identifies transcriptional modules associated with genomic alterations in neuroblastoma.

PubMed

Yang, Liulin; Li, Yun; Wei, Zhi; Chang, Xiao

2018-06-01

Neuroblastoma is a highly complex and heterogeneous cancer in children. Acquired genomic alterations including MYCN amplification, 1p deletion and 11q deletion are important risk factors and biomarkers in neuroblastoma. Here, we performed a co-expression-based gene network analysis to study the intrinsic association between specific genomic changes and transcriptome organization. We identified multiple gene coexpression modules which are recurrent in two independent datasets and associated with functional pathways including nervous system development, cell cycle, immune system process and extracellular matrix/space. Our results also indicated that modules involved in nervous system development and cell cycle are highly associated with MYCN amplification and 1p deletion, while modules responding to immune system process are associated with MYCN amplification only. In summary, this integrated analysis provides novel insights into molecular heterogeneity and pathogenesis of neuroblastoma. This article is part of a Special Issue entitled: Accelerating Precision Medicine through Genetic and Genomic Big Data Analysis edited by Yudong Cai & Tao Huang. Copyright © 2017. Published by Elsevier B.V.

Field test analysis of concentrator photovoltaic system focusing on average photon energy and temperature

NASA Astrophysics Data System (ADS)

Husna, Husyira Al; Ota, Yasuyuki; Minemoto, Takashi; Nishioka, Kensuke

2015-08-01

The concentrator photovoltaic (CPV) system is unique and different from the common flat-plate PV system. It uses a multi-junction solar cell and a Fresnel lens to concentrate direct solar radiation onto the cell while tracking the sun throughout the day. The cell efficiency could reach over 40% under high concentration ratio. In this study, we analyzed a one year set of environmental condition data of the University of Miyazaki, Japan, where the CPV system was installed. Performance ratio (PR) was discussed to describe the system’s performance. Meanwhile, the average photon energy (APE) was used to describe the spectrum distribution at the site where the CPV system was installed. A circuit simulator network was used to simulate the CPV system electrical characteristics under various environmental conditions. As for the result, we found that the PR of the CPV systems depends on the APE level rather than the cell temperature.

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

PubMed Central

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

2009-01-01

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

Cell Adhesion Molecule and Lymphocyte Activation Marker Expression during Experimental Vaginal Candidiasis

PubMed Central

Wormley, Floyd L.; Chaiban, Joseph; Fidel, Paul L.

2001-01-01

Cell-mediated immunity by Th1-type CD4+ T cells is the predominant host defense mechanism against mucosal candidiasis. However, studies using an estrogen-dependent murine model of vaginal candidiasis have demonstrated little to no change in resident vaginal T cells during infection and no systemic T-cell infiltration despite the presence of Candida-specific systemic Th1-type responses in infected mice. The present study was designed to further investigate these observations by characterizing T-cell activation and cell adhesion molecule expression during primary and secondary C. albicans vaginal infections. While flow cytometry analysis of activation markers showed some evidence for activation of CD3+ draining lymph node and/or vaginal lymphocytes during both primary and secondary vaginal Candida infection, CD3+ cells expressing the homing receptors and integrins α4β7, αM290β7, and α4β1 in draining lymph nodes of mice with primary and secondary infections were reduced compared to results for uninfected mice. At the local level, few vaginal lymphocytes expressed integrins, with only minor changes observed during both primary and secondary infections. On the other hand, immunohistochemical analysis of vaginal cell adhesion molecule expression showed increases in mucosal addressin cell adhesion molecule 1 and vascular cell adhesion molecule 1 expression during both primary and secondary infections. Altogether, these data suggest that although the vaginal tissue is permissive to cellular infiltration during a vaginal Candida infection, the reduced numbers of systemic cells expressing the reciprocal cellular adhesion molecules may preempt cellular infiltration, thereby limiting Candida-specific T-cell responses against infection. PMID:11447188

Hybrid fuel cell/diesel generation total energy system, part 2

NASA Astrophysics Data System (ADS)

Blazek, C. F.

1982-11-01

Meeting the Goldstone Deep Space Communications Complex (DGSCC) electrical and thermal requirements with the existing system was compared with using fuel cells. Fuel cell technology selection was based on a 1985 time frame for installation. The most cost-effective fuel feedstock for fuel cell application was identified. Fuels considered included diesel oil, natural gas, methanol and coal. These fuel feedstocks were considered not only on the cost and efficiency of the fuel conversion process, but also on complexity and integration of the fuel processor on system operation and thermal energy availability. After a review of fuel processor technology, catalytic steam reformer technology was selected based on the ease of integration and the economics of hydrogen production. The phosphoric acid fuel cell was selected for application at the GDSCC due to its commercial readiness for near term application. Fuel cell systems were analyzed for both natural gas and methanol feedstock. The subsequent economic analysis indicated that a natural gas fueled system was the most cost effective of the cases analyzed.

Hybrid fuel cell/diesel generation total energy system, part 2

NASA Technical Reports Server (NTRS)

Blazek, C. F.

1982-01-01

Meeting the Goldstone Deep Space Communications Complex (DGSCC) electrical and thermal requirements with the existing system was compared with using fuel cells. Fuel cell technology selection was based on a 1985 time frame for installation. The most cost-effective fuel feedstock for fuel cell application was identified. Fuels considered included diesel oil, natural gas, methanol and coal. These fuel feedstocks were considered not only on the cost and efficiency of the fuel conversion process, but also on complexity and integration of the fuel processor on system operation and thermal energy availability. After a review of fuel processor technology, catalytic steam reformer technology was selected based on the ease of integration and the economics of hydrogen production. The phosphoric acid fuel cell was selected for application at the GDSCC due to its commercial readiness for near term application. Fuel cell systems were analyzed for both natural gas and methanol feedstock. The subsequent economic analysis indicated that a natural gas fueled system was the most cost effective of the cases analyzed.

Computational Model of Population Dynamics Based on the Cell Cycle and Local Interactions

NASA Astrophysics Data System (ADS)

Oprisan, Sorinel Adrian; Oprisan, Ana

2005-03-01

Our study bridges cellular (mesoscopic) level interactions and global population (macroscopic) dynamics of carcinoma. The morphological differences and transitions between well and smooth defined benign tumors and tentacular malignat tumors suggest a theoretical analysis of tumor invasion based on the development of mathematical models exhibiting bifurcations of spatial patterns in the density of tumor cells. Our computational model views the most representative and clinically relevant features of oncogenesis as a fight between two distinct sub-systems: the immune system of the host and the neoplastic system. We implemented the neoplastic sub-system using a three-stage cell cycle: active, dormant, and necrosis. The second considered sub-system consists of cytotoxic active (effector) cells — EC, with a very broad phenotype ranging from NK cells to CTL cells, macrophages, etc. Based on extensive numerical simulations, we correlated the fractal dimensions for carcinoma, which could be obtained from tumor imaging, with the malignat stage. Our computational model was able to also simulate the effects of surgical, chemotherapeutical, and radiotherapeutical treatments.

The role of radiation hard solar cells in minimizing the costs of global satellite communications systems

NASA Technical Reports Server (NTRS)

Summers, Geoffrey P.; Walters, Robert J.; Messenger, Scott R.; Burke, Edward A.

1995-01-01

An analysis embodied in a PC computer program is presented which quantitatively demonstrates how the availability of radiation hard solar cells can minimize the cost of a global satellite communication system. The chief distinction between the currently proposed systems, such as Iridium Odyssey and Ellipsat, is the number of satellites employed and their operating altitudes. Analysis of the major costs associated with implementing these systems shows that operation within the earth's radiation belts can reduce the total system cost by as much as a factor of two, so long as radiation hard components including solar cells, can be used. A detailed evaluation of several types of planar solar cells is given, including commercially available Si and GaAs/Ge cells, and InP/Si cells which are under development. The computer program calculates the end of life (EOL) power density of solar arrays taking into account the cell geometry, coverglass thickness, support frame, electrical interconnects, etc. The EOL power density can be determined for any altitude from low earth orbit (LEO) to geosynchronous (GEO) and for equatorial to polar planes of inclination. The mission duration can be varied over the entire range planned for the proposed satellite systems. An algorithm is included in the program for determining the degradation of cell efficiency for different cell technologies due to proton and electron irradiation. The program can be used to determine the optimum configuration for any cell technology for a particular orbit and for a specified mission life. Several examples of applying the program are presented, in which it is shown that the EOL power density of different technologies can vary by an order of magnitude for certain missions. Therefore, although a relatively radiation soft technology can be made to provide the required EOL power by simply increasing the size of the array, the impact on the total system budget could be unacceptable, due to increased launch and hardware costs. In aggregate these factors can account for more than a 10% increase in the total system cost. Since the estimated total costs of proposed global coverage systems range from $1 Billion to $9 Billion, the availability of radiation hard solar cells could make a decisive difference in the selection of a particular constellation architecture.

Chemokine receptor binding and signal transduction in native cells of the central nervous system.

PubMed

Davis, Christopher N; Chen, Shuzhen; Boehme, Stefen A; Bacon, Kevin B; Harrison, Jeffrey K

2003-04-01

Chemokine receptors belong to the superfamily of seven-transmembrane-spanning, G-protein-coupled receptors, and their expression by central nervous system cells is clearly documented. As this gene family has become the target of novel therapeutic development, the analysis of these receptors requires radioligand binding techniques as well as methods that entail assessing receptor stimulation of signal transduction pathways. Herein, we describe specific protocols for measuring radiolabeled chemokine binding to their cognate receptors on cultured glial cells as well as to receptors expressed in heterologous cell systems. Multiple downstream signaling pathways, including intracellular calcium influx and receptor-dependent kinase activation, are associated with chemokine receptor stimulation. Protocols for measuring these signaling events in chemokine-receptor-expressing cells are also presented.

New technologies for the human cytome project.

PubMed

Tárnok, A

2004-01-01

Cytomes or cell systems are composed of various kinds of single-cells and constitute the elementary building units of organs and organisms. Their individualised (cytomic) analysis overcomes the problem of averaged results from cell and tissue homogenates where molecular changes in low frequency cell populations may be hidden and wrongly interpreted. Analysis of the cytome is of pivotal importance in basic research for the understanding of cells and their interrelations in complex environments like tissues and in predictive medicine where it is a prerequisite for individualised preventive therapy. Analysis of molecular phenotypes requires instrumentation that on the one hand provides high-throughput measurement of individual cells and is on the other hand highly multiplexed, enabling the simultaneous acquisition of many parameters on the single cell level. Upcoming technology suitable to this task, such as slide based cytometry is available or under development. The realisation of cytomic technology is important for the realisation of the human cytome project.

Computational spatiotemporal analysis identifies WAVE2 and Cofilin as joint regulators of costimulation-mediated T cell actin dynamics

PubMed Central

Roybal, Kole T.; Buck, Taráz E.; Ruan, Xiongtao; Cho, Baek Hwan; Clark, Danielle J.; Ambler, Rachel; Tunbridge, Helen M.; Zhang, Jianwei; Verkade, Paul; Wülfing, Christoph; Murphy, Robert F.

2016-01-01

Fluorescence microscopy is one of the most important tools in cell biology research and it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells; however, given extensive cell-to-cell variation, methods do not currently exist to assemble these data into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. Here, we have developed one such method and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28 and we have determined how CD28 modulates actin dynamics. We imaged actin and eight core actin regulators under conditions where CD28 in the context of a strong TCR signal was engaged or blocked to yield over a thousand movies. Our computational analysis identified diminished recruitment of the activator of actin nucleation WAVE2 and the actin severing protein cofilin to F-actin as the dominant difference upon costimulation blockade. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics upon costimulation blockade. Thus we have developed and validated an approach to quantify protein distributions in time and space for analysis of complex regulatory systems. PMID:27095595

Flow Cytometric Analysis of Mononuclear Phagocytes in Nondiseased Human Lung and Lung-Draining Lymph Nodes.

PubMed

Desch, A Nicole; Gibbings, Sophie L; Goyal, Rajni; Kolde, Raivo; Bednarek, Joe; Bruno, Tullia; Slansky, Jill E; Jacobelli, Jordan; Mason, Robert; Ito, Yoko; Messier, Elise; Randolph, Gwendalyn J; Prabagar, Miglena; Atif, Shaikh M; Segura, Elodie; Xavier, Ramnik J; Bratton, Donna L; Janssen, William J; Henson, Peter M; Jakubzick, Claudia V

2016-03-15

The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors. Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs. We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood. Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics.

DIELECTROPHORESIS-BASED MICROFLUIDIC SEPARATION AND DETECTION SYSTEMS

PubMed Central

Yang, Jun; Vykoukal, Jody; Noshari, Jamileh; Becker, Frederick; Gascoyne, Peter; Krulevitch, Peter; Fuller, Chris; Ackler, Harold; Hamilton, Julie; Boser, Bernhard; Eldredge, Adam; Hitchens, Duncan; Andrews, Craig

2009-01-01

Diagnosis and treatment of human diseases frequently requires isolation and detection of certain cell types from a complex mixture. Compared with traditional separation and detection techniques, microfluidic approaches promise to yield easy-to-use diagnostic instruments tolerant of a wide range of operating environments and capable of accomplishing automated analyses. These approaches will enable diagnostic advances to be disseminated from sophisticated clinical laboratories to the point-of-care. Applications will include the separation and differential analysis of blood cell subpopulations for host-based detection of blood cell changes caused by disease, infection, or exposure to toxins, and the separation and analysis of surface-sensitized, custom dielectric beads for chemical, biological, and biomolecular targets. Here we report a new particle separation and analysis microsystem that uses dielectrophoretic field-flow fractionation (DEP-FFF). The system consists of a microfluidic chip with integrated sample injector, a DEP-FFF separator, and an AC impedance sensor. We show the design of a miniaturized impedance sensor integrated circuit (IC) with improved sensitivity, a new packaging approach for micro-flumes that features a slide-together compression package and novel microfluidic interconnects, and the design, control, integration and packaging of a fieldable prototype. Illustrative applications will be shown, including the separation of different sized beads and different cell types, blood cell differential analysis, and impedance sensing results for beads, spores and cells. PMID:22025905

Neil Popovich | NREL

Science.gov Websites

(SEPIO). His areas of expertise include low temperature geothermal power production systems; mineral recovery from geothermal brines; fuel cell systems analysis/manufacturing/engineering; instrumentation

VoroTop: Voronoi cell topology visualization and analysis toolkit

NASA Astrophysics Data System (ADS)

Lazar, Emanuel A.

2018-01-01

This paper introduces a new open-source software program called VoroTop, which uses Voronoi topology to analyze local structure in atomic systems. Strengths of this approach include its abilities to analyze high-temperature systems and to characterize complex structure such as grain boundaries. This approach enables the automated analysis of systems and mechanisms previously not possible.

A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis.

PubMed

Schmitter, Daniel; Wachowicz, Paulina; Sage, Daniel; Chasapi, Anastasia; Xenarios, Ioannis; Simanis; Unser, Michael

2013-01-01

The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. (Continued on next page) (Continued from previous page). "RodCellJ" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells. RodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html.

Systems analysis of apoptotic priming in ovarian cancer identifies vulnerabilities and predictors of drug response.

PubMed

Zervantonakis, Ioannis K; Iavarone, Claudia; Chen, Hsing-Yu; Selfors, Laura M; Palakurthi, Sangeetha; Liu, Joyce F; Drapkin, Ronny; Matulonis, Ursula; Leverson, Joel D; Sampath, Deepak; Mills, Gordon B; Brugge, Joan S

2017-08-28

The lack of effective chemotherapies for high-grade serous ovarian cancers (HGS-OvCa) has motivated a search for alternative treatment strategies. Here, we present an unbiased systems-approach to interrogate a panel of 14 well-annotated HGS-OvCa patient-derived xenografts for sensitivity to PI3K and PI3K/mTOR inhibitors and uncover cell death vulnerabilities. Proteomic analysis reveals that PI3K/mTOR inhibition in HGS-OvCa patient-derived xenografts induces both pro-apoptotic and anti-apoptotic signaling responses that limit cell killing, but also primes cells for inhibitors of anti-apoptotic proteins. In-depth quantitative analysis of BCL-2 family proteins and other apoptotic regulators, together with computational modeling and selective anti-apoptotic protein inhibitors, uncovers new mechanistic details about apoptotic regulators that are predictive of drug sensitivity (BIM, caspase-3, BCL-X L ) and resistance (MCL-1, XIAP). Our systems-approach presents a strategy for systematic analysis of the mechanisms that limit effective tumor cell killing and the identification of apoptotic vulnerabilities to overcome drug resistance in ovarian and other cancers.High-grade serous ovarian cancers (HGS-OvCa) frequently develop chemotherapy resistance. Here, the authors through a systematic analysis of proteomic and drug response data of 14 HGS-OvCa PDXs demonstrate that targeting apoptosis regulators can improve response of these tumors to inhibitors of the PI3K/mTOR pathway.

Molecular and cell biological effects of 3,5,3'-triiodothyronine on progenitor cells of the enteric nervous system in vitro.

PubMed

Mohr, Roland; Neckel, Peter; Zhang, Ying; Stachon, Susanne; Nothelfer, Katharina; Schaeferhoff, Karin; Obermayr, Florian; Bonin, Michael; Just, Lothar

2013-11-01

Thyroid hormones play important roles in the development of neural cells in the central nervous system. Even minor changes to normal thyroid hormone levels affect dendritic and axonal outgrowth, sprouting and myelination and might even lead to irreversible damages such as cretinism. Despite our knowledge of the influence on the mammalian CNS, the role of thyroid hormones in the development of the enteric nervous system (ENS) still needs to be elucidated. In this study we have analyzed for the first time the influence of 3,5,3'-triiodothyronine (T3) on ENS progenitor cells using cell biological assays and a microarray technique. In our in vitro model, T3 inhibited cell proliferation and stimulated neurite outgrowth of differentiating ENS progenitor cells. Microarray analysis revealed a group of 338 genes that were regulated by T3 in differentiating enterospheres. 67 of these genes are involved in function and development of the nervous system. 14 of them belong to genes that are involved in axonal guidance or neurite outgrowth. Interestingly, T3 regulated the expression of netrin G1 and endothelin 3, two guidance molecules that are involved in human enteric dysganglionoses. The results of our study give first insights how T3 may affect the enteric nervous system. T3 is involved in proliferation and differentiation processes in enterospheres. Microarray analysis revealed several interesting gene candidates that might be involved in the observed effects on enterosphere differentiation. Future studies need to be conducted to better understand the gene to gene interactions. © 2013.

Image analysis driven single-cell analytics for systems microbiology.

PubMed

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

2017-04-04

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

Redox flow cell development and demonstration project, calendar year 1976

NASA Technical Reports Server (NTRS)

1977-01-01

The major focus of the effort was the key technology issues that directly influence the fundamental feasibility of the overall redox concept. These issues were the development of a suitable semipermeable separator membrane for the system, the screening and study of candidate redox couples to achieve optimum cell performance, and the carrying out of systems analysis and modeling to develop system performance goals and cost estimates.

Modern Topics in Energy and Power Technical Meeting

DTIC Science & Technology

2016-09-01

systems are abysmally low, primarily due to their poor electronic structure. The III-V-based solar cells show the highest solar PV efficiency and thus are...initiatives include creating jet fuel based on seawater, research on photovoltaics ( PVs ) of different types, lightweight fuel cell systems for unmanned air...technoeconomic analysis studies indicate that a 20% solar -to-hydrogen PEC conversion efficiency is necessary for a commercially viable system. Additional

Theory and applications for optimization of every part of a photovoltaic system

NASA Technical Reports Server (NTRS)

Redfield, D.

1978-01-01

A general method is presented for quantitatively optimizing the design of every part and fabrication step of an entire photovoltaic system, based on the criterion of minimum cost/Watt for the system output power. It is shown that no element or process step can be optimized properly by considering only its own cost and performance. Moreover, a fractional performance loss at any fabrication step within the cell or array produces the same fractional increase in the cost/Watt of the entire array, but not of the full system. One general equation is found to be capable of optimizing all parts of a system, although the cell and array steps are basically different from the power-handling elements. Applications of this analysis are given to show (1) when Si wafers should be cut to increase their packing fraction; and (2) what the optimum dimensions for solar cell metallizations are. The optimum shadow fraction of the fine grid is shown to be independent of metal cost and resistivity as well as cell size. The optimum thicknesses of both the fine grid and the bus bar are substantially greater than the values in general use, and the total array cost has a major effect on these values. By analogy, this analysis is adaptable to other solar energy systems.

High Myeloperoxidase Positive Cell Infiltration in Colorectal Cancer Is an Independent Favorable Prognostic Factor

PubMed Central

Eppenberger-Castori, Serenella; Zlobec, Inti; Viehl, Carsten T.; Frey, Daniel M.; Nebiker, Christian A.; Rosso, Raffaele; Zuber, Markus; Amicarella, Francesca; Iezzi, Giandomenica; Sconocchia, Giuseppe; Heberer, Michael; Lugli, Alessandro; Tornillo, Luigi; Oertli, Daniel

2013-01-01

Background Colorectal cancer (CRC) infiltration by adaptive immune system cells correlates with favorable prognosis. The role of the innate immune system is still debated. Here we addressed the prognostic impact of CRC infiltration by neutrophil granulocytes (NG). Methods A TMA including healthy mucosa and clinically annotated CRC specimens (n = 1491) was stained with MPO and CD15 specific antibodies. MPO+ and CD15+ positive immune cells were counted by three independent observers. Phenotypic profiles of CRC infiltrating MPO+ and CD15+ cells were validated by flow cytometry on cell suspensions derived from enzymatically digested surgical specimens. Survival analysis was performed by splitting randomized data in training and validation subsets. Results MPO+ and CD15+ cell infiltration were significantly correlated (p<0.0001; r = 0.76). However, only high density of MPO+ cell infiltration was associated with significantly improved survival in training (P = 0.038) and validation (P = 0.002) sets. In multivariate analysis including T and N stage, vascular invasion, tumor border configuration and microsatellite instability status, MPO+ cell infiltration proved an independent prognostic marker overall (P = 0.004; HR = 0.65; CI:±0.15) and in both training (P = 0.048) and validation (P = 0.036) sets. Flow-cytometry analysis of CRC cell suspensions derived from clinical specimens showed that while MPO+ cells were largely CD15+/CD66b+, sizeable percentages of CD15+ and CD66b+ cells were MPO−. Conclusions High density MPO+ cell infiltration is a novel independent favorable prognostic factor in CRC. PMID:23734221

Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics.

PubMed

Roybal, Kole T; Buck, Taráz E; Ruan, Xiongtao; Cho, Baek Hwan; Clark, Danielle J; Ambler, Rachel; Tunbridge, Helen M; Zhang, Jianwei; Verkade, Paul; Wülfing, Christoph; Murphy, Robert F

2016-04-19

Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems. Copyright © 2016, American Association for the Advancement of Science.

Tilted pillar array fabrication by the combination of proton beam writing and soft lithography for microfluidic cell capture Part 2: Image sequence analysis based evaluation and biological application.

PubMed

Járvás, Gábor; Varga, Tamás; Szigeti, Márton; Hajba, László; Fürjes, Péter; Rajta, István; Guttman, András

2018-02-01

As a continuation of our previously published work, this paper presents a detailed evaluation of a microfabricated cell capture device utilizing a doubly tilted micropillar array. The device was fabricated using a novel hybrid technology based on the combination of proton beam writing and conventional lithography techniques. Tilted pillars offer unique flow characteristics and support enhanced fluidic interaction for improved immunoaffinity based cell capture. The performance of the microdevice was evaluated by an image sequence analysis based in-house developed single-cell tracking system. Individual cell tracking allowed in-depth analysis of the cell-chip surface interaction mechanism from hydrodynamic point of view. Simulation results were validated by using the hybrid device and the optimized surface functionalization procedure. Finally, the cell capture capability of this new generation microdevice was demonstrated by efficiently arresting cells from a HT29 cell-line suspension. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Soluble Protein Analysis using a Compact Bench-top Flow Cytometer

NASA Technical Reports Server (NTRS)

Pappas, Dimitri; Kao, Shib-Hsin; Cyr, Johnathan

2004-01-01

Future space exploration missions will require analytical technology capable of providing both autonomous medical care to the crew and investigative capabilities to researchers. While several promising candidate technologies exist for further development, flow cytometry is an attractive technology as it offers both crew health (blood cell count, leukocyte differential, etc.) and a wide array of biochemistry and immunology assays. research settings, the application of this technique to soluble protein analysis is also possible. Proteomic beads using fluorescent dyes for optical encoding were used to monitor six cytokines simultaneously in cell medium of cell cultures in stationary and rotating cell culture systems. The results of this work demonstrate that a compact flow cytometer, such as a system proposed for space flight, can detect a variety of soluble proteins for crew health and biotechnology experiments during long-term missions.

Establishment of an in vitro transcription system for Peste des petits ruminant virus.

PubMed

Yunus, Mohammad; Shaila, Melkote S

2012-12-05

Peste-des-petits ruminants virus (PPRV) is a non segmented negative strand RNA virus of the genus Morbillivirus within Paramyxoviridae family. Negative strand RNA viruses are known to carry nucleocapsid (N) protein, phospho (P) protein and RNA polymerase (L protein) packaged within the virion which possess all activities required for transcription, post-transcriptional modification of mRNA and replication. In order to understand the mechanism of transcription and replication of the virus, an in vitro transcription reconstitution system is required. In the present work, an in vitro transcription system has been developed with ribonucleoprotein (RNP) complex purified from virus infected cells as well as partially purified recombinant polymerase (L-P) complex from insect cells along with N-RNA (genomic RNA encapsidated by N protein) template isolated from virus infected cells. RNP complex isolated from virus infected cells and recombinant L-P complex purified from insect cells was used to reconstitute transcription on N-RNA template. The requirement for this transcription reconstitution has been defined. Transcription of viral genes in the in vitro system was confirmed by PCR amplification of cDNAs corresponding to individual transcripts using gene specific primers. In order to measure the relative expression level of viral transcripts, real time PCR analysis was carried out. qPCR analysis of the transcription products made in vitro showed a gradient of polarity of transcription from 3' end to 5' end of the genome similar to that exhibited by the virus in infected cells. This report describes for the first time, the development of an in vitro transcription reconstitution system for PPRV with RNP complex purified from infected cells and recombinant L-P complex expressed in insect cells. Both the complexes were able to synthesize all the mRNA species in vitro, exhibiting a gradient of polarity in transcription.

Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria

PubMed Central

Wienecke, Sarah; Ishwarbhai, Alka; Tsipa, Argyro; Aw, Rochelle; Kylilis, Nicolas; Bell, David J.; McClymont, David W.; Jensen, Kirsten; Biedendieck, Rebekka

2018-01-01

Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications. PMID:29666238

Setting the proportion of CD4+ and CD8+ T-cells co-cultured with canine macrophages infected with Leishmania chagasi.

PubMed

Viana, Kelvinson Fernandes; Aguiar-Soares, Rodrigo Dian Oliveira; Ker, Henrique Gama; Resende, Lucilene Aparecida; Souza-Fagundes, Elaine Maria; Dutra, Walderez Ornelas; Fujiwara, Ricardo Toshio; da Silveira-Lemos, Denise; Sant'Ana, Rita de Cássia Oliveira; Wardini, Amanda Brito; Araújo, Márcio Sobreira Silva; Martins-Filho, Olindo Assis; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro

2015-07-30

New methods for evaluating the canine immune system are necessary, not only to monitor immunological disorders, but also to provide insights for vaccine evaluations and therapeutic interventions, reducing the costs of assays using dog models, and provide a more rational way for analyzing the canine immune response. The present study intended to establish an in vitro toll to assess the parasitological/immunological status of dogs, applicable in pre-clinical trials of vaccinology, prognosis follow-up and therapeutics analysis of canine visceral leishmaniasis. We have evaluated the performance of co-culture systems of canine Leishmania chagasi-infected macrophages with different cell ratios of total lymphocytes or purified CD4(+) and CD8(+) T-cells. Peripheral blood mononuclear cells from uninfected dogs were used for the system set up. Employing the co-culture systems of L. chagasi-infected macrophages and purified CD4(+) or CD8(+) T-cell subsets we observed a microenvironment compatible with the expected status of the analyzed dogs. In this context, it was clearly demonstrated that, at this selected T-cell:target ratio, the adaptive immune response of uninfected dogs, composed by L. chagasi-unprimed T-cells was not able to perform the in vitro killing of L. chagasi-infected macrophages. Our data demonstrated that the co-culture system with T-cells from uninfected dogs at 1:5 and 1:2 ratio did not control the infection, yielding to patent in vitro parasitism (≥ 80%), low NO production (≤ 5 μM) and IL-10 modulated (IFN-γ/IL-10 ≤ 2) immunological profile in vitro. CD4(+) or CD8(+) T-cells at 1:5 or 1:2 ratio to L. chagasi-infected macrophages seems to be ideal for in vitro assays. This co-culture system may have great potential as a canine immunological analysis method, as well as in vaccine evaluations, prognosis follow-up and therapeutic interventions. Copyright © 2015 Elsevier B.V. All rights reserved.

Deciphering defective amelogenesis using in vitro culture systems.

PubMed

Arinawati, Dian Yosi; Miyoshi, Keiko; Tanimura, Ayako; Horiguchi, Taigo; Hagita, Hiroko; Noma, Takafumi

2018-04-01

The conventional two-dimensional (2D) in vitro culture system is frequently used to analyze the gene expression with or without extracellular signals. However, the cells derived from primary culture and cell lines frequently deviate the gene expression profile compared to the corresponding in vivo samples, which sometimes misleads the actual gene regulation in vivo. To overcome this gap, we developed the comparative 2D and 3D in vitro culture systems and applied them to the genetic study of amelogenesis imperfecta (AI) as a model. Recently, we found specificity protein 6 (Sp6) mutation in an autosomal-recessive AI rat that was previously named AMI. We constructed 3D structure of ARE-B30 cells (AMI-derived rat dental epithelial cells) or G5 (control wild type cells) combined with RPC-C2A cells (rat pulp cell line) separated by the collagen membrane, while in 2D structure, ARE-B30 or G5 was cultured with or without the collagen membrane. Comparative analysis of amelogenesis-related gene expression in ARE-B30 and G5 using our 2D and 3D in vitro systems revealed distinct expression profiles, showing the causative outcomes. Bone morphogenetic protein 2 and follistatin were reciprocally expressed in G5, but not in ARE-B30 cells. All-or-none expression of amelotin, kallikrein-related peptidase 4, and nerve growth factor receptor was observed in both cell types. In conclusion, our in vitro culture systems detected the phenotypical differences in the expression of the stage-specific amelogenesis-related genes. Parallel analysis with 2D and 3D culture systems may provide a platform to understand the molecular basis for defective amelogenesis caused by Sp6 mutation. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

[Morphometric analysis of lymphocyte nuclei in chronic lymphocytic leukemia].

PubMed

Ostapenko, V A; Kruchinskiĭ, N G; Smirnova, L A; Cherednik, A B; Nesterov, V N; Tepliakov, A I

1994-01-01

This work is dedicated to the study of use of quantitative analysis of cell nucleus structure for the analysis of peripheral blood lymphocytes in patients with chronic lymphocytic leukaemia. The structure of lymphocytic nuclei of healthy donors was evaluated by means of staining by toluidine blue purified cell suspensions smears. The preparations were analysed on the television measuring system "omnicon" with measurements of the following parameters: square of the nucleus, euchromatin, heterochromatin, and the ratio of heterochromatin and euchromatin squares. Actuarial analysis and nuclei classification of the previously mentioned parameters showed, that in peripheral blood of patients with chronic lymphocytic leukemia a large amount of atypical lymphocytes is present with reduced nucleus sizes. Atypical cells retain the ratio of structural components of chromatine, characteristic to normal cells, which show their low proliferative activity.

Automated measurement of cell motility and proliferation

PubMed Central

Bahnson, Alfred; Athanassiou, Charalambos; Koebler, Douglas; Qian, Lei; Shun, Tongying; Shields, Donna; Yu, Hui; Wang, Hong; Goff, Julie; Cheng, Tao; Houck, Raymond; Cowsert, Lex

2005-01-01

Background Time-lapse microscopic imaging provides a powerful approach for following changes in cell phenotype over time. Visible responses of whole cells can yield insight into functional changes that underlie physiological processes in health and disease. For example, features of cell motility accompany molecular changes that are central to the immune response, to carcinogenesis and metastasis, to wound healing and tissue regeneration, and to the myriad developmental processes that generate an organism. Previously reported image processing methods for motility analysis required custom viewing devices and manual interactions that may introduce bias, that slow throughput, and that constrain the scope of experiments in terms of the number of treatment variables, time period of observation, replication and statistical options. Here we describe a fully automated system in which images are acquired 24/7 from 384 well plates and are automatically processed to yield high-content motility and morphological data. Results We have applied this technology to study the effects of different extracellular matrix compounds on human osteoblast-like cell lines to explore functional changes that may underlie processes involved in bone formation and maintenance. We show dose-response and kinetic data for induction of increased motility by laminin and collagen type I without significant effects on growth rate. Differential motility response was evident within 4 hours of plating cells; long-term responses differed depending upon cell type and surface coating. Average velocities were increased approximately 0.1 um/min by ten-fold increases in laminin coating concentration in some cases. Comparison with manual tracking demonstrated the accuracy of the automated method and highlighted the comparative imprecision of human tracking for analysis of cell motility data. Quality statistics are reported that associate with stage noise, interference by non-cell objects, and uncertainty in the outlining and positioning of cells by automated image analysis. Exponential growth, as monitored by total cell area, did not linearly correlate with absolute cell number, but proved valuable for selection of reliable tracking data and for disclosing between-experiment variations in cell growth. Conclusion These results demonstrate the applicability of a system that uses fully automated image acquisition and analysis to study cell motility and growth. Cellular motility response is determined in an unbiased and comparatively high throughput manner. Abundant ancillary data provide opportunities for uniform filtering according to criteria that select for biological relevance and for providing insight into features of system performance. Data quality measures have been developed that can serve as a basis for the design and quality control of experiments that are facilitated by automation and the 384 well plate format. This system is applicable to large-scale studies such as drug screening and research into effects of complex combinations of factors and matrices on cell phenotype. PMID:15831094

Analyzing the dynamics of cell cycle processes from fixed samples through ergodic principles

PubMed Central

Wheeler, Richard John

2015-01-01

Tools to analyze cyclical cellular processes, particularly the cell cycle, are of broad value for cell biology. Cell cycle synchronization and live-cell time-lapse observation are widely used to analyze these processes but are not available for many systems. Simple mathematical methods built on the ergodic principle are a well-established, widely applicable, and powerful alternative analysis approach, although they are less widely used. These methods extract data about the dynamics of a cyclical process from a single time-point “snapshot” of a population of cells progressing through the cycle asynchronously. Here, I demonstrate application of these simple mathematical methods to analysis of basic cyclical processes—cycles including a division event, cell populations undergoing unicellular aging, and cell cycles with multiple fission (schizogony)—as well as recent advances that allow detailed mapping of the cell cycle from continuously changing properties of the cell such as size and DNA content. This includes examples using existing data from mammalian, yeast, and unicellular eukaryotic parasite cell biology. Through the ongoing advances in high-throughput cell analysis by light microscopy, electron microscopy, and flow cytometry, these mathematical methods are becoming ever more important and are a powerful complementary method to traditional synchronization and time-lapse cell cycle analysis methods. PMID:26543196

In situ electrochemical detection of embryonic stem cell differentiation.

PubMed

Yea, Cheol-Heon; An, Jeung Hee; Kim, Jungho; Choi, Jeong-Woo

2013-06-20

Stem cell sensors have emerged as a promising technique to electrochemically monitor the functional status and viability of stem cells. However, efficient electrochemical analysis techniques are required for the development of effective electrochemical stem cell sensors. In the current study, we report a newly developed electrochemical cyclic voltammetry (CV) system to determine the status of mouse embryonic stem (ES) cells. 1-Naphthly phosphate (1-NP), which was dephosphorylated by alkaline phosphatase into a 1-naphthol on an undifferentiated mouse ES cell, was used as a substrate to electrochemically monitor the differentiation status of mouse ES cells. The peak current in the cyclic voltammetry of 1-NP increased linearly with the concentration of pure 1-NP (R(2)=0.9623). On the other hand, the peak current in the electrochemical responses of 1-NP decreased as the number of undifferentiated ES cells increased. The increased dephosphorylation of 1-NP to 1-naphthol made a decreased electrochemical signal. Non-toxicity of 1-NP was confirmed. In conclusion, the proposed electrochemical analysis system can be applied to an electrical stem cell chip for diagnosis, drug detection and on-site monitoring. Copyright © 2013 Elsevier B.V. All rights reserved.

Skylab program CSM verification analysis report

NASA Technical Reports Server (NTRS)

Schaefer, J. L.; Vanderpol, G. A.

1970-01-01

The application of the SINDA computer program for the transient thermodynamic simulation of the Apollo fuel cell/radiator system for the limit condition of the proposed Skylab mission is described. Results are included for the thermal constraints imposed upon the Pratt and Whitney fuel cell power capability by the Block 2 EPS radiator system operating under the Skylab fixed attitude orbits.

A High-Resolution Minimicroscope System for Wireless Real-Time Monitoring.

PubMed

Wang, Zongjie; Boddeda, Akash; Parker, Benjamin; Samanipour, Roya; Ghosh, Sanjoy; Menard, Frederic; Kim, Keekyoung

2018-07-01

Compact, cost-effective, and high-performance microscope that enables the real-time imaging of cells and lab-on-a-chip devices is highly demanded for cell biology and biomedical engineering. This paper aims to present the design and application of an inexpensive wireless minimicroscope with resolution up to 2592 × 1944 pixels and speed up to 90 f/s. The minimicroscope system was built on a commercial embedded system (Raspberry Pi). We modified a camera module and adopted an inverse dual lens system to obtain the clear field of view and appropriate magnification for tens of micrometer objects. The system was capable of capturing time-lapse images and transferring image data wirelessly. The entire system can be operated wirelessly and cordlessly in a conventional cell culturing incubator. The developed minimicroscope was used to monitor the attachment and proliferation of NIH-3T3 and HEK 293 cells inside an incubator for 50 h. In addition, the minimicroscope was used to monitor a droplet generation process in a microfluidic device. The high-quality images captured by the minimicroscope enabled us an automated analysis of experimental parameters. The successful applications prove the great potential of the developed minimicroscope for monitoring various biological samples and microfluidic devices. This paper presents the design of a high-resolution minimicroscope system that enables the wireless real-time imaging of cells inside the incubator. This system has been verified to be a useful tool to obtain high-quality images and videos for the automated quantitative analysis of biological samples and lab-on-a-chip devices in the long term.

Protein glycosylation in diverse cell systems: implications for modification and analysis of recombinant proteins.

PubMed

Brooks, Susan A

2006-06-01

A major challenge for the biotechnology industry is to engineer the glycosylation pathways of expression systems to synthesize recombinant proteins with human glycosylation. Inappropriate glycosylation can result in reduced activity, limited half-life in circulation and unwanted immunogenicity. In this review, the complexities of glycosylation in human cells are explained and compared with glycosylation in bacteria, yeasts, fungi, insects, plants and nonhuman mammalian species. Key advances in the engineering of the glycosylation of expression systems are highlighted. Advances in the challenging and technically complex field of glycan analysis are also described. The emergence of a new generation of expression systems with sophisticated engineering for humanized glycosylation of glycoproteins appears to be on the horizon.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu Xiaozhong; Hong, Sung Woo; Moreira, Estefania G.

Gonocytes exist in the neonatal testis and represent a transient population of male germ-line stem cells. It has been shown that stem cell self-renewal and progeny production is probably controlled by the neighboring differentiated cells and extracellular matrix (ECM) in vivo known as niches. Recently, we developed an in vitro three-dimensional (3D) Sertoli cell/gonocyte co-culture (SGC) model with ECM overlay, which creates an in vivo-like niche and supports germ-line stem cell functioning within a 3D environment. In this study, we applied morphological and cytotoxicity evaluations, as well as microarray-based gene expression to examine the effects of different phthalate esters (PE)more » on this model. Known in vivo male developmentally toxic PEs (DTPE) and developmentally non-toxic PEs (DNTPE) were evaluated. We observed that DTPE induced significantly greater dose-dependent morphological changes, a decrease in cell viability and an increase in cytotoxicity compared to those treated with DNTPE. Moreover, the gene expression was more greatly altered by DTPE than by DNTPE and non-supervised cluster analysis allowed the discrimination of DTPE from the DNTPE. Our systems-based GO-Quant analysis showed significant alterations in the gene pathways involved in cell cycle, phosphate transport and apoptosis regulation with DTPE but not with DNTPE treatment. Disruptions of steroidogenesis related-gene expression such as Star, Cyp19a1, Hsd17b8, and Nr4a3 were observed in the DTPE group, but not in the DNTPE group. In summary, our observation on cell viability, cytotoxicity, and microarray-based gene expression analysis induced by PEs demonstrate that our in vitro 3D-SGC system mimicked in vivo responses for PEs and suggests that the 3D-SGC system might be useful in identifying developmental reproductive toxicants.« less

Selective heterogeneity in exoprotease production by Bacillus subtilis.

PubMed

Davidson, Fordyce A; Seon-Yi, Chung; Stanley-Wall, Nicola R

2012-01-01

Bacteria have elaborate signalling mechanisms to ensure a behavioural response that is most likely to enhance survival in a changing environment. It is becoming increasingly apparent that as part of this response, bacteria are capable of cell differentiation and can generate multiple, mutually exclusive co-existing cell states. These cell states are often associated with multicellular processes that bring benefit to the community as a whole but which may be, paradoxically, disadvantageous to an individual subpopulation. How this process of cell differentiation is controlled is intriguing and remains a largely open question. In this paper, we consider an important aspect of cell differentiation that is known to occur in the gram-positive bacterium Bacillus subtilis: we investigate the role of two master regulators DegU and Spo0A in the control of extra-cellular protease production. Recent work in this area focussed the on role of DegU in this process and suggested that transient effects in protein production were the drivers of cell-response heterogeneity. Here, using a combination of mathematical modelling, analysis and stochastic simulations, we provide a complementary analysis of this regulatory system that investigates the roles of both DegU and Spo0A in extra-cellular protease production. In doing so, we present a mechanism for bimodality, or system heterogeneity, without the need for a bistable switch in the underlying regulatory network. Moreover, our analysis leads us to conclude that this heterogeneity is in fact a persistent, stable feature. Our results suggest that system response is divided into three zones: low and high signal levels induce a unimodal or undifferentiated response from the cell population with all cells OFF and ON, respectively for exoprotease production. However, for intermediate levels of signal, a heterogeneous response is predicted with a spread of activity levels, representing typical "bet-hedging" behaviour.

Analysis of in vivo single cell behavior by high throughput, human-in-the-loop segmentation of three-dimensional images.

PubMed

Chiang, Michael; Hallman, Sam; Cinquin, Amanda; de Mochel, Nabora Reyes; Paz, Adrian; Kawauchi, Shimako; Calof, Anne L; Cho, Ken W; Fowlkes, Charless C; Cinquin, Olivier

2015-11-25

Analysis of single cells in their native environment is a powerful method to address key questions in developmental systems biology. Confocal microscopy imaging of intact tissues, followed by automatic image segmentation, provides a means to conduct cytometric studies while at the same time preserving crucial information about the spatial organization of the tissue and morphological features of the cells. This technique is rapidly evolving but is still not in widespread use among research groups that do not specialize in technique development, perhaps in part for lack of tools that automate repetitive tasks while allowing experts to make the best use of their time in injecting their domain-specific knowledge. Here we focus on a well-established stem cell model system, the C. elegans gonad, as well as on two other model systems widely used to study cell fate specification and morphogenesis: the pre-implantation mouse embryo and the developing mouse olfactory epithelium. We report a pipeline that integrates machine-learning-based cell detection, fast human-in-the-loop curation of these detections, and running of active contours seeded from detections to segment cells. The procedure can be bootstrapped by a small number of manual detections, and outperforms alternative pieces of software we benchmarked on C. elegans gonad datasets. Using cell segmentations to quantify fluorescence contents, we report previously-uncharacterized cell behaviors in the model systems we used. We further show how cell morphological features can be used to identify cell cycle phase; this provides a basis for future tools that will streamline cell cycle experiments by minimizing the need for exogenous cell cycle phase labels. High-throughput 3D segmentation makes it possible to extract rich information from images that are routinely acquired by biologists, and provides insights - in particular with respect to the cell cycle - that would be difficult to derive otherwise.

System-wide analysis of the transcriptional network of human myelomonocytic leukemia cells predicts attractor structure and phorbol-ester-induced differentiation and dedifferentiation transitions

NASA Astrophysics Data System (ADS)

Sakata, Katsumi; Ohyanagi, Hajime; Sato, Shinji; Nobori, Hiroya; Hayashi, Akiko; Ishii, Hideshi; Daub, Carsten O.; Kawai, Jun; Suzuki, Harukazu; Saito, Toshiyuki

2015-02-01

We present a system-wide transcriptional network structure that controls cell types in the context of expression pattern transitions that correspond to cell type transitions. Co-expression based analyses uncovered a system-wide, ladder-like transcription factor cluster structure composed of nearly 1,600 transcription factors in a human transcriptional network. Computer simulations based on a transcriptional regulatory model deduced from the system-wide, ladder-like transcription factor cluster structure reproduced expression pattern transitions when human THP-1 myelomonocytic leukaemia cells cease proliferation and differentiate under phorbol myristate acetate stimulation. The behaviour of MYC, a reprogramming Yamanaka factor that was suggested to be essential for induced pluripotent stem cells during dedifferentiation, could be interpreted based on the transcriptional regulation predicted by the system-wide, ladder-like transcription factor cluster structure. This study introduces a novel system-wide structure to transcriptional networks that provides new insights into network topology.

Single-cell resolution of intracellular T cell Ca2+ dynamics in response to frequency-based H2O2 stimulation.

PubMed

Kniss-James, Ariel S; Rivet, Catherine A; Chingozha, Loice; Lu, Hang; Kemp, Melissa L

2017-03-01

Adaptive immune cells, such as T cells, integrate information from their extracellular environment through complex signaling networks with exquisite sensitivity in order to direct decisions on proliferation, apoptosis, and cytokine production. These signaling networks are reliant on the interplay between finely tuned secondary messengers, such as Ca 2+ and H 2 O 2 . Frequency response analysis, originally developed in control engineering, is a tool used for discerning complex networks. This analytical technique has been shown to be useful for understanding biological systems and facilitates identification of the dominant behaviour of the system. We probed intracellular Ca 2+ dynamics in the frequency domain to investigate the complex relationship between two second messenger signaling molecules, H 2 O 2 and Ca 2+ , during T cell activation with single cell resolution. Single-cell analysis provides a unique platform for interrogating and monitoring cellular processes of interest. We utilized a previously developed microfluidic device to monitor individual T cells through time while applying a dynamic input to reveal a natural frequency of the system at approximately 2.78 mHz stimulation. Although our network was much larger with more unknown connections than previous applications, we are able to derive features from our data, observe forced oscillations associated with specific amplitudes and frequencies of stimuli, and arrive at conclusions about potential transfer function fits as well as the underlying population dynamics.

Evaluation of the automated hematology analyzer ADVIA® 120 for cerebrospinal fluid analysis and usage of unique hemolysis reagent.

PubMed

Tanada, H; Ikemoto, T; Masutani, R; Tanaka, H; Takubo, T

2014-02-01

In this study, we evaluated the performance of the ADVIA 120 hematology system for cerebrospinal fluid (CSF) assay. Cell counts and leukocyte differentials in CSF were examined with the ADVIA 120 hematology system, while simultaneously confirming an effective hemolysis agent for automated CSF cell counts. The detection limits of both white blood cell (WBC) counts and red blood cell (RBC) counts on the measurement of CSF cell counts by the ADVIA 120 hematology system were superior at 2 cells/μL (10(-6) L). The WBC count was linear up to 9.850 cells/μL, and the RBC count was linear up to approximately 20 000 cells/μL. The intrarun reproducibility indicated good precision. The leukocyte differential of CSF cells, performed by the ADVIA120 hematology system, showed good correlation with the microscopic procedure. The VersaLyse hemolysis solution efficiently lysed the samples without interfering with cell counts and leukocyte differential, even in a sample that included approximately 50 000/μL RBC. These data show the ADVIA 120 hematology system correctly measured the WBC count and leukocyte differential in CSF. The VersaLyse hemolysis solution is considered to be optimal for hemolysis treatment of CSF when measuring cell counts and differentials by the ADVIA 120 hematology system. © 2013 John Wiley & Sons Ltd.

Validation of cytogenetic risk groups according to International Prognostic Scoring Systems by peripheral blood CD34+FISH: results from a German diagnostic study in comparison with an international control group

PubMed Central

Braulke, Friederike; Platzbecker, Uwe; Müller-Thomas, Catharina; Götze, Katharina; Germing, Ulrich; Brümmendorf, Tim H.; Nolte, Florian; Hofmann, Wolf-Karsten; Giagounidis, Aristoteles A. N.; Lübbert, Michael; Greenberg, Peter L.; Bennett, John M.; Solé, Francesc; Mallo, Mar; Slovak, Marilyn L.; Ohyashiki, Kazuma; Le Beau, Michelle M.; Tüchler, Heinz; Pfeilstöcker, Michael; Nösslinger, Thomas; Hildebrandt, Barbara; Shirneshan, Katayoon; Aul, Carlo; Stauder, Reinhard; Sperr, Wolfgang R.; Valent, Peter; Fonatsch, Christa; Trümper, Lorenz; Haase, Detlef; Schanz, Julie

2015-01-01

International Prognostic Scoring Systems are used to determine the individual risk profile of myelodysplastic syndrome patients. For the assessment of International Prognostic Scoring Systems, an adequate chromosome banding analysis of the bone marrow is essential. Cytogenetic information is not available for a substantial number of patients (5%–20%) with dry marrow or an insufficient number of metaphase cells. For these patients, a valid risk classification is impossible. In the study presented here, the International Prognostic Scoring Systems were validated based on fluorescence in situ hybridization analyses using extended probe panels applied to cluster of differentiation 34 positive (CD34+) peripheral blood cells of 328 MDS patients of our prospective multicenter German diagnostic study and compared to chromosome banding results of 2902 previously published patients with myelodysplastic syndromes. For cytogenetic risk classification by fluorescence in situ hybridization analyses of CD34+ peripheral blood cells, the groups differed significantly for overall and leukemia-free survival by uni- and multivariate analyses without discrepancies between treated and untreated patients. Including cytogenetic data of fluorescence in situ hybridization analyses of peripheral CD34+ blood cells (instead of bone marrow banding analysis) into the complete International Prognostic Scoring System assessment, the prognostic risk groups separated significantly for overall and leukemia-free survival. Our data show that a reliable stratification to the risk groups of the International Prognostic Scoring Systems is possible from peripheral blood in patients with missing chromosome banding analysis by using a comprehensive probe panel (clinicaltrials.gov identifier:01355913). PMID:25344522

Laser capture microdissection: should an ultraviolet or infrared laser be used?

PubMed

Vandewoestyne, Mado; Goossens, Karen; Burvenich, Christian; Van Soom, Ann; Peelman, Luc; Deforce, Dieter

2013-08-15

Laser capture microdissection (LCM) is a well-established cell separation technique. It combines microscopy with laser beam technology and allows targeting of specific cells or tissue regions that need to be separated from others. Consequently, this biological material can be used for genome or transcriptome analyses. Appropriate methods of sample preparation, however, are crucial for the success of downstream molecular analysis. The aim of this study was to objectively compare the two main LCM systems, one based on an ultraviolet (UV) laser and the other based on an infrared (IR) laser, on different criteria ranging from user-friendliness to sample quality. The comparison was performed on two types of samples: peripheral blood mononuclear cells and blastocysts. The UV laser LCM system had several advantages over the IR laser LCM system. Not only does the UV system allow faster and more precise sample collection, but also the obtained samples-even single cell samples-can be used for DNA extraction and downstream polymerase chain reaction (PCR) applications. RNA-based applications are more challenging for both LCM systems. Although sufficient RNA can be extracted from as few as 10 cells for reverse transcription quantitative PCR (RT-qPCR) analysis, the low RNA quality should be taken into account when designing the RT-qPCR assays. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Trends in the development of microfluidic cell biochips for in vitro hepatotoxicity.

PubMed

Baudoin, Régis; Corlu, Anne; Griscom, Laurent; Legallais, Cécile; Leclerc, Eric

2007-06-01

Current developments in the technological fields of liver tissue engineering, bioengineering, biomechanics, microfabrication and microfluidics have lead to highly complex and pertinent new tools called "cell biochips" for in vitro toxicology. The purpose of "cell biochips" is to mimic organ tissues in vitro in order to partially reduce the amount of in vivo testing. These "cell biochips" consist of microchambers containing engineered tissue and living cell cultures interconnected by a microfluidic network, which allows the control of microfluidic flows for dynamic cultures, by continuous feeding of nutrients to cultured cells and waste removal. Cell biochips also allow the control of physiological contact times of diluted molecules with the tissues and cells, for rapid testing of sample preparations or specific addressing. Cell biochips can be situated between in vitro and in vivo testing. These types of systems can enhance functionality of cells by mimicking the tissue architecture complexities when compared to in vitro analysis but at the same time present a more rapid and simple process when compared to in vivo testing procedures. In this paper, we first introduce the concepts of microfluidic and biochip systems based on recent progress in microfabrication techniques used to mimic liver tissue in vitro. This includes progress and understanding in biomaterials science (cell culture substrate), biomechanics (dynamic cultures conditions) and biology (tissue engineering). The development of new "cell biochips" for chronic toxicology analysis of engineered tissues can be achieved through the combination of these research domains. Combining these advanced research domains, we then present "cell biochips" that allow liver chronic toxicity analysis in vitro on engineered tissues. An extension of the "cell biochip" idea has also allowed "organ interactions on chip", which can be considered as a first step towards the replacement of animal testing using a combined liver/lung organ model.

BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models

PubMed Central

Bilgin, Cemal Cagatay; Fontenay, Gerald; Cheng, Qingsu; Chang, Hang; Han, Ju; Parvin, Bahram

2016-01-01

BioSig3D is a computational platform for high-content screening of three-dimensional (3D) cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i) morphogenesis of a panel of human mammary epithelial cell lines (HMEC), and (ii) heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation. PMID:26978075

Soft-state biomicrofluidic pulse generator for single cell analysis

NASA Astrophysics Data System (ADS)

Sabounchi, Poorya; Ionescu-Zanetti, Cristian; Chen, Roger; Karandikar, Manjiree; Seo, Jeonggi; Lee, Luke P.

2006-05-01

We present the design, fabrication, and characterization of a soft-state biomicrofluidic pulse generator for single cell analysis. Hydrodynamic cell trapping via lateral microfluidic junctions allows the trapping of single cells from a bulk suspension. Microfluidic injection sites adjacent to the cell-trapping channels enable the pulsed delivery of nanoliter volumes of biochemical reagent. We demonstrated the application and removal of reagent at a frequency of 10Hz with a rise time of less than 33ms and a reagent consumption rate of 0.2nL/s. It is shown that this system operates as a low-pass filter with a cutoff frequency of 7Hz.

Significantly improved precision of cell migration analysis in time-lapse video microscopy through use of a fully automated tracking system

PubMed Central

2010-01-01

Background Cell motility is a critical parameter in many physiological as well as pathophysiological processes. In time-lapse video microscopy, manual cell tracking remains the most common method of analyzing migratory behavior of cell populations. In addition to being labor-intensive, this method is susceptible to user-dependent errors regarding the selection of "representative" subsets of cells and manual determination of precise cell positions. Results We have quantitatively analyzed these error sources, demonstrating that manual cell tracking of pancreatic cancer cells lead to mis-calculation of migration rates of up to 410%. In order to provide for objective measurements of cell migration rates, we have employed multi-target tracking technologies commonly used in radar applications to develop fully automated cell identification and tracking system suitable for high throughput screening of video sequences of unstained living cells. Conclusion We demonstrate that our automatic multi target tracking system identifies cell objects, follows individual cells and computes migration rates with high precision, clearly outperforming manual procedures. PMID:20377897

Magnetomicrofluidics Circuits for Organizing Bioparticle Arrays

NASA Astrophysics Data System (ADS)

Abedini-Nassab, Roozbeh

Single-cell analysis (SCA) tools have important applications in the analysis of phenotypic heterogeneity, which is difficult or impossible to analyze in bulk cell culture or patient samples. SCA tools thus have a myriad of applications ranging from better credentialing of drug therapies to the analysis of rare latent cells harboring HIV infection or in Cancer. However, existing SCA systems usually lack the required combination of programmability, flexibility, and scalability necessary to enable the study of cell behaviors and cell-cell interactions at the scales sufficient to analyze extremely rare events. To advance the field, I have developed a novel, programmable, and massively-parallel SCA tool which is based on the principles of computer circuits. By integrating these magnetic circuits with microfluidics channels, I developed a platform that can organize a large number of single particles into an array in a controlled manner. My magnetophoretic circuits use passive elements constructed in patterned magnetic thin films to move cells along programmed tracks with an external rotating magnetic field. Cell motion along these tracks is analogous to the motion of charges in an electrical conductor, following a rule similar to Ohm's law. I have also developed asymmetric conductors, similar to electrical diodes, and storage sites for cells that behave similarly to electrical capacitors. I have also developed magnetophoretic circuits which use an overlaid pattern of microwires to switch single cells between different tracks. This switching mechanism, analogous to the operation of electronic transistors, is achieved by establishing a semiconducting gap in the magnetic pattern which can be changed from an insulating state to a conducting state by application of electrical current to an overlaid electrode. I performed an extensive study on the operation of transistors to optimize their geometry and minimize the required gate currents. By combining these elements into integrated circuits, I have built devices which are capable of organizing a precise number of cells into individually addressable array sites, similar to how a random access memory (RAM) stores electronic data. My programmable magnetic circuits allow for the organization of both cells and single-cell pairs into large arrays. Single cells can also potentially be retrieved for downstream high-throughput genomic analysis. In order to enhance the efficiency of the tool and to increase the delivery speed of the particles, I have also developed microfluidics systems that are combined with the magnetophoretic circuits. This hybrid system, called magnetomicrofluidics, is capable of rapidly organizing an array of particles and cells with the high precision and control. I have also shown that cells can be grown inside these chips for multiple days, enabling the long-term phenotypic analysis of rare cellular events. These types of studies can reveal important insights about the intercellular signaling networks and answer crucial questions in biology and immunology.

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

PubMed

Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

2017-06-15

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. Copyright © 2017 American Society for Microbiology.

Discovery of functional interactions among actin regulators by analysis of image fluctuations in an unperturbed motile cell system.

PubMed

Isogai, Tadamoto; Danuser, Gaudenz

2018-05-26

Cell migration is driven by propulsive forces derived from polymerizing actin that pushes and extends the plasma membrane. The underlying actin network is constantly undergoing adaptation to new mechano-chemical environments and intracellular conditions. As such, mechanisms that regulate actin dynamics inherently contain multiple feedback loops and redundant pathways. Given the highly adaptable nature of such a system, studies that use only perturbation experiments (e.g. knockdowns, overexpression, pharmacological activation/inhibition, etc.) are challenged by the nonlinearity and redundancy of the pathway. In these pathway configurations, perturbation experiments at best describe the function(s) of a molecular component in an adapting (e.g. acutely drug-treated) or fully adapted (e.g. permanent gene silenced) cell system, where the targeted component now resides in a non-native equilibrium. Here, we propose how quantitative live-cell imaging and analysis of constitutive fluctuations of molecular activities can overcome these limitations. We highlight emerging actin filament barbed-end biology as a prime example of a complex, nonlinear molecular process that requires a fluctuation analytic approach, especially in an unperturbed cellular system, to decipher functional interactions of barbed-end regulators, actin polymerization and membrane protrusion.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

High-efficiency concentration/multi-solar-cell system for orbital power generation

NASA Technical Reports Server (NTRS)

Onffroy, J. R.; Stoltzmann, D. E.; Lin, R. J. H.; Knowles, G. R.

1980-01-01

An analysis was performed to determine the economic feasibility of a concentrating spectrophotovoltaic orbital electrical power generation system. In this system dichroic beam-splitting mirrors are used to divide the solar spectrum into several wavebands. Absorption of these wavebands by solar cells with matched energy bandgaps increases the cell efficiency while decreasing the amount of heat which must be rejected. The optical concentration is performed in two stages. The first concentration stage employs a Cassegrain-type telescope, resulting in a short system length. The output from this stage is directed to compound parabolic concentrators which comprise the second stage of concentration. Ideal efficiencies for one-, two-, three-, and four-cell systems were calculated under 1000 sun, AMO conditions, and optimum energy bands were determined. Realistic efficiencies were calculated for various combinations of Si, GaAs, Ge and GaP. Efficiencies of 32 to 33 percent were obtained with the multicell systems. The optimum system consists of an f/3.5 optical system, a beam splitter to divide the spectrum at 0.9 microns, and two solar cell arrays, GaAs and Si.

Visual analysis of mass cytometry data by hierarchical stochastic neighbour embedding reveals rare cell types.

PubMed

van Unen, Vincent; Höllt, Thomas; Pezzotti, Nicola; Li, Na; Reinders, Marcel J T; Eisemann, Elmar; Koning, Frits; Vilanova, Anna; Lelieveldt, Boudewijn P F

2017-11-23

Mass cytometry allows high-resolution dissection of the cellular composition of the immune system. However, the high-dimensionality, large size, and non-linear structure of the data poses considerable challenges for the data analysis. In particular, dimensionality reduction-based techniques like t-SNE offer single-cell resolution but are limited in the number of cells that can be analyzed. Here we introduce Hierarchical Stochastic Neighbor Embedding (HSNE) for the analysis of mass cytometry data sets. HSNE constructs a hierarchy of non-linear similarities that can be interactively explored with a stepwise increase in detail up to the single-cell level. We apply HSNE to a study on gastrointestinal disorders and three other available mass cytometry data sets. We find that HSNE efficiently replicates previous observations and identifies rare cell populations that were previously missed due to downsampling. Thus, HSNE removes the scalability limit of conventional t-SNE analysis, a feature that makes it highly suitable for the analysis of massive high-dimensional data sets.

Extracting topographic structure from digital elevation data for geographic information-system analysis

USGS Publications Warehouse

Jenson, Susan K.; Domingue, Julia O.

1988-01-01

The first phase of analysis is a conditioning phase that generates three data sets: the original OEM with depressions filled, a data set indicating the flow direction for each cell, and a flow accumulation data set in which each cell receives a value equal to the total number of cells that drain to it. The original OEM and these three derivative data sets can then be processed in a variety of ways to optionally delineate drainage networks, overland paths, watersheds for userspecified locations, sub-watersheds for the major tributaries of a drainage network, or pour point linkages between watersheds. The computer-generated drainage lines and watershed polygons and the pour point linkage information can be transferred to vector-based geographic information systems for futher analysis. Comparisons between these computergenerated features and their manually delineated counterparts generally show close agreement, indicating that these software tools will save analyst time spent in manual interpretation and digitizing.

Quantitative assessment of neurite outgrowth in human embryonic stem cell derived hN2 cells using automated high-content image analysis

EPA Science Inventory

Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary ne...

Analysis of Normal Human Mammary Epigenomes Reveals Cell-Specific Active Enhancer States and Associated Transcription Factor Networks.

PubMed

Pellacani, Davide; Bilenky, Misha; Kannan, Nagarajan; Heravi-Moussavi, Alireza; Knapp, David J H F; Gakkhar, Sitanshu; Moksa, Michelle; Carles, Annaick; Moore, Richard; Mungall, Andrew J; Marra, Marco A; Jones, Steven J M; Aparicio, Samuel; Hirst, Martin; Eaves, Connie J

2016-11-15

The normal adult human mammary gland is a continuous bilayered epithelial system. Bipotent and myoepithelial progenitors are prominent and unique components of the outer (basal) layer. The inner (luminal) layer includes both luminal-restricted progenitors and a phenotypically separable fraction that lacks progenitor activity. We now report an epigenomic comparison of these three subsets with one another, with their associated stromal cells, and with three immortalized, non-tumorigenic human mammary cell lines. Each genome-wide analysis contains profiles for six histone marks, methylated DNA, and RNA transcripts. Analysis of these datasets shows that each cell type has unique features, primarily within genomic regulatory regions, and that the cell lines group together. Analyses of the promoter and enhancer profiles place the luminal progenitors in between the basal cells and the non-progenitor luminal subset. Integrative analysis reveals networks of subset-specific transcription factors. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy.

PubMed

Wang, Quanli; Niemi, Jarad; Tan, Chee-Meng; You, Lingchong; West, Mike

2010-01-01

An increasingly common component of studies in synthetic and systems biology is analysis of dynamics of gene expression at the single-cell level, a context that is heavily dependent on the use of time-lapse movies. Extracting quantitative data on the single-cell temporal dynamics from such movies remains a major challenge. Here, we describe novel methods for automating key steps in the analysis of single-cell, fluorescent images-segmentation and lineage reconstruction-to recognize and track individual cells over time. The automated analysis iteratively combines a set of extended morphological methods for segmentation, and uses a neighborhood-based scoring method for frame-to-frame lineage linking. Our studies with bacteria, budding yeast and human cells, demonstrate the portability and usability of these methods, whether using phase, bright field or fluorescent images. These examples also demonstrate the utility of our integrated approach in facilitating analyses of engineered and natural cellular networks in diverse settings. The automated methods are implemented in freely available, open-source software.

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Economics of Direct Hydrogen Polymer Electrolyte Membrane Fuel Cell Systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mahadevan, Kathyayani

Battelle's Economic Analysis of PEM Fuel Cell Systems project was initiated in 2003 to evaluate the technology and markets that are near-term and potentially could support the transition to fuel cells in automotive markets. The objective of Battelle?s project was to assist the DOE in developing fuel cell systems for pre-automotive applications by analyzing the technical, economic, and market drivers of direct hydrogen PEM fuel cell adoption. The project was executed over a 6-year period (2003 to 2010) and a variety of analyses were completed in that period. The analyses presented in the final report include: Commercialization scenarios for stationarymore » generation through 2015 (2004); Stakeholder feedback on technology status and performance status of fuel cell systems (2004); Development of manufacturing costs of stationary PEM fuel cell systems for backup power markets (2004); Identification of near-term and mid-term markets for PEM fuel cells (2006); Development of the value proposition and market opportunity of PEM fuel cells in near-term markets by assessing the lifecycle cost of PEM fuel cells as compared to conventional alternatives used in the marketplace and modeling market penetration (2006); Development of the value proposition of PEM fuel cells in government markets (2007); Development of the value proposition and opportunity for large fuel cell system application at data centers and wastewater treatment plants (2008); Update of the manufacturing costs of PEM fuel cells for backup power applications (2009).« less

High purity microfluidic sorting and analysis of circulating tumor cells: towards routine mutation detection.

PubMed

Autebert, Julien; Coudert, Benoit; Champ, Jérôme; Saias, Laure; Guneri, Ezgi Tulukcuoglu; Lebofsky, Ronald; Bidard, François-Clément; Pierga, Jean-Yves; Farace, Françoise; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis

2015-05-07

A new generation of the Ephesia cell capture technology optimized for CTC capture and genetic analysis is presented, characterized in depth and compared with the CellSearch system as a reference. This technology uses magnetic particles bearing tumour-cell specific EpCAM antibodies, self-assembled in a regular array in a microfluidic flow cell. 48,000 high aspect-ratio columns are generated using a magnetic field in a high throughput (>3 ml h(-1)) device and act as sieves to specifically capture the cells of interest through antibody-antigen interactions. Using this device optimized for CTC capture and analysis, we demonstrated the capture of epithelial cells with capture efficiency above 90% for concentrations as low as a few cells per ml. We showed the high specificity of capture with only 0.26% of non-epithelial cells captured for concentrations above 10 million cells per ml. We investigated the capture behavior of cells in the device, and correlated the cell attachment rate with the EpCAM expression on the cell membranes for six different cell lines. We developed and characterized a two-step blood processing method to allow for rapid processing of 10 ml blood tubes in less than 4 hours, and showed a capture rate of 70% for as low as 25 cells spiked in 10 ml blood tubes, with less than 100 contaminating hematopoietic cells. Using this device and procedure, we validated our system on patient samples using an automated cell immunostaining procedure and a semi-automated cell counting method. Our device captured CTCs in 75% of metastatic prostate cancer patients and 80% of metastatic breast cancer patients, and showed similar or better results than the CellSearch device in 10 out of 13 samples. Finally, we demonstrated the possibility of detecting cancer-related PIK3CA gene mutation in 20 cells captured in the chip with a good correlation between the cell count and the quantitation value Cq of the post-capture qPCR.

Banning standard cell engineering notebook

NASA Technical Reports Server (NTRS)

1976-01-01

A family of standardized thick-oxide P-MOS building blocks (standard cells) is described. The information is presented in a form useful for systems designs, logic design, and the preparation of inputs to both sets of Design Automation programs for array design and analysis. A data sheet is provided for each cell and gives the cell name, the cell number, its logic symbol, Boolean equation, truth table, circuit schematic circuit composite, input-output capacitances, and revision date. The circuit type file, also given for each cell, together with the logic drawing contained on the data sheet provides all the information required to prepare input data files for the Design Automation Systems. A detailed description of the electrical design procedure is included.

Single-Cell RNA Sequencing of Glioblastoma Cells.

PubMed

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

2018-01-01

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

Mathematical modeling of solid cancer growth with angiogenesis

PubMed Central

2012-01-01

Background Cancer arises when within a single cell multiple malfunctions of control systems occur, which are, broadly, the system that promote cell growth and the system that protect against erratic growth. Additional systems within the cell must be corrupted so that a cancer cell, to form a mass of any real size, produces substances that promote the growth of new blood vessels. Multiple mutations are required before a normal cell can become a cancer cell by corruption of multiple growth-promoting systems. Methods We develop a simple mathematical model to describe the solid cancer growth dynamics inducing angiogenesis in the absence of cancer controlling mechanisms. Results The initial conditions supplied to the dynamical system consist of a perturbation in form of pulse: The origin of cancer cells from normal cells of an organ of human body. Thresholds of interacting parameters were obtained from the steady states analysis. The existence of two equilibrium points determine the strong dependency of dynamical trajectories on the initial conditions. The thresholds can be used to control cancer. Conclusions Cancer can be settled in an organ if the following combination matches: better fitness of cancer cells, decrease in the efficiency of the repairing systems, increase in the capacity of sprouting from existing vascularization, and higher capacity of mounting up new vascularization. However, we show that cancer is rarely induced in organs (or tissues) displaying an efficient (numerically and functionally) reparative or regenerative mechanism. PMID:22300422

High content analysis of phagocytic activity and cell morphology with PuntoMorph.

PubMed

Al-Ali, Hassan; Gao, Han; Dalby-Hansen, Camilla; Peters, Vanessa Ann; Shi, Yan; Brambilla, Roberta

2017-11-01

Phagocytosis is essential for maintenance of normal homeostasis and healthy tissue. As such, it is a therapeutic target for a wide range of clinical applications. The development of phenotypic screens targeting phagocytosis has lagged behind, however, due to the difficulties associated with image-based quantification of phagocytic activity. We present a robust algorithm and cell-based assay system for high content analysis of phagocytic activity. The method utilizes fluorescently labeled beads as a phagocytic substrate with defined physical properties. The algorithm employs statistical modeling to determine the mean fluorescence of individual beads within each image, and uses the information to conduct an accurate count of phagocytosed beads. In addition, the algorithm conducts detailed and sophisticated analysis of cellular morphology, making it a standalone tool for high content screening. We tested our assay system using microglial cultures. Our results recapitulated previous findings on the effects of microglial stimulation on cell morphology and phagocytic activity. Moreover, our cell-level analysis revealed that the two phenotypes associated with microglial activation, specifically cell body hypertrophy and increased phagocytic activity, are not highly correlated. This novel finding suggests the two phenotypes may be under the control of distinct signaling pathways. We demonstrate that our assay system outperforms preexisting methods for quantifying phagocytic activity in multiple dimensions including speed, accuracy, and resolution. We provide a framework to facilitate the development of high content assays suitable for drug screening. For convenience, we implemented our algorithm in a standalone software package, PuntoMorph. Copyright © 2017 Elsevier B.V. All rights reserved.

Droplet microfluidics for single-cell analysis.

PubMed

Brouzes, Eric

2012-01-01

This book chapter aims at providing an overview of all the aspects and procedures needed to develop a droplet-based workflow for single-cell analysis (see Fig. 10.1). The surfactant system used to stabilize droplets is a critical component of droplet microfluidics; its properties define the type of droplet-based assays and workflows that can be developed. The scope of this book chapter is limited to fluorinated surfactant systems that have proved to generate extremely stable droplets and allow to easily retrieve the encapsulated material. The formulation section discusses how the experimental parameters influence the choice of the surfactant system to use. The circuit design section presents recipes to design and integrate different droplet modules into a whole assay. The fabrication section describes the manufacturing of microfluidic chip including the surface treatment which is pivotal in droplet microfluidics. Finally, the last section reviews the experimental setup for fluorescence detection with an emphasis on cell injection and incubation.

Analysis of regenerative fuel cells

NASA Technical Reports Server (NTRS)

Gross, S.

1982-01-01

The concept of a rechargeable fuel cell (RFC) system is considered. A newer type of rechargeable battery, the nickel hydrogen (Ni-H2) battery, is also evaluated. A review was made of past studies which showed large variations in weight, cost, and efficiency. Hydrogen-bromine and hydrogen-chlorine regenerable fuel cells were studied, and were found to have a potential for higher energy storage efficiency then the hydrogen-oxygen system. A reduction of up to 15 percent in solar array size may be possible as a result. These systems are not yet developed, but further study of them is recommended.

Influence of zero-G on single-cell systems and zero-G fermenter design concepts

NASA Technical Reports Server (NTRS)

Mayeux, J. V.

1977-01-01

An analysis was made to identify potential gravity-sensitive mechanisms that may be present in the single-cell growth system. Natural convection (density gradients, induced sedimentation, and buoyancy) is important in microbial systems. The absence of natural convection in the space-flight environment could provide an opportunity for new approaches for developments in industrial fermentation and agriculture. Some of the potential influences of gravity (i.e., convection, sedimentation, etc.) on the cell were discussed to provide insight into what experimental areas may be pursued in future space-flight research programs.

Distinguishing autocrine and paracrine signals in hematopoietic stem cell culture using a biofunctional microcavity platform

NASA Astrophysics Data System (ADS)

Müller, Eike; Wang, Weijia; Qiao, Wenlian; Bornhäuser, Martin; Zandstra, Peter W.; Werner, Carsten; Pompe, Tilo

2016-08-01

Homeostasis of hematopoietic stem cells (HSC) in the mammalian bone marrow stem cell niche is regulated by signals of the local microenvironment. Besides juxtacrine, endocrine and metabolic cues, paracrine and autocrine signals are involved in controlling quiescence, proliferation and differentiation of HSC with strong implications on expansion and differentiation ex vivo as well as in vivo transplantation. Towards this aim, a cell culture analysis on a polymer microcavity carrier platform was combined with a partial least square analysis of a mechanistic model of cell proliferation. We could demonstrate the discrimination of specific autocrine and paracrine signals from soluble factors as stimulating and inhibitory effectors in hematopoietic stem and progenitor cell culture. From that we hypothesize autocrine signals to be predominantly involved in maintaining the quiescent state of HSC in single-cell niches and advocate our analysis platform as an unprecedented option for untangling convoluted signaling mechanisms in complex cell systems being it of juxtacrine, paracrine or autocrine origin.

Application of laser-capture microdissection to analysis of gene expression in the testis.

PubMed

Sluka, Pavel; O'Donnell, Liza; McLachlan, Robert I; Stanton, Peter G

2008-01-01

The isolation and molecular analysis of highly purified cell populations from complex, heterogeneous tissues has been a challenge for many years. Spermatogenesis in the testis is a particularly difficult process to study given the unique multiple cellular associations within the seminiferous epithelium, making the isolation of specific cell types difficult. Laser-capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. This technology has enhanced our ability to directly examine gene expression in enriched testicular cell populations by routine methods of gene expression analysis, such as real-time RT-PCR, differential display, and gene microarrays. The application of LCM has however introduced methodological hurdles that have not been encountered with more conventional molecular analyses of whole tissue. In particular, tissue handling (i.e. fixation, storage, and staining), consumables (e.g. slide choice), staining reagents (conventional H&E vs. fluorescence), extraction methods, and downstream applications have all required re-optimisation to facilitate differential gene expression analysis using the small amounts of material obtained using LCM. This review will discuss three critical issues that are essential for successful procurement of cells from testicular tissue sections; tissue morphology, capture success, and maintenance of molecular integrity. The importance of these issues will be discussed with specific reference to the two most commonly used LCM systems; the Arcturus PixCell IIe and PALM systems. The rat testis will be used as a model, and emphasis will be placed on issues of tissue handling, processing, and staining methods, including the application of fluorescence techniques to assist in the identification of cells of interest for the purposes of mRNA expression analysis.

Identification Of Cells With A Compact Microscope Imaging System With Intelligent Controls

NASA Technical Reports Server (NTRS)

McDowell, Mark (Inventor)

2006-01-01

A Microscope Imaging System (CMIS) with intelligent controls is disclosed that provides techniques for scanning, identifying, detecting and tracking mic?oscopic changes in selected characteristics or features of various surfaces including, but not limited to, cells, spheres, and manufactured products subject to difficult-to-see imperfections. The practice of the present invention provides applications that include colloidal hard spheres experiments, biological cell detection for patch clamping, cell movement and tracking, as well as defect identification in products, such as semiconductor devices, where surface damage can be significant, but difficult to detect. The CMIS system is a machine vision system, which combines intelligent image processing with remote control capabilities and provides the ability to autofocus on a microscope sample, automatically scan an image, and perform machine vision analysis on multiple samples simultaneously.

Transcriptomic study on persistence and survival of Listeria monocytogenes following lethal treatment with Nisin.

PubMed

Wu, Shuyan; Yu, Pak-Lam; Wheeler, Dave; Flint, Steve

2018-06-19

The aim of this study was to determine the gene expression associated with the persistence of a Listeria monocytogenes stationary phase population when facing lethal nisin treatment METHODS: RNA Seq analysis was used for gene expression profiling of the persister cells in rich medium (persister TN) compared with untreated cells (non-persister).The results were confirmed using RT PCR. Functional genes associated with the persister populations were identified in multiple systems, such as heat shock related stress response, cell wall synthesis, ATP-binding cassette (ABC) transport system, phosphotransferase system (PTS system), and SOS/DNA repair. This study pointed to genetic regulation of persister cells exposed to lethal nisin and provides some insight into possible mechanisms of impeding bacterial persistence. Copyright © 2018. Published by Elsevier Ltd.

Tracking of Cells with a Compact Microscope Imaging System with Intelligent Controls

NASA Technical Reports Server (NTRS)

McDowell, Mark (Inventor)

2007-01-01

A Microscope Imaging System (CMIS) with intelligent controls is disclosed that provides techniques for scanning, identifying, detecting and tracking microscopic changes in selected characteristics or features of various surfaces including, but not limited to, cells, spheres, and manufactured products subject to difficult-to-see imperfections. The practice of the present invention provides applications that include colloidal hard spheres experiments, biological cell detection for patch clamping, cell movement and tracking, as well as defect identification in products, such as semiconductor devices, where surface damage can be significant, but difficult to detect. The CMIS system is a machine vision system, which combines intelligent image processing with remote control capabilities and provides the ability to autofocus on a microscope sample, automatically scan an image, and perform machine vision analysis on multiple samples simultaneously

Tracking of cells with a compact microscope imaging system with intelligent controls

NASA Technical Reports Server (NTRS)

McDowell, Mark (Inventor)

2007-01-01

A Microscope Imaging System (CMIS) with intelligent controls is disclosed that provides techniques for scanning, identifying, detecting and tracking microscopic changes in selected characteristics or features of various surfaces including, but not limited to, cells, spheres, and manufactured products subject to difficult-to-see imperfections. The practice of the present invention provides applications that include colloidal hard spheres experiments, biological cell detection for patch clamping, cell movement and tracking, as well as defect identification in products, such as semiconductor devices, where surface damage can be significant, but difficult to detect. The CMIS system is a machine vision system, which combines intelligent image processing with remote control capabilities and provides the ability to auto-focus on a microscope sample, automatically scan an image, and perform machine vision analysis on multiple samples simultaneously.

Integrating cell phone imaging with magnetic levitation (i-LEV) for label-free blood analysis at the point-of-living

PubMed Central

Durmus, Naside Gozde; Davis, Ronald W.; Steinmetz, Lars M.; Demirci, Utkan

2016-01-01

There is an emerging need for portable, robust, inexpensive and easy-to-use disease diagnosis and prognosis monitoring platforms to share health information at the point-of-living, including clinical and home settings. Recent advances in digital health technologies have improved early diagnosis, drug treatment, and personalized medicine. Smartphones with high-resolution cameras and high data processing power enable intriguing biomedical applications when integrated with diagnostic devices. Further, these devices have immense potential to contribute to public health in resource-limited settings where there is a particular need for portable, rapid, label-free, easy-to-use and affordable biomedical devices to diagnose and continuously monitor patients for precision medicine, especially those suffering from rare diseases, such as sickle cell anemia, thalassemia and chronic fatigue syndrome. Here, we present a magnetic levitation-based diagnosis system in which different cell types (i.e., white and red blood cells) are levitated in a magnetic gradient and separated due to their unique densities. Moreover, we introduce an easy-to-use, smartphone incorporated levitation system for cell analysis. Using our portable imaging magnetic levitation (i-LEV) system, we show that white and red blood cells can be identified and cell numbers can be quantified without using any labels. In addition, cells levitated in i-LEV can be distinguished at single cell resolution, potentially enabling diagnosis and monitoring, as well as clinical and research applications. PMID:26523938

Integrating Cell Phone Imaging with Magnetic Levitation (i-LEV) for Label-Free Blood Analysis at the Point-of-Living.

PubMed

Baday, Murat; Calamak, Semih; Durmus, Naside Gozde; Davis, Ronald W; Steinmetz, Lars M; Demirci, Utkan

2016-03-02

There is an emerging need for portable, robust, inexpensive, and easy-to-use disease diagnosis and prognosis monitoring platforms to share health information at the point-of-living, including clinical and home settings. Recent advances in digital health technologies have improved early diagnosis, drug treatment, and personalized medicine. Smartphones with high-resolution cameras and high data processing power enable intriguing biomedical applications when integrated with diagnostic devices. Further, these devices have immense potential to contribute to public health in resource-limited settings where there is a particular need for portable, rapid, label-free, easy-to-use, and affordable biomedical devices to diagnose and continuously monitor patients for precision medicine, especially those suffering from rare diseases, such as sickle cell anemia, thalassemia, and chronic fatigue syndrome. Here, a magnetic levitation-based diagnosis system is presented in which different cell types (i.e., white and red blood cells) are levitated in a magnetic gradient and separated due to their unique densities. Moreover, an easy-to-use, smartphone incorporated levitation system for cell analysis is introduced. Using our portable imaging magnetic levitation (i-LEV) system, it is shown that white and red blood cells can be identified and cell numbers can be quantified without using any labels. In addition, cells levitated in i-LEV can be distinguished at single-cell resolution, potentially enabling diagnosis and monitoring, as well as clinical and research applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of Passive Fuel Cell Thermal Management Technology

NASA Technical Reports Server (NTRS)

Burke, Kenneth A.; Jakupca, Ian; Colozza, Anthony

2011-01-01

The NASA Glenn Research Center is developing advanced passive thermal management technology to reduce the mass and improve the reliability of space fuel cell systems for the NASA exploration program. The passive thermal management system relies on heat conduction within the cooling plate to move the heat from the central portion of the cell stack out to the edges of the fuel cell stack rather than using a pumped loop cooling system to convectively remove the heat. Using the passive approach eliminates the need for a coolant pump and other cooling loop components which reduces fuel cell system mass and improves overall system reliability. Previous analysis had identified that low density, ultra-high thermal conductivity materials would be needed for the cooling plates in order to achieve the desired reductions in mass and the highly uniform thermal heat sink for each cell within a fuel cell stack. A pyrolytic graphite material was identified and fabricated into a thin plate using different methods. Also a development project with Thermacore, Inc. resulted in a planar heat pipe. Thermal conductivity tests were done using these materials. The results indicated that lightweight passive fuel cell cooling is feasible.

Clinicopathological categorization of Epstein-Barr virus-positive T/NK-cell lymphoproliferative disease: an analysis of 42 cases with an emphasis on prognostic implications.

PubMed

Paik, Jin Ho; Choe, Ji-Young; Kim, Hyojin; Lee, Jeong-Ok; Kang, Hyoung Jin; Shin, Hee Young; Lee, Dong Soon; Heo, Dae Seog; Kim, Chul-Woo; Cho, Kwang-Hyun; Kim, Tae Min; Jeon, Yoon Kyung

2017-01-01

Epstein-Barr virus-positive T/NK-cell lymphoproliferative diseases (EBV-T/NK-LPDs) include several overlapping EBV-related conditions with variably aggressive courses. For prognostic categorization, we retrospectively analyzed 42 EBV-T/NK-LPD cases. Male (79% [33/42]), young (≤40 years; 83% [35/42]) patients and T-cell lineage (81% [34/42]; CD8/CD4 = 1.8) were predominant. Clinicopathologically, three systemic and one cutaneous category were developed: hemophagocytic lymphohistiocytosis (HLH; 26% [11/42]), chronic active EBV infection (CAEBV; 31% [13/42]), systemic unclassifiable disease (24% [10/42]), and hydroa vacciniforme/hydroa vacciniforme-like lymphoma (HV/HVL; 19% [8/42]). Prognostically, cutaneous disease (HV/HVL) was better than systemic disease (p = 0.014; median, 285 vs. 10 months). In systemic diseases, HLH was worst (p = 0.002; 3[HLH] vs. 4[unclassifiable] vs. not reached [CAEBV]). Univariate survival analysis (n = 42) revealed cytopenia (≥one lineage; p < 0.001), onset age (>40 years; p = 0.001), T-cell lineage (p = 0.041), hemophagocytic histiocytes (p = 0.031), elevated lactate dehydrogenase (p = 0.020), and liver dysfunction (p = 0.023) predicted shorter survival. In multivariate analysis, T-cell lineage (p = 0.025 [HR =11.3]) and cytopenia (p = 0.028 [HR =5.4]) were independent prognostic factors. Therefore, EBV-T/NK-LPD could be classified into four prognostic categories.

Ellipsoidal cell flow system

DOEpatents

Salzman, Gary C.; Mullaney, Paul F.

1976-01-01

The disclosure relates to a system incorporating an ellipsoidal flow chamber having light reflective walls for low level light detection in practicing cellular analysis. The system increases signal-to-noise ratio by a factor of ten over prior art systems. In operation, laser light passes through the primary focus of the ellipsoid. A controlled flow of cells simultaneously passes through this focus so that the laser light impinges on the cells and is modulated by the cells. The reflective walls of the ellipsoid reflect the cell-modulated light to the secondary focus of the ellipsoid. A tapered light guide at the secondary focus picks up a substantial portion of modulated reflective light and directs it onto a light detector to produce a signal. The signal is processed to obtain the intensity distribution of the modulated light and hence sought after characteristics of the cells. In addition, cells may be dyed so as to fluoresce in response to the laser light and their fluorescence may be processed as cell-modulated light above described. A light discriminating filter would be used to distinguish reflected modulated laser light from reflected fluorescent light.

Identifying viable regulatory and innovation pathways for regenerative medicine: a case study of cultured red blood cells.

PubMed

Mittra, J; Tait, J; Mastroeni, M; Turner, M L; Mountford, J C; Bruce, K

2015-01-25

The creation of red blood cells for the blood transfusion markets represents a highly innovative application of regenerative medicine with a medium term (5-10 year) prospect for first clinical studies. This article describes a case study analysis of a project to derive red blood cells from human embryonic stem cells, including the systemic challenges arising from (i) the selection of appropriate and viable regulatory protocols and (ii) technological constraints related to stem cell manufacture and scale up to clinical Good Manufacturing Practice (GMP) standard. The method used for case study analysis (Analysis of Life Science Innovation Systems (ALSIS)) is also innovative, demonstrating a new approach to social and natural science collaboration to foresight product development pathways. Issues arising along the development pathway include cell manufacture and scale-up challenges, affected by regulatory demands emerging from the innovation ecosystem (preclinical testing and clinical trials). Our discussion reflects on the efforts being made by regulators to adapt the current pharmaceuticals-based regulatory model to an allogeneic regenerative medicine product and the broader lessons from this case study for successful innovation and translation of regenerative medicine therapies, including the role of methodological and regulatory innovation in future development in the field. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics.

PubMed

Tung, Yi-Chung; Torisawa, Yu-suke; Futai, Nobuyuki; Takayama, Shuichi

2007-11-01

This paper describes a micro flow cytometer system designed for efficient and non-damaging analysis of samples with small numbers of precious cells. The system utilizes actuation of Braille-display pins for micro-scale fluid manipulation and a fluorescence microscope with a CCD camera for optical detection. The microfluidic chip is fully disposable and is composed of a polydimethylsiloxane (PDMS) slab with microchannel features sealed against a thin deformable PDMS membrane. The channels are designed with diffusers to alleviate pulsatile flow behaviors inherent in pin actuator-based peristaltic pumping schemes to maximize hydrodynamic focusing of samples with minimal disturbances in the laminar streams within the channel. A funnel connected to the microfluidic channel is designed for efficient loading of samples with small number of cells and is also positioned on the chip to prevent physical damages of the samples by the squeezing actions of Braille pins during actuation. The sample loading scheme was characterized by both computational fluidic dynamics (CFD) simulation and experimental observation. A fluorescein solution was first used for flow field investigation, followed by use of fluorescence beads with known relative intensities for optical detection performance calibration. Murine myoblast cells (C2C12) were exploited to investigate cell viability for the sample loading scheme of the device. Furthermore, human promyelocytic leukemia (HL60) cells stained by hypotonic DNA staining buffer were also tested in the system for cell cycle analysis. The ability to efficiently analyze cellular samples where the number of cells is small was demonstrated by analyzing cells from a single embryoid body derived from mouse embryonic stem cells. Consequently, the designed microfluidic device reported in this paper is promising for easy-to-use, small sample size flow cytometric analysis, and has potential to be further integrated with other Braille display-based microfluidic devices to facilitate a multi-functional lab-on-a-chip for mammalian cell manipulations.

Image processing and machine learning in the morphological analysis of blood cells.

PubMed

Rodellar, J; Alférez, S; Acevedo, A; Molina, A; Merino, A

2018-05-01

This review focuses on how image processing and machine learning can be useful for the morphological characterization and automatic recognition of cell images captured from peripheral blood smears. The basics of the 3 core elements (segmentation, quantitative features, and classification) are outlined, and recent literature is discussed. Although red blood cells are a significant part of this context, this study focuses on malignant lymphoid cells and blast cells. There is no doubt that these technologies may help the cytologist to perform efficient, objective, and fast morphological analysis of blood cells. They may also help in the interpretation of some morphological features and may serve as learning and survey tools. Although research is still needed, it is important to define screening strategies to exploit the potential of image-based automatic recognition systems integrated in the daily routine of laboratories along with other analysis methodologies. © 2018 John Wiley & Sons Ltd.

A cell-based systems biology assessment of human blood to monitor immune responses after influenza vaccination.

PubMed

Hoek, Kristen L; Samir, Parimal; Howard, Leigh M; Niu, Xinnan; Prasad, Nripesh; Galassie, Allison; Liu, Qi; Allos, Tara M; Floyd, Kyle A; Guo, Yan; Shyr, Yu; Levy, Shawn E; Joyce, Sebastian; Edwards, Kathryn M; Link, Andrew J

2015-01-01

Systems biology is an approach to comprehensively study complex interactions within a biological system. Most published systems vaccinology studies have utilized whole blood or peripheral blood mononuclear cells (PBMC) to monitor the immune response after vaccination. Because human blood is comprised of multiple hematopoietic cell types, the potential for masking responses of under-represented cell populations is increased when analyzing whole blood or PBMC. To investigate the contribution of individual cell types to the immune response after vaccination, we established a rapid and efficient method to purify human T and B cells, natural killer (NK) cells, myeloid dendritic cells (mDC), monocytes, and neutrophils from fresh venous blood. Purified cells were fractionated and processed in a single day. RNA-Seq and quantitative shotgun proteomics were performed to determine expression profiles for each cell type prior to and after inactivated seasonal influenza vaccination. Our results show that transcriptomic and proteomic profiles generated from purified immune cells differ significantly from PBMC. Differential expression analysis for each immune cell type also shows unique transcriptomic and proteomic expression profiles as well as changing biological networks at early time points after vaccination. This cell type-specific information provides a more comprehensive approach to monitor vaccine responses.

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level.

PubMed

Zhu, Zhi; Zhang, Wenhua; Leng, Xuefei; Zhang, Mingxia; Guan, Zhichao; Lu, Jiangquan; Yang, Chaoyong James

2012-10-21

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100,000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.

Cell-to-Cell Communication Circuits: Quantitative Analysis of Synthetic Logic Gates

PubMed Central

Hoffman-Sommer, Marta; Supady, Adriana; Klipp, Edda

2012-01-01

One of the goals in the field of synthetic biology is the construction of cellular computation devices that could function in a manner similar to electronic circuits. To this end, attempts are made to create biological systems that function as logic gates. In this work we present a theoretical quantitative analysis of a synthetic cellular logic-gates system, which has been implemented in cells of the yeast Saccharomyces cerevisiae (Regot et al., 2011). It exploits endogenous MAP kinase signaling pathways. The novelty of the system lies in the compartmentalization of the circuit where all basic logic gates are implemented in independent single cells that can then be cultured together to perform complex logic functions. We have constructed kinetic models of the multicellular IDENTITY, NOT, OR, and IMPLIES logic gates, using both deterministic and stochastic frameworks. All necessary model parameters are taken from literature or estimated based on published kinetic data, in such a way that the resulting models correctly capture important dynamic features of the included mitogen-activated protein kinase pathways. We analyze the models in terms of parameter sensitivity and we discuss possible ways of optimizing the system, e.g., by tuning the culture density. We apply a stochastic modeling approach, which simulates the behavior of whole populations of cells and allows us to investigate the noise generated in the system; we find that the gene expression units are the major sources of noise. Finally, the model is used for the design of system modifications: we show how the current system could be transformed to operate on three discrete values. PMID:22934039

MAPPI-DAT: data management and analysis for protein-protein interaction data from the high-throughput MAPPIT cell microarray platform.

PubMed

Gupta, Surya; De Puysseleyr, Veronic; Van der Heyden, José; Maddelein, Davy; Lemmens, Irma; Lievens, Sam; Degroeve, Sven; Tavernier, Jan; Martens, Lennart

2017-05-01

Protein-protein interaction (PPI) studies have dramatically expanded our knowledge about cellular behaviour and development in different conditions. A multitude of high-throughput PPI techniques have been developed to achieve proteome-scale coverage for PPI studies, including the microarray based Mammalian Protein-Protein Interaction Trap (MAPPIT) system. Because such high-throughput techniques typically report thousands of interactions, managing and analysing the large amounts of acquired data is a challenge. We have therefore built the MAPPIT cell microArray Protein Protein Interaction-Data management & Analysis Tool (MAPPI-DAT) as an automated data management and analysis tool for MAPPIT cell microarray experiments. MAPPI-DAT stores the experimental data and metadata in a systematic and structured way, automates data analysis and interpretation, and enables the meta-analysis of MAPPIT cell microarray data across all stored experiments. MAPPI-DAT is developed in Python, using R for data analysis and MySQL as data management system. MAPPI-DAT is cross-platform and can be ran on Microsoft Windows, Linux and OS X/macOS. The source code and a Microsoft Windows executable are freely available under the permissive Apache2 open source license at https://github.com/compomics/MAPPI-DAT. jan.tavernier@vib-ugent.be or lennart.martens@vib-ugent.be. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

Isolation and characterization of circulating tumor cells using a novel workflow combining the CellSearch® system and the CellCelector™.

PubMed

Neumann, Martin Horst Dieter; Schneck, Helen; Decker, Yvonne; Schömer, Susanne; Franken, André; Endris, Volker; Pfarr, Nicole; Weichert, Wilko; Niederacher, Dieter; Fehm, Tanja; Neubauer, Hans

2017-01-01

Circulating tumor cells (CTC) are rare cells which have left the primary tumor to enter the blood stream. Although only a small CTC subgroup is capable of extravasating, the presence of CTCs is associated with an increased risk of metastasis and a shorter overall survival. Understanding the heterogeneous CTC biology will optimize treatment decisions and will thereby improve patient outcome. For this, robust workflows for detection and isolation of CTCs are urgently required. Here, we present a workflow to characterize CTCs by combining the advantages of both the CellSearch ® and the CellCelector™ micromanipulation system. CTCs were isolated from CellSearch ® cartridges using the CellCelector™ system and were deposited into PCR tubes for subsequent molecular analysis (whole genome amplification (WGA) and massive parallel multigene sequencing). By a CellCelector™ screen we reidentified 97% of CellSearch ® SKBR-3 cells. Furthermore, we isolated 97% of CellSearch ® -proven patient CTCs using the CellCelector™ system. Therein, we found an almost perfect correlation of R 2  = 0.98 (Spearman's rho correlation, n = 20, p < 0.00001) between the CellSearch ® CTC count (n = 271) and the CellCelector™ detected CTCs (n = 252). Isolated CTCs were analyzed by WGA and massive parallel multigene sequencing. In total, single nucleotide polymorphisms (SNPs) could be detected in 50 genes in seven CTCs, 12 MCF-7, and 3 T47D cells, respectively. Taken together, CTC quantification via the CellCelector™ system ensures a comprehensive detection of CTCs preidentified by the CellSearch ® system. Moreover, the isolation of CTCs after CellSearch ® using the CellCelector™ system guarantees for CTC enrichment without any contaminants enabling subsequent high throughput genomic analyses on single cell level. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:125-132, 2017. © 2016 American Institute of Chemical Engineers.

Connecting Neuronal Cell Protective Pathways and Drug Combinations in a Huntington's Disease Model through the Application of Quantitative Systems Pharmacology.

PubMed

Pei, Fen; Li, Hongchun; Henderson, Mark J; Titus, Steven A; Jadhav, Ajit; Simeonov, Anton; Cobanoglu, Murat Can; Mousavi, Seyed H; Shun, Tongying; McDermott, Lee; Iyer, Prema; Fioravanti, Michael; Carlisle, Diane; Friedlander, Robert M; Bahar, Ivet; Taylor, D Lansing; Lezon, Timothy R; Stern, Andrew M; Schurdak, Mark E

2017-12-19

Quantitative Systems Pharmacology (QSP) is a drug discovery approach that integrates computational and experimental methods in an iterative way to gain a comprehensive, unbiased understanding of disease processes to inform effective therapeutic strategies. We report the implementation of QSP to Huntington's Disease, with the application of a chemogenomics platform to identify strategies to protect neuronal cells from mutant huntingtin induced death. Using the STHdh Q111 cell model, we investigated the protective effects of small molecule probes having diverse canonical modes-of-action to infer pathways of neuronal cell protection connected to drug mechanism. Several mechanistically diverse protective probes were identified, most of which showed less than 50% efficacy. Specific combinations of these probes were synergistic in enhancing efficacy. Computational analysis of these probes revealed a convergence of pathways indicating activation of PKA. Analysis of phospho-PKA levels showed lower cytoplasmic levels in STHdh Q111 cells compared to wild type STHdh Q7 cells, and these levels were increased by several of the protective compounds. Pharmacological inhibition of PKA activity reduced protection supporting the hypothesis that protection may be working, in part, through activation of the PKA network. The systems-level studies described here can be broadly applied to any discovery strategy involving small molecule modulation of disease phenotype.

Thermo-electrochemical evaluation of lithium-ion batteries for space applications

NASA Astrophysics Data System (ADS)

Walker, W.; Yayathi, S.; Shaw, J.; Ardebili, H.

2015-12-01

Advanced energy storage and power management systems designed through rigorous materials selection, testing and analysis processes are essential to ensuring mission longevity and success for space exploration applications. Comprehensive testing of Boston Power Swing 5300 lithium-ion (Li-ion) cells utilized by the National Aeronautics and Space Administration (NASA) to power humanoid robot Robonaut 2 (R2) is conducted to support the development of a test-correlated Thermal Desktop (TD) Systems Improved Numerical Differencing Analyzer (SINDA) (TD-S) model for evaluation of power system thermal performance. Temperature, current, working voltage and open circuit voltage measurements are taken during nominal charge-discharge operations to provide necessary characterization of the Swing 5300 cells for TD-S model correlation. Building from test data, embedded FORTRAN statements directly simulate Ohmic heat generation of the cells during charge-discharge as a function of surrounding temperature, local cell temperature and state of charge. The unique capability gained by using TD-S is demonstrated by simulating R2 battery thermal performance in example orbital environments for hypothetical extra-vehicular activities (EVA) exterior to a small satellite. Results provide necessary demonstration of this TD-S technique for thermo-electrochemical analysis of Li-ion cells operating in space environments.

Metabolic cancer biology: structural-based analysis of cancer as a metabolic disease, new sights and opportunities for disease treatment.

PubMed

Masoudi-Nejad, Ali; Asgari, Yazdan

2015-02-01

The cancer cell metabolism or the Warburg effect discovery goes back to 1924 when, for the first time Otto Warburg observed, in contrast to the normal cells, cancer cells have different metabolism. With the initiation of high throughput technologies and computational systems biology, cancer cell metabolism renaissances and many attempts were performed to revise the Warburg effect. The development of experimental and analytical tools which generate high-throughput biological data including lots of information could lead to application of computational models in biological discovery and clinical medicine especially for cancer. Due to the recent availability of tissue-specific reconstructed models, new opportunities in studying metabolic alteration in various kinds of cancers open up. Structural approaches at genome-scale levels seem to be suitable for developing diagnostic and prognostic molecular signatures, as well as in identifying new drug targets. In this review, we have considered these recent advances in structural-based analysis of cancer as a metabolic disease view. Two different structural approaches have been described here: topological and constraint-based methods. The ultimate goal of this type of systems analysis is not only the discovery of novel drug targets but also the development of new systems-based therapy strategies. Copyright © 2014 Elsevier Ltd. All rights reserved.

System level modeling and component level control of fuel cells

NASA Astrophysics Data System (ADS)

Xue, Xingjian

This dissertation investigates the fuel cell systems and the related technologies in three aspects: (1) system-level dynamic modeling of both PEM fuel cell (PEMFC) and solid oxide fuel cell (SOFC); (2) condition monitoring scheme development of PEM fuel cell system using model-based statistical method; and (3) strategy and algorithm development of precision control with potential application in energy systems. The dissertation first presents a system level dynamic modeling strategy for PEM fuel cells. It is well known that water plays a critical role in PEM fuel cell operations. It makes the membrane function appropriately and improves the durability. The low temperature operating conditions, however, impose modeling difficulties in characterizing the liquid-vapor two phase change phenomenon, which becomes even more complex under dynamic operating conditions. This dissertation proposes an innovative method to characterize this phenomenon, and builds a comprehensive model for PEM fuel cell at the system level. The model features the complete characterization of multi-physics dynamic coupling effects with the inclusion of dynamic phase change. The model is validated using Ballard stack experimental result from open literature. The system behavior and the internal coupling effects are also investigated using this model under various operating conditions. Anode-supported tubular SOFC is also investigated in the dissertation. While the Nernst potential plays a central role in characterizing the electrochemical performance, the traditional Nernst equation may lead to incorrect analysis results under dynamic operating conditions due to the current reverse flow phenomenon. This dissertation presents a systematic study in this regard to incorporate a modified Nernst potential expression and the heat/mass transfer into the analysis. The model is used to investigate the limitations and optimal results of various operating conditions; it can also be utilized to perform the optimal design of tubular SOFC. With the system-level dynamic model as a basis, a framework for the robust, online monitoring of PEM fuel cell is developed in the dissertation. The monitoring scheme employs the Hotelling T2 based statistical scheme to handle the measurement noise and system uncertainties and identifies the fault conditions through a series of self-checking and conformal testing. A statistical sampling strategy is also utilized to improve the computation efficiency. Fuel/gas flow control is the fundamental operation for fuel cell energy systems. In the final part of the dissertation, a high-precision and robust tracking control scheme using piezoelectric actuator circuit with direct hysteresis compensation is developed. The key characteristic of the developed control algorithm includes the nonlinear continuous control action with the adaptive boundary layer strategy.

Raman-Activated Droplet Sorting (RADS) for Label-Free High-Throughput Screening of Microalgal Single-Cells.

PubMed

Wang, Xixian; Ren, Lihui; Su, Yetian; Ji, Yuetong; Liu, Yaoping; Li, Chunyu; Li, Xunrong; Zhang, Yi; Wang, Wei; Hu, Qiang; Han, Danxiang; Xu, Jian; Ma, Bo

2017-11-21

Raman-activated cell sorting (RACS) has attracted increasing interest, yet throughput remains one major factor limiting its broader application. Here we present an integrated Raman-activated droplet sorting (RADS) microfluidic system for functional screening of live cells in a label-free and high-throughput manner, by employing AXT-synthetic industrial microalga Haematococcus pluvialis (H. pluvialis) as a model. Raman microspectroscopy analysis of individual cells is carried out prior to their microdroplet encapsulation, which is then directly coupled to DEP-based droplet sorting. To validate the system, H. pluvialis cells containing different levels of AXT were mixed and underwent RADS. Those AXT-hyperproducing cells were sorted with an accuracy of 98.3%, an enrichment ratio of eight folds, and a throughput of ∼260 cells/min. Of the RADS-sorted cells, 92.7% remained alive and able to proliferate, which is equivalent to the unsorted cells. Thus, the RADS achieves a much higher throughput than existing RACS systems, preserves the vitality of cells, and facilitates seamless coupling with downstream manipulations such as single-cell sequencing and cultivation.

Enhanced reactive oxygen species overexpression by CuO nanoparticles in poorly differentiated hepatocellular carcinoma cells

NASA Astrophysics Data System (ADS)

Kung, Mei-Lang; Hsieh, Shu-Ling; Wu, Chih-Chung; Chu, Tian-Huei; Lin, Yu-Chun; Yeh, Bi-Wen; Hsieh, Shuchen

2015-01-01

Copper oxide nanoparticles (CuO NPs) are known to exhibit toxic effects on a variety of cell types and organs. To determine the oxidative impact of CuO NPs on hepatocellular carcinoma (HCC) cells, well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) cells were exposed to CuO NPs. Cell viability assay showed that the median inhibition concentration (IC50) for SK-Hep-1 and HepG2 cells was 25 μg ml-1 and 85 μg ml-1, respectively. Cellular fluorescence intensity using DCFH-DA staining analysis revealed significant intracellular reactive oxygen species (ROS) generation of up to 242% in SK-Hep-1 cells, compared with 86% in HepG2 cells. HPLC analysis demonstrated that a CuO NP treatment caused cellular GSH depletion of 58% and a GSH/GSSG ratio decrease to ~0.1 in SK-Hep-1 cells. The oxidative stress caused by enhanced superoxide anion production was observed in both HepG2 (146%) and SK-Hep-1 (192%) cells. The Griess assay verified that CuO NPs induced NO production (170%) in SK-Hep-1 cells. Comet assay and western blot further demonstrated that CuO NPs induced severe DNA strand breakage (70%) in SK-Hep-1 cells and caused DNA damage via increased γ-H2AX levels. These results suggest that well-differentiated HepG2 cells possess a robust antioxidant defense system against CuO NP-induced ROS stress and exhibit more tolerance to oxidative stress. Conversely, poorly differentiated SK-Hep-1 cells exhibited a deregulated antioxidant defense system that allowed accumulation of CuO NP-induced ROS and resulted in severe cytotoxicity.Copper oxide nanoparticles (CuO NPs) are known to exhibit toxic effects on a variety of cell types and organs. To determine the oxidative impact of CuO NPs on hepatocellular carcinoma (HCC) cells, well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) cells were exposed to CuO NPs. Cell viability assay showed that the median inhibition concentration (IC50) for SK-Hep-1 and HepG2 cells was 25 μg ml-1 and 85 μg ml-1, respectively. Cellular fluorescence intensity using DCFH-DA staining analysis revealed significant intracellular reactive oxygen species (ROS) generation of up to 242% in SK-Hep-1 cells, compared with 86% in HepG2 cells. HPLC analysis demonstrated that a CuO NP treatment caused cellular GSH depletion of 58% and a GSH/GSSG ratio decrease to ~0.1 in SK-Hep-1 cells. The oxidative stress caused by enhanced superoxide anion production was observed in both HepG2 (146%) and SK-Hep-1 (192%) cells. The Griess assay verified that CuO NPs induced NO production (170%) in SK-Hep-1 cells. Comet assay and western blot further demonstrated that CuO NPs induced severe DNA strand breakage (70%) in SK-Hep-1 cells and caused DNA damage via increased γ-H2AX levels. These results suggest that well-differentiated HepG2 cells possess a robust antioxidant defense system against CuO NP-induced ROS stress and exhibit more tolerance to oxidative stress. Conversely, poorly differentiated SK-Hep-1 cells exhibited a deregulated antioxidant defense system that allowed accumulation of CuO NP-induced ROS and resulted in severe cytotoxicity. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05843g

Testing of Lightweight Fuel Cell Vehicles System at Low Speeds with Energy Efficiency Analysis

NASA Astrophysics Data System (ADS)

Mustaffa, Muhammad Rizuwan B.; Mohamed, Wan Ahmad Najmi B. Wan

2013-12-01

A fuel cell vehicle power train mini test bench was developed which consists of a 1 kW open cathode hydrogen fuel cell, electric motor, wheel, gearing system, DC/DC converter and vehicle control system (VCS). Energy efficiency identification and energy flow evaluation is a useful tool in identifying a detail performance of each component and sub-systems in a fuel cell vehicle system configuration. Three artificial traction loads was simulated at 30 kg, 40 kg and 50 kg force on a single wheel drive configuration. The wheel speed range reported here covers from idle to 16 km/h (low speed range) as a preliminary input in the research work frame. The test result shows that the system efficiency is 84.5 percent when the energy flow is considered from the fuel cell to the wheel and 279 watts of electrical power was produced by the fuel cell during that time. Dynamic system responses was also identified as the load increases beyond the motor traction capabilities where the losses at the converter and motor controller increased significantly as it tries to meet the motor traction power demands. This work is currently being further expanded within the work frame of developing a road-worthy fuel cell vehicle.

Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells.

PubMed

Kuriyama, Kentaro; Shintaku, Hirofumi; Santiago, Juan G

2015-07-01

There is a substantial need for simultaneous analyses of RNA and DNA from individual single cells. Such analysis provides unique evidence of cell-to-cell differences and the correlation between gene expression and genomic mutation in highly heterogeneous cell populations. We present a novel microfluidic system that leverages isotachophoresis to fractionate and isolate cytoplasmic RNA and genomic DNA (gDNA) from single cells. The system uniquely enables independent, sequence-specific analyses of these critical markers. Our system uses a microfluidic chip with a simple geometry and four end-channel electrodes, and completes the entire process in <5 min, including lysis, purification, fractionation, and delivery to DNA and RNA output reservoirs, each containing high quality and purity aliquots with no measurable cross-contamination of cytoplasmic RNA versus gDNA. We demonstrate our system with simultaneous, sequence-specific quantitation using off-chip RT-qPCR and qPCR for simultaneous cytoplasmic RNA and gDNA analyses, respectively. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Radar-based alert system to operate a sewerage network: relevance and operational effectiveness after several years of use.

PubMed

Faure, D; Payrastre, O; Auchet, P

2005-01-01

Since January 2000, the sewerage network of a very urbanised catchment area in the Greater Nancy Urban Community has been operated according to the alarms generated in real time by a storm alert system using weather radar data. This alert system is based on an automatic identification of intense rain cells in the radar images. This paper presents the characteristics of this alert system and synthesises the main results of two complementary studies realised in 2002 in order to estimate the relevance and the operational effectiveness of the alert system. The first study consisted in an off-line analysis of almost 50,000 intense rain cells detected in four years of historical radar data. The second study was an analysis of the experience feedback after two years of operational use of this alert system. The results of these studies are discussed in function of the initial operational objectives.

4E analysis and multi objective optimization of a micro gas turbine and solid oxide fuel cell hybrid combined heat and power system

NASA Astrophysics Data System (ADS)

Sanaye, Sepehr; Katebi, Arash

2014-02-01

Energy, exergy, economic and environmental (4E) analysis and optimization of a hybrid solid oxide fuel cell and micro gas turbine (SOFC-MGT) system for use as combined generation of heat and power (CHP) is investigated in this paper. The hybrid system is modeled and performance related results are validated using available data in literature. Then a multi-objective optimization approach based on genetic algorithm is incorporated. Eight system design parameters are selected for the optimization procedure. System exergy efficiency and total cost rate (including capital or investment cost, operational cost and penalty cost of environmental emissions) are the two objectives. The effects of fuel unit cost, capital investment and system power output on optimum design parameters are also investigated. It is observed that the most sensitive and important design parameter in the hybrid system is fuel cell current density which has a significant effect on the balance between system cost and efficiency. The selected design point from the Pareto distribution of optimization results indicates a total system exergy efficiency of 60.7%, with estimated electrical energy cost 0.057 kW-1 h-1, and payback period of about 6.3 years for the investment.

Impedance spectroscopy with field-effect transistor arrays for the analysis of anti-cancer drug action on individual cells.

PubMed

Susloparova, A; Koppenhöfer, D; Vu, X T; Weil, M; Ingebrandt, S

2013-02-15

In this study, impedance spectroscopy measurements of silicon-based open-gate field-effect transistor (FET) devices were utilized to study the adhesion status of cancer cells at a single cell level. We developed a trans-impedance amplifier circuit for the FETs with a higher bandwidth compared to a previously described system. The new system was characterized with a fast lock-in amplifier, which enabled measuring of impedance spectra up to 50 MHz. We studied cellular activities, including cell adhesion and anti-cancer drug induced apoptosis of human embryonic kidney (HEK293) and human lung adenocarcinoma epithelial (H441) cells. A well-known chemotherapeutic drug, topotecan hydrochloride, was used to investigate the effect of this drug to tumor cells cultured on the FET devices. The presence of the drug resulted in a 20% change in the amplitude of the impedance spectra at 200 kHz as a result of the induced apoptosis process. Real-time impedance measurements were performed inside an incubator at a constant frequency. The experimental results can be interpreted with an equivalent electronic circuit to resolve the influence of the system parameters. The developed method could be applied for the analysis of the specificity and efficacy of novel anti-cancer drugs in cancer therapy research on a single cell level in parallelized measurements. Copyright © 2012 Elsevier B.V. All rights reserved.

Mathematical Modeling and Nonlinear Dynamical Analysis of Cell Growth in Response to Antibiotics

NASA Astrophysics Data System (ADS)

Jin, Suoqin; Niu, Lili; Wang, Gang; Zou, Xiufen

2015-06-01

This study is devoted to the revelation of the dynamical mechanisms of cell growth in response to antibiotics. We establish a mathematical model of ordinary differential equations for an antibiotic-resistant growth system with one positive feedback loop. We perform a dynamical analysis of the behavior of this model system. We present adequate sets of conditions that can guarantee the existence and stability of biologically-reasonable steady states. Using bifurcation analysis and numerical simulation, we show that the relative growth rate, which is defined as the ratio of the cell growth rate to the basal cell growth rate in the absence of antibiotics, can exhibit bistable behavior in an extensive range of parameters that correspond to a growth state and a nongrowth state in biology. We discover that both antibiotic and antibiotic resistance genes can cooperatively enhance bistability, whereas the cooperative coefficient of feedback can contribute to the onset of bistability. These results would contribute to a better understanding of not only the evolution of antibiotics but also the emergence of drug resistance in other diseases.

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column.

PubMed

Jiang, Xiaogang; Feng, Shun; Tian, Ruijun; Han, Guanghui; Jiang, Xinning; Ye, Mingliang; Zou, Hanfa

2007-02-01

An approach was developed to automate sample introduction for nanoflow LC-MS/MS (microLC-MS/MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 microm id x 2 cm SCX trap column and a 75 microm id x 12 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two-dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC-7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current microLC-MS/MS system. This system represented a promising platform for routine proteome analysis.

Therapeutic application of extracellular vesicles in acute and chronic renal injury.

PubMed

Rovira, Jordi; Diekmann, Fritz; Campistol, Josep M; Ramírez-Bajo, María José

A new cell-to-cell communication system was discovered in the 1990s, which involves the release of vesicles into the extracellular space. These vesicles shuttle bioactive particles, including proteins, mRNA, miRNA, metabolites, etc. This particular communication has been conserved throughout evolution, which explains why most cell types are capable of producing vesicles. Extracellular vesicles (EVs) are involved in the regulation of different physiological processes, as well as in the development and progression of several diseases. EVs have been widely studied over recent years, especially those produced by embryonic and adult stem cells, blood cells, immune system and nervous system cells, as well as tumour cells. EV analysis from bodily fluids has been used as a diagnostic tool for cancer and recently for different renal diseases. However, this review analyses the importance of EVs generated by stem cells, their function and possible clinical application in renal diseases and kidney transplantation. Copyright © 2016. Published by Elsevier España, S.L.U.

Multi-scale imaging and informatics pipeline for in situ pluripotent stem cell analysis.

PubMed

Gorman, Bryan R; Lu, Junjie; Baccei, Anna; Lowry, Nathan C; Purvis, Jeremy E; Mangoubi, Rami S; Lerou, Paul H

2014-01-01

Human pluripotent stem (hPS) cells are a potential source of cells for medical therapy and an ideal system to study fate decisions in early development. However, hPS cells cultured in vitro exhibit a high degree of heterogeneity, presenting an obstacle to clinical translation. hPS cells grow in spatially patterned colony structures, necessitating quantitative single-cell image analysis. We offer a tool for analyzing the spatial population context of hPS cells that integrates automated fluorescent microscopy with an analysis pipeline. It enables high-throughput detection of colonies at low resolution, with single-cellular and sub-cellular analysis at high resolutions, generating seamless in situ maps of single-cellular data organized by colony. We demonstrate the tool's utility by analyzing inter- and intra-colony heterogeneity of hPS cell cycle regulation and pluripotency marker expression. We measured the heterogeneity within individual colonies by analyzing cell cycle as a function of distance. Cells loosely associated with the outside of the colony are more likely to be in G1, reflecting a less pluripotent state, while cells within the first pluripotent layer are more likely to be in G2, possibly reflecting a G2/M block. Our multi-scale analysis tool groups colony regions into density classes, and cells belonging to those classes have distinct distributions of pluripotency markers and respond differently to DNA damage induction. Lastly, we demonstrate that our pipeline can robustly handle high-content, high-resolution single molecular mRNA FISH data by using novel image processing techniques. Overall, the imaging informatics pipeline presented offers a novel approach to the analysis of hPS cells that includes not only single cell features but also colony wide, and more generally, multi-scale spatial configuration.

Stationary Fuel Cell Systems Analysis | Hydrogen and Fuel Cells | NREL

Science.gov Websites

Post, Presented at the 2016 DOE Annual Merit Review Meeting (June 2016) Transportation Big Data Ainscough, Sam Sprik, and Matt Post (December 2015) Stationary Fuel Cell Evaluation: 2015 Annual Merit Quarter 4 of 2014, Genevieve Saur, Jennifer Kurtz, Chris Ainscough, Sam Sprik, and Matt Post (April 2015

2015 Annual Progress Report: DOE Hydrogen and Fuel Cells Program

DOE Office of Scientific and Technical Information (OSTI.GOV)

None, None

The 2015 Annual Progress Report summarizes fiscal year 2015 activities and accomplishments by projects funded by the DOE Hydrogen and Fuel Cells Program. It covers the program areas of hydrogen production; hydrogen delivery; hydrogen storage; fuel cells; manufacturing R&D; technology validation; safety, codes and standards; systems analysis; and market transformation.

DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array.

PubMed

Douglas, Erik S; Hsiao, Sonny C; Onoe, Hiroaki; Bertozzi, Carolyn R; Francis, Matthew B; Mathies, Richard A

2009-07-21

A microdevice is developed for DNA-barcode directed capture of single cells on an array of pH-sensitive microelectrodes for metabolic analysis. Cells are modified with membrane-bound single-stranded DNA, and specific single-cell capture is directed by the complementary strand bound in the sensor area of the iridium oxide pH microelectrodes within a microfluidic channel. This bifunctional microelectrode array is demonstrated for the pH monitoring and differentiation of primary T cells and Jurkat T lymphoma cells. Single Jurkat cells exhibited an extracellular acidification rate of 11 milli-pH min(-1), while primary T cells exhibited only 2 milli-pH min(-1). This system can be used to capture non-adherent cells specifically and to discriminate between visually similar healthy and cancerous cells in a heterogeneous ensemble based on their altered metabolic properties.

DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array

PubMed Central

Douglas, Erik S.; Hsiao, Sonny C.; Onoe, Hiroaki; Bertozzi, Carolyn R.; Francis, Matthew B.; Mathies, Richard A.

2010-01-01

A microdevice is developed for DNA-barcode directed capture of single cells on an array of pH-sensitive microelectrodes for metabolic analysis. Cells are modified with membrane-bound single-stranded DNA, and specific single-cell capture is directed by the complementary strand bound in the sensor area of the iridium oxide pH microelectrodes within a microfluidic channel. This bifunctional microelectrode array is demonstrated for the pH monitoring and differentiation of primary T cells and Jurkat T lymphoma cells. Single Jurkat cells exhibited an extracellular acidification rate of 11 milli-pH min−1, while primary T cells exhibited only 2 milli-pH min−1. This system can be used to capture non-adherent cells specifically and to discriminate between visually similar healthy and cancerous cells in a heterogeneous ensemble based on their altered metabolic properties. PMID:19568668

Study of living single cells in culture: automated recognition of cell behavior.

PubMed

Bodin, P; Papin, S; Meyer, C; Travo, P

1988-07-01

An automated system capable of analyzing the behavior, in real time, of single living cells in culture, in a noninvasive and nondestructive way, has been developed. A large number of cell positions in single culture dishes were recorded using a computer controlled, robotized microscope. During subsequent observations, binary images obtained from video image analysis of the microscope visual field allowed the identification of the recorded cells. These cells could be revisited automatically every few minutes. Long-term studies of the behavior of cells make possible the analysis of cellular locomotary and mitotic activities as well as determination of cell shape (chosen from a defined library) for several hours or days in a fully automated way with observations spaced up to 30 minutes. Short-term studies of the behavior of cells permit the study, in a semiautomatic way, of acute effects of drugs (5 to 15 minutes) on changes of surface area and length of cells.

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

PubMed Central

Wu, Jincheng; Tzanakakis, Emmanuel S.

2014-01-01

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

Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; Faley, Shannon; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell death is a stochastic process, often initiated and/or executed in a multi-pathway/multi-organelle fashion. Therefore, high-throughput single-cell analysis platforms are required to provide detailed characterization of kinetics and mechanisms of cell death in heterogeneous cell populations. However, there is still a largely unmet need for inert fluorescent probes, suitable for prolonged kinetic studies. Here, we compare the use of innovative adaptation of unsymmetrical SYTO dyes for dynamic real-time analysis of apoptosis in conventional as well as microfluidic chip-based systems. We show that cyanine SYTO probes allow non-invasive tracking of intracellular events over extended time. Easy handling and “stain–no wash” protocols open up new opportunities for high-throughput analysis and live-cell sorting. Furthermore, SYTO probes are easily adaptable for detection of cell death using automated microfluidic chip-based cytometry. Overall, the combined use of SYTO probes and state-of-the-art Lab-on-a-Chip platform emerges as a cost effective solution for automated drug screening compared to conventional Annexin V or TUNEL assays. In particular, it should allow for dynamic analysis of samples where low cell number has so far been an obstacle, e.g. primary cancer stems cells or circulating minimal residual tumors. PMID:19298813

Emission spectra profiling of fluorescent proteins in living plant cells

PubMed Central

2013-01-01

Background Fluorescence imaging at high spectral resolution allows the simultaneous recording of multiple fluorophores without switching optical filters, which is especially useful for time-lapse analysis of living cells. The collected emission spectra can be used to distinguish fluorophores by a computation analysis called linear unmixing. The availability of accurate reference spectra for different fluorophores is crucial for this type of analysis. The reference spectra used by plant cell biologists are in most cases derived from the analysis of fluorescent proteins in solution or produced in animal cells, although these spectra are influenced by both the cellular environment and the components of the optical system. For instance, plant cells contain various autofluorescent compounds, such as cell wall polymers and chlorophyll, that affect the spectral detection of some fluorophores. Therefore, it is important to acquire both reference and experimental spectra under the same biological conditions and through the same imaging systems. Results Entry clones (pENTR) of fluorescent proteins (FPs) were constructed in order to create C- or N-terminal protein fusions with the MultiSite Gateway recombination technology. The emission spectra for eight FPs, fused C-terminally to the A- or B-type cyclin dependent kinases (CDKA;1 and CDKB1;1) and transiently expressed in epidermal cells of tobacco (Nicotiana benthamiana), were determined by using the Olympus FluoView™ FV1000 Confocal Laser Scanning Microscope. These experimental spectra were then used in unmixing experiments in order to separate the emission of fluorophores with overlapping spectral properties in living plant cells. Conclusions Spectral imaging and linear unmixing have a great potential for efficient multicolor detection in living plant cells. The emission spectra for eight of the most commonly used FPs were obtained in epidermal cells of tobacco leaves and used in unmixing experiments. The generated set of FP Gateway entry vectors represents a valuable resource for plant cell biologists. PMID:23552272

Blockade of the Programmed Death-1 Pathway Restores Sarcoidosis CD4+ T-Cell Proliferative Capacity

PubMed Central

Braun, Nicole A.; Celada, Lindsay J.; Herazo-Maya, Jose D.; Abraham, Susamma; Shaginurova, Guzel; Sevin, Carla M.; Grutters, Jan; Culver, Daniel A.; Dworski, Ryszard; Sheller, James; Massion, Pierre P.; Polosukhin, Vasiliy V.; Johnson, Joyce E.; Kaminski, Naftali; Wilkes, David S.; Oswald-Richter, Kyra A.

2014-01-01

Rationale: Effective therapeutic interventions for chronic, idiopathic lung diseases remain elusive. Normalized T-cell function is an important contributor to spontaneous resolution of pulmonary sarcoidosis. Up-regulation of inhibitor receptors, such as programmed death-1 (PD-1) and its ligand, PD-L1, are important inhibitors of T-cell function. Objectives: To determine the effects of PD-1 pathway blockade on sarcoidosis CD4+ T-cell proliferative capacity. Methods: Gene expression profiles of sarcoidosis and healthy control peripheral blood mononuclear cells were analyzed at baseline and follow-up. Flow cytometry was used to measure ex vivo expression of PD-1 and PD-L1 on systemic and bronchoalveolar lavage–derived cells of subjects with sarcoidosis and control subjects, as well as the effects of PD-1 pathway blockade on cellular proliferation after T-cell receptor stimulation. Immunohistochemistry analysis for PD-1/PD-L1 expression was conducted on sarcoidosis, malignant, and healthy control lung specimens. Measurements and Main Results: Microarray analysis demonstrates longitudinal increase in PDCD1 gene expression in sarcoidosis peripheral blood mononuclear cells. Immunohistochemistry analysis revealed increased PD-L1 expression within sarcoidosis granulomas and lung malignancy, but this was absent in healthy lungs. Increased numbers of sarcoidosis PD-1+ CD4+ T cells are present systemically, compared with healthy control subjects (P < 0.0001). Lymphocytes with reduced proliferative capacity exhibited increased proliferation with PD-1 pathway blockade. Longitudinal analysis of subjects with sarcoidosis revealed reduced PD-1+ CD4+ T cells with spontaneous clinical resolution but not with disease progression. Conclusions: Analogous to the effects in other chronic lung diseases, these findings demonstrate that the PD-1 pathway is an important contributor to sarcoidosis CD4+ T-cell proliferative capacity and clinical outcome. Blockade of the PD-1 pathway may be a viable therapeutic target to optimize clinical outcomes. PMID:25073001

Vision-based Nano Robotic System for High-throughput Non-embedded Cell Cutting

NASA Astrophysics Data System (ADS)

Shang, Wanfeng; Lu, Haojian; Wan, Wenfeng; Fukuda, Toshio; Shen, Yajing

2016-03-01

Cell cutting is a significant task in biology study, but the highly productive non-embedded cell cutting is still a big challenge for current techniques. This paper proposes a vision-based nano robotic system and then realizes automatic non-embedded cell cutting with this system. First, the nano robotic system is developed and integrated with a nanoknife inside an environmental scanning electron microscopy (ESEM). Then, the positions of the nanoknife and the single cell are recognized, and the distance between them is calculated dynamically based on image processing. To guarantee the positioning accuracy and the working efficiency, we propose a distance-regulated speed adapting strategy, in which the moving speed is adjusted intelligently based on the distance between the nanoknife and the target cell. The results indicate that the automatic non-embedded cutting is able to be achieved within 1-2 mins with low invasion benefiting from the high precise nanorobot system and the sharp edge of nanoknife. This research paves a way for the high-throughput cell cutting at cell’s natural condition, which is expected to make significant impact on the biology studies, especially for the in-situ analysis at cellular and subcellular scale, such as cell interaction investigation, neural signal transduction and low invasive cell surgery.

1990 fuel cell seminar: Program and abstracts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1990-12-31

This volume contains author prepared short resumes of the presentations at the 1990 Fuel Cell Seminar held November 25-28, 1990 in Phoenix, Arizona. Contained herein are 134 short descriptions organized into topic areas entitled An Environmental Overview, Transportation Applications, Technology Advancements for Molten Carbonate Fuel Cells, Technology Advancements for Solid Fuel Cells, Component Technologies and Systems Analysis, Stationary Power Applications, Marine and Space Applications, Technology Advancements for Acid Type Fuel Cells, and Technology Advancement for Solid Oxide Fuel Cells.

Comparative proteomics analysis of oral cancer cell lines: identification of cancer associated proteins

PubMed Central

2014-01-01

Background A limiting factor in performing proteomics analysis on cancerous cells is the difficulty in obtaining sufficient amounts of starting material. Cell lines can be used as a simplified model system for studying changes that accompany tumorigenesis. This study used two-dimensional gel electrophoresis (2DE) to compare the whole cell proteome of oral cancer cell lines vs normal cells in an attempt to identify cancer associated proteins. Results Three primary cell cultures of normal cells with a limited lifespan without hTERT immortalization have been successfully established. 2DE was used to compare the whole cell proteome of these cells with that of three oral cancer cell lines. Twenty four protein spots were found to have changed in abundance. MALDI TOF/TOF was then used to determine the identity of these proteins. Identified proteins were classified into seven functional categories – structural proteins, enzymes, regulatory proteins, chaperones and others. IPA core analysis predicted that 18 proteins were related to cancer with involvements in hyperplasia, metastasis, invasion, growth and tumorigenesis. The mRNA expressions of two proteins – 14-3-3 protein sigma and Stress-induced-phosphoprotein 1 – were found to correlate with the corresponding proteins’ abundance. Conclusions The outcome of this analysis demonstrated that a comparative study of whole cell proteome of cancer versus normal cell lines can be used to identify cancer associated proteins. PMID:24422745

Flow cells for bioanalytical and bioprocess applications with optimized dynamic response and flow characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lancaster, V.R.; Modlin, D.N.

1994-12-31

In this study, the authors present a method for design and characterization of flow cells developed for minimum flow volume and optimal dynamic response with a given central observation area. The dynamic response of a circular shaped dual ported flow cell was compared to that obtained from a flow cell whose optimized shape was determined using this method. In the optimized flow cell design, the flow rate at the nominal operating pressure increased by 50% whereas the flow cell volume was reduced by 70%. In addition, the dynamic response of the new flow cell was found to be 200% fastermore » than the circular flow cell. The fluid dynamic analysis included simple graphical techniques utilizing free stream vorticity functions and Hagen-Poiseuille relationships. The flow cell dynamic response was measured using a fluorescence detection system. The fluoresce in emission from a 400{micro}m spot located at the exit port was measured as a function of time after switching the input to the flow cell between fluorescent and non-fluorescent solutions. Analysis of results revealed the system could be reasonably characterized as a first order dynamic system. Although some evidence of second order behavior was also observed, it is reasonable to assume that a first order model will provide adequate predictive capability for many real world applications. Given a set of flow cell requirements, the methods presented in this study can be used to design and characterize flow cells with lower reagent consumption and reduced purging times. These improvements can be readily translated into reduced process times and/or lower usage of high cost reagents.« less

Integrated microfluidic systems for cell lysis, mixing/pumping and DNA amplification

NASA Astrophysics Data System (ADS)

Lee, Chia-Yen; Lee, Gwo-Bin; Lin, Jr-Lung; Huang, Fu-Chun; Liao, Chia-Sheng

2005-06-01

The present paper reports a fully automated microfluidic system for the DNA amplification process by integrating an electroosmotic pump, an active micromixer and an on-chip temperature control system. In this DNA amplification process, the cell lysis is initially performed in a micro cell lysis reactor. Extracted DNA samples, primers and reagents are then driven electroosmotically into a mixing region where they are mixed by the active micromixer. The homogeneous mixture is then thermally cycled in a micro-PCR (polymerase chain reaction) chamber to perform DNA amplification. Experimental results show that the proposed device can successfully automate the sample pretreatment operation for DNA amplification, thereby delivering significant time and effort savings. The new microfluidic system, which facilitates cell lysis, sample driving/mixing and DNA amplification, could provide a significant contribution to ongoing efforts to miniaturize bio-analysis systems by utilizing a simple fabrication process and cheap materials.

Circulating and disseminated tumor cells: diagnostic tools and therapeutic targets in motion

PubMed Central

Lin, Peter P.; Gires, Olivier

2017-01-01

Enumeration of circulating tumor cells (CTCs) in peripheral blood with the gold standard CellSearchTM has proven prognostic value for tumor recurrence and progression of metastatic disease. Therefore, the further molecular characterization of isolated CTCs might have clinical relevance as liquid biopsy for therapeutic decision-making and to monitor disease progression. The direct analysis of systemic cancer appears particularly important in view of the known disparity in expression of therapeutic targets as well as epithelial-to-mesenchymal transition (EMT)-based heterogeneity between primary and systemic tumor cells, which all substantially complicate monitoring and therapeutic targeting at present. Since CTCs are the potential precursor cells of metastasis, their in-depth molecular profiling should also provide a useful resource for target discovery. The present review will discuss the use of systemically spread cancer cells as liquid biopsy and focus on potential target antigens. PMID:27683128

Efficiency and cost analysis of cell saver auto transfusion system in total knee arthroplasty.

PubMed

Bilgili, Mustafa Gökhan; Erçin, Ersin; Peker, Gökhan; Kural, Cemal; Başaran, Serdar Hakan; Duramaz, Altuğ; Avkan, Cevdet

2014-06-01

Blood loss and replacement is still a controversial issue in major orthopaedic surgery. Allogenic blood transfusion may cause legal problems and concerns regarding the transmission of transfusion-related diseases. Cellsaver Systems (CSS) were developed as an alternative to allogenic transfusion but CSS transfusion may cause coagulation, infection and haemodynamic instability. Our aim was to analyse the efficiency and cost analysis of a cell saver auto-transfusion system in the total knee arthroplasty procedure. Retrospective comparative study. Those patients who were operated on by unilateral, cemented total knee arthroplasty (TKA) were retrospectively evaluated. Group 1 included 37 patients who were treated using the cell saver system, and Group 2 involved 39 patients who were treated by allogenic blood transfusion. The groups were compared in terms of preoperative haemoglobin and haematocrit levels, blood loss and transfusion amount, whether allogenic transfusion was made, degree of deformity, body mass index and cost. No significant results could be obtained in the statistical comparisons made in terms of the demographic properties, deformity properties, preoperative laboratory values, transfusion amount and length of hospital stay of the groups. Average blood loss was calculated to be less in Group 1 (p<0.05) and cost was higher in Group 1 (p<0.05). Cell saver systems do not decrease the amount of allogenic blood transfusion and costs more. Therefore, the routine usage of the auto-transfusion systems is a controversial issue. Cell saver system usage does not affect allogenic blood transfusion incidence or allogenic blood transfusion volume. It was found that preoperative haemoglobin and body mass index rates may affect allogenic blood transfusion. Therefore, it is foreseen that auto-transfusion systems could be useful in patients with low haemoglobin level and body mass index.

The role of radiation hard solar cells in minimizing the costs of global satellite communication systems

NASA Technical Reports Server (NTRS)

Summers, Geoffrey P.; Walters, Robert J.; Messenger, Scott R.; Burke, Edward A.

1996-01-01

An analysis embodied in a PC computer program is presented, which quantitatively demonstrates how the availability of radiation hard solar cells can help minimize the cost of a global satellite communications system. An important distinction between the currently proposed systems, such as Iridium, Odyssey and Ellipsat, is the number of satellites employed and their operating altitudes. Analysis of the major costs associated with implementing these systems shows that operation at orbital altitudes within the earth's radiation belts (10(exp 3) to 10(exp 4)km) can reduce the total cost of a system by several hundred percent, so long as radiation hard components including solar cells can be used. A detailed evaluation of the predicted performance of photovoltaic arrays using several different planar solar cell technologies is given, including commercially available Si and GaAs/Ge, and InP/Si which is currently under development. Several examples of applying the program are given, which show that the end of life (EOL) power density of different technologies can vary by a factor of ten for certain missions. Therefore, although a relatively radiation-soft technology can usually provide the required EOL power by simply increasing the size of the array, the impact upon the total system budget could be unacceptable, due to increased launch and hardware costs. In aggregate, these factors can account for more than a 10% increase in the total system cost. Since the estimated total costs of proposed global-coverage systems range from $1B to $9B, the availability of radiation-hard solar cells could make a decisive difference in the selection of a particular constellation architecture.

HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

NASA Astrophysics Data System (ADS)

Sebesta, Mikael; Egelberg, Peter J.; Langberg, Anders; Lindskov, Jens-Henrik; Alm, Kersti; Janicke, Birgit

2016-03-01

Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system - hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

Performance benchmarking of four cell-free protein expression systems.

PubMed

Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

2016-02-01

Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins. © 2015 Wiley Periodicals, Inc.

Quantitative analysis of gold nanoparticles in single cells by laser ablation inductively coupled plasma-mass spectrometry.

PubMed

Wang, Meng; Zheng, Ling-Na; Wang, Bing; Chen, Han-Qing; Zhao, Yu-Liang; Chai, Zhi-Fang; Reid, Helen J; Sharp, Barry L; Feng, Wei-Yue

2014-10-21

Single cell analysis has become an important field of research in recent years reflecting the heterogeneity of cellular responses in biological systems. Here, we demonstrate a new method, based on laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS), which can quantify in situ gold nanoparticles (Au NPs) in single cells. Dried residues of picoliter droplets ejected by a commercial inkjet printer were used to simulate matrix-matched calibration standards. The gold mass in single cells exposed to 100 nM NIST Au NPs (Reference material 8012, 30 nm) for 4 h showed a log-normal distribution, ranging from 1.7 to 72 fg Au per cell, which approximately corresponds to 9 to 370 Au NPs per cell. The average result from 70 single cells (15 ± 13 fg Au per cell) was in good agreement with the result from an aqua regia digest solution of 1.2 × 10(6) cells (18 ± 1 fg Au per cell). The limit of quantification was 1.7 fg Au. This paper demonstrates the great potential of LA-ICPMS for single cell analysis and the beneficial study of biological responses to metal drugs or NPs at the single cell level.

Design of five-layer gold nanoparticles self-assembled in a liquid open tubular column for ultrasensitive nano-LC-MS/MS proteomic analysis of 80 living cells.

PubMed

Shao, Xi; Zhang, Xiangmin

2017-04-01

In this work, for the first time, a liquid open tubular column modified by five-layer gold nanoparticles and linked with C 18 (GNPs@C 18 ) was designed and fabricated for nano-LC-MS/MS analysis of 80 living cells. Sixty nanometer gold nanoparticles were self-assembled layer by layer on the inner wall of a 20 μm id fused-silica capillary. C 18 was then linked on the gold nanoparticles to make the liquid open tubular column show hydrophobic character. Enough loading capacities for analysis of 80 living cells, ∼100 fmol for pk-10 and ∼30 fmol for insulin, were obtained with the 2 m × 20 μm id five-layer GNPs@C 18 open tubular column. The open tubular column was used in an online pretreatment and direct nano-LC-MS/MS analysis system to analyze 80 living HepG2 cells. In total, 650 proteins were identified in triplicate runs. The subcellular localization of the identified proteins showed that our system had no bias toward different cellular compartments. Protein copy number per cell of the identified proteins showed that the detection limit could reach 50 zmol and the abundance of the identified proteins could cover a dynamic range of 6 orders. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Current-Controlled Electrical Point-Source Stimulation of Embryonic Stem Cells

PubMed Central

Chen, Michael Q.; Xie, Xiaoyan; Wilson, Kitchener D.; Sun, Ning; Wu, Joseph C.; Giovangrandi, Laurent; Kovacs, Gregory T. A.

2010-01-01

Stem cell therapy is emerging as a promising clinical approach for myocardial repair. However, the interactions between the graft and host, resulting in inconsistent levels of integration, remain largely unknown. In particular, the influence of electrical activity of the surrounding host tissue on graft differentiation and integration is poorly understood. In order to study this influence under controlled conditions, an in vitro system was developed. Electrical pacing of differentiating murine embryonic stem (ES) cells was performed at physiologically relevant levels through direct contact with microelectrodes, simulating the local activation resulting from contact with surrounding electroactive tissue. Cells stimulated with a charged balanced voltage-controlled current source for up to 4 days were analyzed for cardiac and ES cell gene expression using real-time PCR, immunofluorescent imaging, and genome microarray analysis. Results varied between ES cells from three progressive differentiation stages and stimulation amplitudes (nine conditions), indicating a high sensitivity to electrical pacing. Conditions that maximally encouraged cardiomyocyte differentiation were found with Day 7 EBs stimulated at 30 µA. The resulting gene expression included a sixfold increase in troponin-T and a twofold increase in β-MHCwithout increasing ES cell proliferation marker Nanog. Subsequent genome microarray analysis revealed broad transcriptome changes after pacing. Concurrent to upregulation of mature gene programs including cardiovascular, neurological, and musculoskeletal systems is the apparent downregulation of important self-renewal and pluripotency genes. Overall, a robust system capable of long-term stimulation of ES cells is demonstrated, and specific conditions are outlined that most encourage cardiomyocyte differentiation. PMID:20652088

Resolving stem and progenitor cells in the adult mouse incisor through gene co-expression analysis

PubMed Central

Seidel, Kerstin; Marangoni, Pauline; Tang, Cynthia; Houshmand, Bahar; Du, Wen; Maas, Richard L; Murray, Steven; Oldham, Michael C; Klein, Ophir D

2017-01-01

Investigations into stem cell-fueled renewal of an organ benefit from an inventory of cell type-specific markers and a deep understanding of the cellular diversity within stem cell niches. Using the adult mouse incisor as a model for a continuously renewing organ, we performed an unbiased analysis of gene co-expression relationships to identify modules of co-expressed genes that represent differentiated cells, transit-amplifying cells, and residents of stem cell niches. Through in vivo lineage tracing, we demonstrated the power of this approach by showing that co-expression module members Lrig1 and Igfbp5 define populations of incisor epithelial and mesenchymal stem cells. We further discovered that two adjacent mesenchymal tissues, the periodontium and dental pulp, are maintained by distinct pools of stem cells. These findings reveal novel mechanisms of incisor renewal and illustrate how gene co-expression analysis of intact biological systems can provide insights into the transcriptional basis of cellular identity. DOI: http://dx.doi.org/10.7554/eLife.24712.001 PMID:28475038

Sensing in tissue bioreactors

NASA Astrophysics Data System (ADS)

Rolfe, P.

2006-03-01

Specialized sensing and measurement instruments are under development to aid the controlled culture of cells in bioreactors for the fabrication of biological tissues. Precisely defined physical and chemical conditions are needed for the correct culture of the many cell-tissue types now being studied, including chondrocytes (cartilage), vascular endothelial cells and smooth muscle cells (blood vessels), fibroblasts, hepatocytes (liver) and receptor neurones. Cell and tissue culture processes are dynamic and therefore, optimal control requires monitoring of the key process variables. Chemical and physical sensing is approached in this paper with the aim of enabling automatic optimal control, based on classical cell growth models, to be achieved. Non-invasive sensing is performed via the bioreactor wall, invasive sensing with probes placed inside the cell culture chamber and indirect monitoring using analysis within a shunt or a sampling chamber. Electroanalytical and photonics-based systems are described. Chemical sensing for gases, ions, metabolites, certain hormones and proteins, is under development. Spectroscopic analysis of the culture medium is used for measurement of glucose and for proteins that are markers of cell biosynthetic behaviour. Optical interrogation of cells and tissues is also investigated for structural analysis based on scatter.

Validation of cytogenetic risk groups according to International Prognostic Scoring Systems by peripheral blood CD34+FISH: results from a German diagnostic study in comparison with an international control group.

PubMed

Braulke, Friederike; Platzbecker, Uwe; Müller-Thomas, Catharina; Götze, Katharina; Germing, Ulrich; Brümmendorf, Tim H; Nolte, Florian; Hofmann, Wolf-Karsten; Giagounidis, Aristoteles A N; Lübbert, Michael; Greenberg, Peter L; Bennett, John M; Solé, Francesc; Mallo, Mar; Slovak, Marilyn L; Ohyashiki, Kazuma; Le Beau, Michelle M; Tüchler, Heinz; Pfeilstöcker, Michael; Nösslinger, Thomas; Hildebrandt, Barbara; Shirneshan, Katayoon; Aul, Carlo; Stauder, Reinhard; Sperr, Wolfgang R; Valent, Peter; Fonatsch, Christa; Trümper, Lorenz; Haase, Detlef; Schanz, Julie

2015-02-01

International Prognostic Scoring Systems are used to determine the individual risk profile of myelodysplastic syndrome patients. For the assessment of International Prognostic Scoring Systems, an adequate chromosome banding analysis of the bone marrow is essential. Cytogenetic information is not available for a substantial number of patients (5%-20%) with dry marrow or an insufficient number of metaphase cells. For these patients, a valid risk classification is impossible. In the study presented here, the International Prognostic Scoring Systems were validated based on fluorescence in situ hybridization analyses using extended probe panels applied to cluster of differentiation 34 positive (CD34(+)) peripheral blood cells of 328 MDS patients of our prospective multicenter German diagnostic study and compared to chromosome banding results of 2902 previously published patients with myelodysplastic syndromes. For cytogenetic risk classification by fluorescence in situ hybridization analyses of CD34(+) peripheral blood cells, the groups differed significantly for overall and leukemia-free survival by uni- and multivariate analyses without discrepancies between treated and untreated patients. Including cytogenetic data of fluorescence in situ hybridization analyses of peripheral CD34(+) blood cells (instead of bone marrow banding analysis) into the complete International Prognostic Scoring System assessment, the prognostic risk groups separated significantly for overall and leukemia-free survival. Our data show that a reliable stratification to the risk groups of the International Prognostic Scoring Systems is possible from peripheral blood in patients with missing chromosome banding analysis by using a comprehensive probe panel (clinicaltrials.gov identifier:01355913). Copyright© Ferrata Storti Foundation.

Innovation in stem cell advocacy: you only get what you can measure.

PubMed

Jakimo, Alan L; Fernandez, Alan C

2011-11-01

We propose that stem cell advocacy must engage in self-analysis to determine how to be maximally effective. For this analysis, eight advocacy elements can be measured: agitation, legislation, regulation, litigation, policy development, collaboration, education and innovation. For several of these elements, we show that stem cell advocates, particularly advocates for human embryonic stem cell research, have been matched by their opponents. This demonstrates the need for combining innovation and collaboration with advocacy-oriented education. To pursue innovative and collaborative education, we propose a 'bench-to-public knowledge' model and present some preliminary observations made with this model for different stem cell types. We also propose development of a semantic web information system to be operated within Internet Cloud/Apps/Social Media. We call this system the 'Stem Cell Information Technology Accelerator Platform'. Toward its construction, we propose formation of a working group to conceive semantic web ontology for stem cell science and its clinical translation into medicine. This ontology would function as a map of the relationships between and among the various informational components comprising discourse on stem cell research and its clinical translation, and would allow various stakeholders to contribute to evolving models of that science and translation. These models could, in turn, support an innovative and collaborative approach to education in furtherance of stem cell advocacy.

Time-Resolved Analysis of Cytosolic and Surface-Associated Proteins of Staphylococcus aureus HG001 under Planktonic and Biofilm Conditions.

PubMed

Moche, Martin; Schlüter, Rabea; Bernhardt, Jörg; Plate, Kristina; Riedel, Katharina; Hecker, Michael; Becher, Dörte

2015-09-04

Staphylococcal biofilms are associated with persistent infections due to their capacity to protect bacteria against the host's immune system and antibiotics. Cell-surface-associated proteins are of great importance during biofilm formation. In the present study, an optimized biotinylation approach for quantitative GeLC-MS-based analysis of the staphylococcal cell-surface proteome was applied and the cytoplasmic protein fraction was analyzed to elucidate proteomic differences between colony biofilms and planktonic cells. The experimental setup enabled a time-resolved monitoring of the proteome under both culture conditions and the comparison of biofilm cells to planktonic cells at several time points. This allowed discrimination of differences attributed to delayed growth phases from responses provoked by biofilm conditions. Biofilm cells expressed CcpA-dependent catabolic proteins earlier than planktonic cells and strongly accumulated proteins that belong to the SigB stress regulon. The amount of the cell-surface protein and virulence gene regulator Rot decreased within biofilms and MgrA-dependent regulations appeared more pronounced. Biofilm cells simultaneously up-regulated activators (e.g., SarZ) as well as repressors (e.g., SarX) of RNAIII. A decreased amount of high-affinity iron uptake systems and an increased amount of the iron-storage protein FtnA possibly indicated a lower demand of iron in biofilms.

A novel portable filtration system for sampling and concentration of microorganisms: Demonstration on marine microalgae with subsequent quantification using IC-NASBA.

PubMed

Loukas, Christos-Moritz; Mowlem, Matthew C; Tsaloglou, Maria-Nefeli; Green, Nicolas G

2018-05-01

This paper presents a novel portable sample filtration/concentration system, designed for use on samples of microorganisms with very low cell concentrations and large volumes, such as water-borne parasites, pathogens associated with faecal matter, or toxic phytoplankton. The example application used for demonstration was the in-field collection and concentration of microalgae from seawater samples. This type of organism is responsible for Harmful Algal Blooms (HABs), an example of which is commonly referred to as "red tides", which are typically the result of rapid proliferation and high biomass accumulation of harmful microalgal species in the water column or at the sea surface. For instance, Karenia brevis red tides are the cause of aquatic organism mortality and persistent blooms may cause widespread die-offs of populations of other organisms including vertebrates. In order to respond to, and adequately manage HABs, monitoring of toxic microalgae is required and large-volume sample concentrators would be a useful tool for in situ monitoring of HABs. The filtering system presented in this work enables consistent sample collection and concentration from 1 L to 1 mL in five minutes, allowing for subsequent benchtop sample extraction and analysis using molecular methods such as NASBA and IC-NASBA. The microalga Tetraselmis suecica was successfully detected at concentrations ranging from 2 × 10 5  cells/L to 20 cells/L. Karenia brevis was also detected and quantified at concentrations between 10 cells/L and 10 6  cells/L. Further analysis showed that the filter system, which concentrates cells from very large volumes with consequently more reliable sampling, produced samples that were more consistent than the independent non-filtered samples (benchtop controls), with a logarithmic dependency on increasing cell numbers. This filtering system provides simple, rapid, and consistent sample collection and concentration for further analysis, and could be applied to a wide range of different samples and target organisms in situations lacking laboratories. Copyright © 2018. Published by Elsevier B.V.

Nanoscale live cell optical imaging of the dynamics of intracellular microvesicles in neural cells.

PubMed

Lee, Sohee; Heo, Chaejeong; Suh, Minah; Lee, Young Hee

2013-11-01

Recent advances in biotechnology and imaging technology have provided great opportunities to investigate cellular dynamics. Conventional imaging methods such as transmission electron microscopy, scanning electron microscopy, and atomic force microscopy are powerful techniques for cellular imaging, even at the nanoscale level. However, these techniques have limitations applications in live cell imaging because of the experimental preparation required, namely cell fixation, and the innately small field of view. In this study, we developed a nanoscale optical imaging (NOI) system that combines a conventional optical microscope with a high resolution dark-field condenser (Cytoviva, Inc.) and halogen illuminator. The NOI system's maximum resolution for live cell imaging is around 100 nm. We utilized NOI to investigate the dynamics of intracellular microvesicles of neural cells without immunocytological analysis. In particular, we studied direct, active random, and moderate random dynamic motions of intracellular microvesicles and visualized lysosomal vesicle changes after treatment of cells with a lysosomal inhibitor (NH4Cl). Our results indicate that the NOI system is a feasible, high-resolution optical imaging system for live small organelles that does not require complicated optics or immunocytological staining processes.

Simulation of a 250 kW diesel fuel processor/PEM fuel cell system

NASA Astrophysics Data System (ADS)

Amphlett, J. C.; Mann, R. F.; Peppley, B. A.; Roberge, P. R.; Rodrigues, A.; Salvador, J. P.

Polymer-electrolyte membrane (PEM) fuel cell systems offer a potential power source for utility and mobile applications. Practical fuel cell systems use fuel processors for the production of hydrogen-rich gas. Liquid fuels, such as diesel or other related fuels, are attractive options as feeds to a fuel processor. The generation of hydrogen gas for fuel cells, in most cases, becomes the crucial design issue with respect to weight and volume in these applications. Furthermore, these systems will require a gas clean-up system to insure that the fuel quality meets the demands of the cell anode. The endothermic nature of the reformer will have a significant affect on the overall system efficiency. The gas clean-up system may also significantly effect the overall heat balance. To optimize the performance of this integrated system, therefore, waste heat must be used effectively. Previously, we have concentrated on catalytic methanol-steam reforming. A model of a methanol steam reformer has been previously developed and has been used as the basis for a new, higher temperature model for liquid hydrocarbon fuels. Similarly, our fuel cell evaluation program previously led to the development of a steady-state electrochemical fuel cell model (SSEM). The hydrocarbon fuel processor model and the SSEM have now been incorporated in the development of a process simulation of a 250 kW diesel-fueled reformer/fuel cell system using a process simulator. The performance of this system has been investigated for a variety of operating conditions and a preliminary assessment of thermal integration issues has been carried out. This study demonstrates the application of a process simulation model as a design analysis tool for the development of a 250 kW fuel cell system.

FLOCK cluster analysis of plasma cell flow cytometry data predicts bone marrow involvement by plasma cell neoplasia.

PubMed

Dorfman, David M; LaPlante, Charlotte D; Li, Betty

2016-09-01

We analyzed plasma cell populations in bone marrow samples from 353 patients with possible bone marrow involvement by a plasma cell neoplasm, using FLOCK (FLOw Clustering without K), an unbiased, automated, computational approach to identify cell subsets in multidimensional flow cytometry data. FLOCK identified discrete plasma cell populations in the majority of bone marrow specimens found by standard histologic and immunophenotypic criteria to be involved by a plasma cell neoplasm (202/208 cases; 97%), including 34 cases that were negative by standard flow cytometric analysis that included clonality assessment. FLOCK identified discrete plasma cell populations in only a minority of cases negative for involvement by a plasma cell neoplasm by standard histologic and immunophenotypic criteria (38/145 cases; 26%). Interestingly, 55% of the cases negative by standard analysis, but containing a FLOCK-identified discrete plasma cell population, were positive for monoclonal gammopathy by serum protein electrophoresis and immunofixation. FLOCK-identified and quantitated plasma cell populations accounted for 3.05% of total cells on average in cases positive for involvement by a plasma cell neoplasm by standard histologic and immunophenotypic criteria, and 0.27% of total cells on average in cases negative for involvement by a plasma cell neoplasm by standard histologic and immunophenotypic criteria (p<0.0001; area under the curve by ROC analysis=0.96). The presence of a FLOCK-identified discrete plasma cell population was predictive of the presence of plasma cell neoplasia with a sensitivity of 97%, compared with only 81% for standard flow cytometric analysis, and had specificity of 74%, PPV of 84% and NPV of 95%. FLOCK analysis, which has been shown to provide useful diagnostic information for evaluating patients with suspected systemic mastocytosis, is able to identify neoplastic plasma cell populations analyzed by flow cytometry, and may be helpful in the diagnostic evaluation of bone marrow samples for involvement by plasma cell neoplasia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Concanavalin A-mediated polyclonal helper assay of normal thymocytes and its use for the analysis of ontogeny.

PubMed

Kina, T; Nishikawa, S; Amagai, T; Katsura, Y

1987-01-01

A concanavalin A (Con A)-mediated polyclonal helper assay system was established by using the thymus cells or splenic T cells as a source of helper T cells. When splenic B cells were cocultured with thymus cells or splenic Lyt-2- T cells in the presence of an optimal dose of Con A, B Cells were polyclonally activated and differentiated into immunoglobulin-secreting cells. This Con A-mediated helper activity was completely inhibited by the addition of alpha-methyl-D-mannoside and could not be substituted by culture supernatant of Con A-stimulated thymocytes or splenic T cells. Almost all the activity of the thymus cells was carried by peanut agglutinin low binding population. Genetic restriction between T and B cells was not observed in this helper function. In ontogeny, Con A-mediated helper activity in the thymus was first detected at a few days after birth and reached the adult level at about 1 week of age. The polyclonal helper assay system developed in the present study provides a sensitive system to analyse the helper function of thymus cells and also to delineate the early phase of the differentiation of helper T cell population.

Detection of early changes in lung cell cytology by flow-systems analysis techniques. Progress report, October 1, 1976--June 30, 1977. [Damage induced by exposure to toxic agents associated with production of synthetic fuels from oil shale and coal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinkamp, J.A.; Hansen, K.M.; Wilson, J.S.

1977-07-01

This report summarizes results of continuing experiments to develop cytological and biochemical indicators for estimating damage to respiratory tract cells in animals exposed to toxic agents associated with production of synthetic fuels from oil shale and coal, the specific goal being the application of advanced flow-systems technologies to the detection of early atypical cellular changes in lung epithelium. The objectives of the program during the past 6 months were: to develop standard methods for lavaging lungs of several rodent species (hamster, rat, and mouse) to increase cell yield; initiate oil shale exposures in hamsters and rats; study the effects ofmore » macrophage mobility in the presence of oil shale; and determine the effects of different fixatives on lung cell morphology using electron microscopy. To develop standard methods for lavaging the respiratory tract of test animals, experiments were devised to increase cell yield with minimal debris and blood. Proteolytic enzymes such as trypsin were also tested but produced excessive amounts of fibrinated blood. Experimental animals were exposed to raw and spent oil shale particulates to determine if changes in lung cell differential counts and/or atypical cellular changes were noted. Since the multiparameter cell separator system was inoperative during this reporting period due to major modifications, including the addition of an uv krypton laser, emphasis was primarily on cytological techniques. As the flow-systems instrumentation becomes fully operational during the next month, automated analysis of respiratory tract cells and measurement of physical and biochemical properties as a function of exposure to toxic agents will continue.« less

An accelerated framework for the classification of biological targets from solid-state micropore data.

PubMed

Hanif, Madiha; Hafeez, Abdul; Suleman, Yusuf; Mustafa Rafique, M; Butt, Ali R; Iqbal, Samir M

2016-10-01

Micro- and nanoscale systems have provided means to detect biological targets, such as DNA, proteins, and human cells, at ultrahigh sensitivity. However, these devices suffer from noise in the raw data, which continues to be significant as newer and devices that are more sensitive produce an increasing amount of data that needs to be analyzed. An important dimension that is often discounted in these systems is the ability to quickly process the measured data for an instant feedback. Realizing and developing algorithms for the accurate detection and classification of biological targets in realtime is vital. Toward this end, we describe a supervised machine-learning approach that records single cell events (pulses), computes useful pulse features, and classifies the future patterns into their respective types, such as cancerous/non-cancerous cells based on the training data. The approach detects cells with an accuracy of 70% from the raw data followed by an accurate classification when larger training sets are employed. The parallel implementation of the algorithm on graphics processing unit (GPU) demonstrates a speedup of three to four folds as compared to a serial implementation on an Intel Core i7 processor. This incredibly efficient GPU system is an effort to streamline the analysis of pulse data in an academic setting. This paper presents for the first time ever, a non-commercial technique using a GPU system for realtime analysis, paired with biological cluster targeting analysis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Solid Oxide Fuel Cell/Gas Turbine Hybrid Cycle Technology for Auxiliary Aerospace Power

NASA Technical Reports Server (NTRS)

Steffen, Christopher J., Jr.; Freeh, Joshua E.; Larosiliere, Louis M.

2005-01-01

A notional 440 kW auxiliary power unit has been developed for 300 passenger commercial transport aircraft in 2015AD. A hybrid engine using solid-oxide fuel cell stacks and a gas turbine bottoming cycle has been considered. Steady-state performance analysis during cruise operation has been presented. Trades between performance efficiency and system mass were conducted with system specific energy as the discriminator. Fuel cell performance was examined with an area specific resistance. The ratio of fuel cell versus turbine power was explored through variable fuel utilization. Area specific resistance, fuel utilization, and mission length had interacting effects upon system specific energy. During cruise operation, the simple cycle fuel cell/gas turbine hybrid was not able to outperform current turbine-driven generators for system specific energy, despite a significant improvement in system efficiency. This was due in part to the increased mass of the hybrid engine, and the increased water flow required for on-board fuel reformation. Two planar, anode-supported cell design concepts were considered. Designs that seek to minimize the metallic interconnect layer mass were seen to have a large effect upon the system mass estimates.

Imaging of blood cells based on snapshot Hyper-Spectral Imaging systems

NASA Astrophysics Data System (ADS)

Robison, Christopher J.; Kolanko, Christopher; Bourlai, Thirimachos; Dawson, Jeremy M.

2015-05-01

Snapshot Hyper-Spectral imaging systems are capable of capturing several spectral bands simultaneously, offering coregistered images of a target. With appropriate optics, these systems are potentially able to image blood cells in vivo as they flow through a vessel, eliminating the need for a blood draw and sample staining. Our group has evaluated the capability of a commercial Snapshot Hyper-Spectral imaging system, the Arrow system from Rebellion Photonics, in differentiating between white and red blood cells on unstained blood smear slides. We evaluated the imaging capabilities of this hyperspectral camera; attached to a microscope at varying objective powers and illumination intensity. Hyperspectral data consisting of 25, 443x313 hyperspectral bands with ~3nm spacing were captured over the range of 419 to 494nm. Open-source hyper-spectral data cube analysis tools, used primarily in Geographic Information Systems (GIS) applications, indicate that white blood cells features are most prominent in the 428-442nm band for blood samples viewed under 20x and 50x magnification over a varying range of illumination intensities. These images could potentially be used in subsequent automated white blood cell segmentation and counting algorithms for performing in vivo white blood cell counting.

Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

PubMed

Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

2010-01-01

Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

Automatic analysis of the micronucleus test in primary human lymphocytes using image analysis.

PubMed

Frieauff, W; Martus, H J; Suter, W; Elhajouji, A

2013-01-01

The in vitro micronucleus test (MNT) is a well-established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and a specific parameter. Various automated systems to replace the tedious and time-consuming visual slide analysis procedure as well as flow cytometric approaches have been discussed. The ROBIAS (Robotic Image Analysis System) for both automatic cytotoxicity assessment and micronucleus detection in human lymphocytes was developed at Novartis where the assay has been used to validate positive results obtained in the MNT in TK6 cells, which serves as the primary screening system for genotoxicity profiling in early drug development. In addition, the in vitro MNT has become an accepted alternative to support clinical studies and will be used for regulatory purposes as well. The comparison of visual with automatic analysis results showed a high degree of concordance for 25 independent experiments conducted for the profiling of 12 compounds. For concentration series of cyclophosphamide and carbendazim, a very good correlation between automatic and visual analysis by two examiners could be established, both for the relative division index used as cytotoxicity parameter, as well as for micronuclei scoring in mono- and binucleated cells. Generally, false-positive micronucleus decisions could be controlled by fast and simple relocation of the automatically detected patterns. The possibility to analyse 24 slides within 65h by automatic analysis over the weekend and the high reproducibility of the results make automatic image processing a powerful tool for the micronucleus analysis in primary human lymphocytes. The automated slide analysis for the MNT in human lymphocytes complements the portfolio of image analysis applications on ROBIAS which is supporting various assays at Novartis.

Economic competitiveness of fuel cell onsite integrated energy systems

NASA Technical Reports Server (NTRS)

Bollenbacher, G.

1983-01-01

The economic competitiveness of fuel cell onsite integrated energy systems (OS/IES) in residential and commercial buildings is examined. The analysis is carried out for three different buildings with each building assumed to be at three geographic locations spanning a range of climatic conditions. Numerous design options and operating strategies are evaluated and two economic criteria are used to measure economic performance. In general the results show that fuel cell OS/IES's are competitive in most regions of the country if the OS/IES is properly designed. The preferred design is grid connected, makes effective use of the fuel cell's thermal output, and has a fuel cell powerplant sized for the building's base electrical load.

Qualification of computerized monitoring systems in a cell therapy facility compliant with the good manufacturing practices.

PubMed

Del Mazo-Barbara, Anna; Mirabel, Clémentine; Nieto, Valentín; Reyes, Blanca; García-López, Joan; Oliver-Vila, Irene; Vives, Joaquim

2016-09-01

Computerized systems (CS) are essential in the development and manufacture of cell-based medicines and must comply with good manufacturing practice, thus pushing academic developers to implement methods that are typically found within pharmaceutical industry environments. Qualitative and quantitative risk analyses were performed by Ishikawa and Failure Mode and Effects Analysis, respectively. A process for qualification of a CS that keeps track of environmental conditions was designed and executed. The simplicity of the Ishikawa analysis permitted to identify critical parameters that were subsequently quantified by Failure Mode Effects Analysis, resulting in a list of test included in the qualification protocols. The approach presented here contributes to simplify and streamline the qualification of CS in compliance with pharmaceutical quality standards.

Detection of early changes in lung cell cytology by flow-systems analysis techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinkamp, J.A.; Hansen, K.M.; Wilson, J.S.

1976-12-01

This report summarizes results of continuing experiments to develop cytological and biochemical indicators for estimating damage to respiratory cells in test animals exposed by inhalation to toxic agents associated with nonnuclear energy production, the specific goal being the application of advanced multiparameter flow-systems technologies to the detection of early atypical cellular changes in lung epithelium. Normal Syrian hamster lung cell samples composed of macrophages, leukocytes, ciliated columnar cells, and epithelial cells were stained with fluorescent dyes specific for different biochemical parameters and were analyzed in liquid suspension as they flowed through a chamber intersecting a laser beam of exciting light.more » Multiple sensors measured the total or two-color fluorescence and light scatter on a cell-by-cell basis. Cellular parameters proportional to optical measurements (i.e., cell size, DNA content, total protein, nonspecific esterase activity, nuclear and cytoplasmic diameters) were displayed as frequency distribution histograms. Lung cell samples were also separated according to various cytological parameters and identified microscopically. The basic operating features of the methodology are discussed briefly, along with specific examples of preliminary results illustrating the initial characterization of exfoliated pulmonary cells from normal hamsters. As the flow technology is adapted further to the analysis of respiratory cells, measurements of changes in physical and biochemical properties as a function of exposure to toxic agents will be performed.« less

Reliability analysis and initial requirements for FC systems and stacks

NASA Astrophysics Data System (ADS)

Åström, K.; Fontell, E.; Virtanen, S.

In the year 2000 Wärtsilä Corporation started an R&D program to develop SOFC systems for CHP applications. The program aims to bring to the market highly efficient, clean and cost competitive fuel cell systems with rated power output in the range of 50-250 kW for distributed generation and marine applications. In the program Wärtsilä focuses on system integration and development. System reliability and availability are key issues determining the competitiveness of the SOFC technology. In Wärtsilä, methods have been implemented for analysing the system in respect to reliability and safety as well as for defining reliability requirements for system components. A fault tree representation is used as the basis for reliability prediction analysis. A dynamic simulation technique has been developed to allow for non-static properties in the fault tree logic modelling. Special emphasis has been placed on reliability analysis of the fuel cell stacks in the system. A method for assessing reliability and critical failure predictability requirements for fuel cell stacks in a system consisting of several stacks has been developed. The method is based on a qualitative model of the stack configuration where each stack can be in a functional, partially failed or critically failed state, each of the states having different failure rates and effects on the system behaviour. The main purpose of the method is to understand the effect of stack reliability, critical failure predictability and operating strategy on the system reliability and availability. An example configuration, consisting of 5 × 5 stacks (series of 5 sets of 5 parallel stacks) is analysed in respect to stack reliability requirements as a function of predictability of critical failures and Weibull shape factor of failure rate distributions.

Final Report - Stationary and Emerging Market Fuel Cell System Cost Assessment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Contini, Vince; Heinrichs, Mike; George, Paul

The U.S. Department of Energy (DOE) is focused on providing a portfolio of technology solutions to meet energy security challenges of the future. Fuel cells are a part of this portfolio of technology offerings. To help meet these challenges and supplement the understanding of the current research, Battelle has executed a five-year program that evaluated the total system costs and total ownership costs of two technologies: (1) an ~80 °C polymer electrolyte membrane fuel cell (PEMFC) technology and (2) a solid oxide fuel cell (SOFC) technology, operating with hydrogen or reformate for different applications. Previous research conducted by Battelle, andmore » more recently by other research institutes, suggests that fuel cells can offer customers significant fuel and emission savings along with other benefits compared to incumbent alternatives. For this project, Battelle has applied a proven cost assessment approach to assist the DOE Fuel Cell Technologies Program in making decisions regarding research and development, scale-up, and deployment of fuel cell technology. The cost studies and subsequent reports provide accurate projections of current system costs and the cost impact of state-of-the-art technologies in manufacturing, increases in production volume, and changes to system design on system cost and life cycle cost for several near-term and emerging fuel cell markets. The studies also provide information on types of manufacturing processes that must be developed to commercialize fuel cells and also provide insights into the optimization needed for use of off-the-shelf components in fuel cell systems. Battelle’s analysis is intended to help DOE prioritize investments in research and development of components to reduce the costs of fuel cell systems while considering systems optimization.« less

Automated microscopy system for detection and genetic characterization of fetal nucleated red blood cells on slides

NASA Astrophysics Data System (ADS)

Ravkin, Ilya; Temov, Vladimir

1998-04-01

The detection and genetic analysis of fetal cells in maternal blood will permit noninvasive prenatal screening for genetic defects. Applied Imaging has developed and is currently evaluating a system for semiautomatic detection of fetal nucleated red blood cells on slides and acquisition of their DNA probe FISH images. The specimens are blood smears from pregnant women (9 - 16 weeks gestation) enriched for nucleated red blood cells (NRBC). The cells are identified by using labeled monoclonal antibodies directed to different types of hemoglobin chains (gamma, epsilon); the nuclei are stained with DAPI. The Applied Imaging system has been implemented with both Olympus BX and Nikon Eclipse series microscopes which were equipped with transmission and fluorescence optics. The system includes the following motorized components: stage, focus, transmission, and fluorescence filter wheels. A video camera with light integration (COHU 4910) permits low light imaging. The software capabilities include scanning, relocation, autofocusing, feature extraction, facilities for operator review, and data analysis. Detection of fetal NRBCs is achieved by employing a combination of brightfield and fluorescence images of nuclear and cytoplasmic markers. The brightfield and fluorescence images are all obtained with a single multi-bandpass dichroic mirror. A Z-stack of DNA probe FISH images is acquired by moving focus and switching excitation filters. This stack is combined to produce an enhanced image for presentation and spot counting.

Using Movies to Analyse Gene Circuit Dynamics in Single Cells

PubMed Central

Locke, James CW; Elowitz, Michael B

2010-01-01

Preface Many bacterial systems rely on dynamic genetic circuits to control critical processes. A major goal of systems biology is to understand these behaviours in terms of individual genes and their interactions. However, traditional techniques based on population averages wash out critical dynamics that are either unsynchronized between cells or driven by fluctuations, or ‘noise,’ in cellular components. Recently, the combination of time-lapse microscopy, quantitative image analysis, and fluorescent protein reporters has enabled direct observation of multiple cellular components over time in individual cells. In conjunction with mathematical modelling, these techniques are now providing powerful insights into genetic circuit behaviour in diverse microbial systems. PMID:19369953

On the interplay between mathematics and biology. Hallmarks toward a new systems biology

NASA Astrophysics Data System (ADS)

Bellomo, Nicola; Elaiw, Ahmed; Althiabi, Abdullah M.; Alghamdi, Mohammed Ali

2015-03-01

This paper proposes a critical analysis of the existing literature on mathematical tools developed toward systems biology approaches and, out of this overview, develops a new approach whose main features can be briefly summarized as follows: derivation of mathematical structures suitable to capture the complexity of biological, hence living, systems, modeling, by appropriate mathematical tools, Darwinian type dynamics, namely mutations followed by selection and evolution. Moreover, multiscale methods to move from genes to cells, and from cells to tissue are analyzed in view of a new systems biology approach.

Theoretical analysis of insulin-dependent glucose uptake heterogeneity in 3D bioreactor cell culture.

PubMed

Magrofuoco, Enrico; Elvassore, Nicola; Doyle, Francis J

2012-01-01

Three-dimensional (3D) cell cultures in bioreactors are becoming relevant as models for biological and physiological in vitro studies. In such systems, mathematical models can assist the experiment design that links the macroscopic properties to single-cell responses. We investigated the relationship between biochemical stimuli and cell response within a 3D cell culture in scaffold with heterogeneous porosity. Specifically, we studied the effect of insulin on the local glucose metabolism as a function of 3D pore size distribution. The multiscale mathematical model combines the mass transport within a 3D scaffold and a signaling pathways model. It considers the scaffold heterogeneity, and it describes spatiotemporal concentration of metabolites, biochemical stimuli, and cell density. The signaling model was integrated into this model, linking the local insulin concentration at cell membrane to the glucose uptake rate through glucose transporter type 4 (GLUT4) translocation from the cytosol to the cell membrane. The integrated model determines the cell response heterogeneities in a single channel, hence the biological response distribution in a 3D system. It also provides macroscopic outcomes to evaluate the feasibility of an experimental measurement of the system response. From our analysis, it became apparent that the flow rate is the most important operative variable, and that an optimum value ensures a fast and detectable cell response. This model on insulin-dependent glucose consumption rate offers insight into the cell metabolism physiology, which is a fundamental requirement for the study metabolic disorder such as Type 2 diabetes mellitus, in which the physiological insulin-dependent glucose metabolism is impaired. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Development of complex-shaped liver multicellular spheroids as a human-based model for nanoparticle toxicity assessment in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dubiak-Szepietowska, Monika, E-mail: Monika.Dubiak-Szepietowska@fh-jena.de; Karczmarczyk, Aleksandra; Jönsson-Niedziółka, Martin

The emergence of human-based models is incontestably required for the study of complex physiological pathways and validation of reliable in vitro methods as alternative for in vivo studies in experimental animals for toxicity assessment. With this objective, we have developed and tested three dimensional environments for cells using different types of hydrogels including transglutaminase-cross-linked gelatin, collagen type I, and growth-factor depleted Matrigel. Cells grown in Matrigel exhibited the greatest cell proliferation and spheroid diameter. Moreover, analysis of urea and albumin biosynthesis revealed that the created system allowed the immortalized liver cell line HepG2 to re-establish normal hepatocyte-like properties which weremore » not observed under the conditions of conventional cell cultures. This study presents a scalable technology for production of complex-shaped liver multicellular spheroids as a system which improves the predictive value of cell-based assays for safety and risk assessment. The time- and dose-dependent toxicity of nanoparticles demonstrates a higher cytotoxic effect when HepG2 cells grown as monolayer than embedded in hydrogels. The experimental setup provided evidence that the cell environment has significant influence on cell sensitivity and that liver spheroid is a useful and novel tool to examine nanoparticle dosing effect even at the level of in vitro studies. Therefore, this system can be applied to a wide variety of potentially hostile compounds in basic screening to provide initial warning of adverse effects and trigger subsequent analysis and remedial actions. - Highlights: • Comparison of HepG2 cells growth in Matrigel, Collagen I gel and gelatin gel. • Examination of nanoparticles (NP) dosing effect at the level of in vitro studies. • Influence of the cell culture media composition on the cytotoxic effect of NP.« less

Quantitative Schlieren analysis applied to holograms of crystals grown on Spacelab 3

NASA Technical Reports Server (NTRS)

Brooks, Howard L.

1986-01-01

In order to extract additional information about crystals grown in the microgravity environment of Spacelab, a quantitative schlieren analysis technique was developed for use in a Holography Ground System of the Fluid Experiment System. Utilizing the Unidex position controller, it was possible to measure deviation angles produced by refractive index gradients of 0.5 milliradians. Additionally, refractive index gradient maps for any recorded time during the crystal growth were drawn and used to create solute concentration maps for the environment around the crystal. The technique was applied to flight holograms of Cell 204 of the Fluid Experiment System that were recorded during the Spacelab 3 mission on STS 51B. A triglycine sulfate crystal was grown under isothermal conditions in the cell and the data gathered with the quantitative schlieren analysis technique is consistent with a diffusion limited growth process.

Carcinogenic Effects of Oil Dispersants: a KEGG Pathway-based RNA-seq Study of Human Airway Epithelial Cells

PubMed Central

Liu, Yao-Zhong; Zhang, Lei; Roy-Engel, Astrid M; Saito, Shigeki; Lasky, Joseph A; Wang, Guangdi; Wang, He

2016-01-01

The health impacts of the BP oil spill are yet to be further revealed as the toxicological effects of oil products and dispersants on human respiratory system may be latent and complex, and hence difficult to study and follow up. Here we performed RNA-seq analyses of a system of human airway epithelial cells treated with the BP crude oil and/or dispersants Corexit 9500 and Corexit 9527 that were used to help break up the oil spill. Based on the RNA-seq data, we then systemically analyzed the transcriptomic perturbations of the cells at the KEGG pathway level using two pathway-based analysis tools, GAGE (generally applicable gene set enrichment) and GSNCA (Gene Sets Net Correlations Analysis). Our results suggested a pattern of change towards carcinogenesis for the treated cells marked by upregulation of ribosomal biosynthesis (hsa03008) (p = 1.97e-13), protein processing (hsa04141) (p = 4.09e-7), Wnt signaling (hsa04310) (p = 6.76e-3), neurotrophin signaling (hsa04722) (p = 7.73e-3) and insulin signaling (hsa04910) (p = 1.16e-2) pathways under the dispersant Corexit 9527 treatment, as identified by GAGE analysis. Furthermore, through GSNCA analysis, we identified gene co-expression changes for several KEGG cancer pathways, including small cell lung cancer pathway (hsa05222, p = 9.99e-5), under various treatments of oil/dispersant, especially the mixture of oil and Corexit 9527. Overall, our results suggested carcinogenic effects of dispersants (in particular Corexit 9527) and their mixtures with the BP crude oil, and provided further support for more stringent safety precautions and regulations for operations involving long-term respiratory exposure to oil and dispersants. PMID:27866042

Analysis of Stationary, Photovoltaic-based Surface Power System Designs at the Lunar South Pole

NASA Technical Reports Server (NTRS)

Freeh, Joshua E.

2009-01-01

Combinations of solar arrays and either batteries or regenerative fuel cells are analyzed for a surface power system module at the lunar south pole. The systems are required to produce 5 kW of net electrical power in sunlight and 2 kW of net electrical power during lunar night periods for a 10-year period between 2020 and 2030. Systems-level models for energy conservation, performance, degradation, and mass are used to compare to various systems. The sensitivities of important and/or uncertain variables including battery specific energy, fuel cell operating voltage, and DC-DC converter efficiency are compared to better understand the system. Switching unit efficiency, battery specific energy, and fuel cell operating voltage appear to be important system-level variables for this system. With reasonably sized solar arrays, the regenerative fuel cell system has significantly lower mass than the battery system based on the requirements and assumptions made herein. The total operational time is estimated at about 10,000 hours in battery discharge/fuel cell mode and about 4,000 and 8,000 hours for the battery charge and electrolyzer modes, respectively. The estimated number of significant depth-of-discharge cycles for either energy storage system is less than 100 for the 10-year period.

Energy efficiency analysis: biomass-to-wheel efficiency related with biofuels production, fuel distribution, and powertrain systems.

PubMed

Huang, Wei-Dong; Zhang, Y-H Percival

2011-01-01

Energy efficiency analysis for different biomass-utilization scenarios would help make more informed decisions for developing future biomass-based transportation systems. Diverse biofuels produced from biomass include cellulosic ethanol, butanol, fatty acid ethyl esters, methane, hydrogen, methanol, dimethyether, Fischer-Tropsch diesel, and bioelectricity; the respective powertrain systems include internal combustion engine (ICE) vehicles, hybrid electric vehicles based on gasoline or diesel ICEs, hydrogen fuel cell vehicles, sugar fuel cell vehicles (SFCV), and battery electric vehicles (BEV). We conducted a simple, straightforward, and transparent biomass-to-wheel (BTW) analysis including three separate conversion elements--biomass-to-fuel conversion, fuel transport and distribution, and respective powertrain systems. BTW efficiency is a ratio of the kinetic energy of an automobile's wheels to the chemical energy of delivered biomass just before entering biorefineries. Up to 13 scenarios were analyzed and compared to a base line case--corn ethanol/ICE. This analysis suggests that BEV, whose electricity is generated from stationary fuel cells, and SFCV, based on a hydrogen fuel cell vehicle with an on-board sugar-to-hydrogen bioreformer, would have the highest BTW efficiencies, nearly four times that of ethanol-ICE. In the long term, a small fraction of the annual US biomass (e.g., 7.1%, or 700 million tons of biomass) would be sufficient to meet 100% of light-duty passenger vehicle fuel needs (i.e., 150 billion gallons of gasoline/ethanol per year), through up to four-fold enhanced BTW efficiencies by using SFCV or BEV. SFCV would have several advantages over BEV: much higher energy storage densities, faster refilling rates, better safety, and less environmental burdens.

Energy Efficiency Analysis: Biomass-to-Wheel Efficiency Related with Biofuels Production, Fuel Distribution, and Powertrain Systems

PubMed Central

Huang, Wei-Dong; Zhang, Y-H Percival

2011-01-01

Background Energy efficiency analysis for different biomass-utilization scenarios would help make more informed decisions for developing future biomass-based transportation systems. Diverse biofuels produced from biomass include cellulosic ethanol, butanol, fatty acid ethyl esters, methane, hydrogen, methanol, dimethyether, Fischer-Tropsch diesel, and bioelectricity; the respective powertrain systems include internal combustion engine (ICE) vehicles, hybrid electric vehicles based on gasoline or diesel ICEs, hydrogen fuel cell vehicles, sugar fuel cell vehicles (SFCV), and battery electric vehicles (BEV). Methodology/Principal Findings We conducted a simple, straightforward, and transparent biomass-to-wheel (BTW) analysis including three separate conversion elements -- biomass-to-fuel conversion, fuel transport and distribution, and respective powertrain systems. BTW efficiency is a ratio of the kinetic energy of an automobile's wheels to the chemical energy of delivered biomass just before entering biorefineries. Up to 13 scenarios were analyzed and compared to a base line case – corn ethanol/ICE. This analysis suggests that BEV, whose electricity is generated from stationary fuel cells, and SFCV, based on a hydrogen fuel cell vehicle with an on-board sugar-to-hydrogen bioreformer, would have the highest BTW efficiencies, nearly four times that of ethanol-ICE. Significance In the long term, a small fraction of the annual US biomass (e.g., 7.1%, or 700 million tons of biomass) would be sufficient to meet 100% of light-duty passenger vehicle fuel needs (i.e., 150 billion gallons of gasoline/ethanol per year), through up to four-fold enhanced BTW efficiencies by using SFCV or BEV. SFCV would have several advantages over BEV: much higher energy storage densities, faster refilling rates, better safety, and less environmental burdens. PMID:21765941

Single-cell analysis and sorting using droplet-based microfluidics.

PubMed

Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

2013-05-01

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.

Single-cell analysis and sorting using droplet-based microfluidics

PubMed Central

Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

2014-01-01

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. as an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. the beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ~200 Hz as well as cell enrichment. the microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ~1 million cells, the microfluidic operations require 2–6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5–7 d. PMID:23558786

Detection of early changes in lung cell cytology by flow-systems analysis techniques. [Rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinkamp, J.A.; Wilson, J.S.; Svitra, Z.V.

1980-03-01

Ongoing experiments designed to develop automated flow-analysis methods for assaying damage to pulmonary lavage cells in experimental animals exposed by inhalation to environmental pollutants are summarized. Pulmonary macrophages were characterized on their ability to phagocytize polystyrene latex fluorescent spheres. Lung cells consisting primarily of macrophages and leukocytes were analyzed for fluorescence (phagocytosis of spheres) and size using flow cytometric methods. Studies also concentrated on combining phagocytosis with other cellular parameters (DNA content, cell viability, and B-glucuronidase activity). As baseline studies are completed in normal animals, experimental animals will be exposed to gaseous and particulate environmental pollutants. (ERB

[An immunocytochemical study of the C-cell function of the thyroid in rats exposed on the Kosmos-2044 biosatellite].

PubMed

Loginov, V I

1993-01-01

Immunocytochemical analysis of thyroid gland C-cells of the rats exposed to a 14-day space flight revealed a decrease in the number of C-cells, volume of their nuclei and a declined percentage of active secretory C-cells, which point to a decline of calcitonin proactive and calcitonin secretory hypofunction of the thyroid C-cells system in flown rats. Tail suspension as a microgravity model caused similar changes in C-cells.

Design, analysis and test verification of advanced encapsulation systems

NASA Technical Reports Server (NTRS)

Garcia, A., III

1983-01-01

A preliminary reduced variable master was constructed for pressure loading. A study of cell thickness versus cell stress was completed. Work is continuing on encapsulation of qualification modules. A 4 ft x 4 ft 'credit card' construction laminate was made.

Design, analysis and test verification of advanced encapsulation systems

NASA Astrophysics Data System (ADS)

Garcia, A., III

1983-02-01

A preliminary reduced variable master was constructed for pressure loading. A study of cell thickness versus cell stress was completed. Work is continuing on encapsulation of qualification modules. A 4 ft x 4 ft 'credit card' construction laminate was made.

Highly accurate apparatus for electrochemical characterization of the felt electrodes used in redox flow batteries

NASA Astrophysics Data System (ADS)

Park, Jong Ho; Park, Jung Jin; Park, O. Ok; Jin, Chang-Soo; Yang, Jung Hoon

2016-04-01

Because of the rise in renewable energy use, the redox flow battery (RFB) has attracted extensive attention as an energy storage system. Thus, many studies have focused on improving the performance of the felt electrodes used in RFBs. However, existing analysis cells are unsuitable for characterizing felt electrodes because of their complex 3-dimensional structure. Analysis is also greatly affected by the measurement conditions, viz. compression ratio, contact area, and contact strength between the felt and current collector. To address the growing need for practical analytical apparatus, we report a new analysis cell for accurate electrochemical characterization of felt electrodes under various conditions, and compare it with previous ones. In this cell, the measurement conditions can be exhaustively controlled with a compression supporter. The cell showed excellent reproducibility in cyclic voltammetry analysis and the results agreed well with actual RFB charge-discharge performance.

Automatic analysis of microscopic images of red blood cell aggregates

NASA Astrophysics Data System (ADS)

Menichini, Pablo A.; Larese, Mónica G.; Riquelme, Bibiana D.

2015-06-01

Red blood cell aggregation is one of the most important factors in blood viscosity at stasis or at very low rates of flow. The basic structure of aggregates is a linear array of cell commonly termed as rouleaux. Enhanced or abnormal aggregation is seen in clinical conditions, such as diabetes and hypertension, producing alterations in the microcirculation, some of which can be analyzed through the characterization of aggregated cells. Frequently, image processing and analysis for the characterization of RBC aggregation were done manually or semi-automatically using interactive tools. We propose a system that processes images of RBC aggregation and automatically obtains the characterization and quantification of the different types of RBC aggregates. Present technique could be interesting to perform the adaptation as a routine used in hemorheological and Clinical Biochemistry Laboratories because this automatic method is rapid, efficient and economical, and at the same time independent of the user performing the analysis (repeatability of the analysis).

Radiation biodosimetry: Applications for spaceflight

NASA Astrophysics Data System (ADS)

Blakely, W. F.; Miller, A. C.; Grace, M. B.; McLeland, C. B.; Luo, L.; Muderhwa, J. M.; Miner, V. L.; Prasanna, P. G. S.

The multiparametric dosimetry system that we are developing for medical radiological defense applications could be adapted for spaceflight environments. The system complements the internationally accepted personnel dosimeters and cytogenetic analysis of chromosome aberrations, considered the best means of documenting radiation doses for health records. Our system consists of a portable hematology analyzer, molecular biodosimetry using nucleic acid and antigen-based diagnostic equipment, and a dose assessment management software application. A dry-capillary tube reagent-based centrifuge blood cell counter (QBC Autoread Plus, Beckon Dickinson Bioscience) measures peripheral blood lymphocytes and monocytes, which could determine radiation dose based on the kinetics of blood cell depletion. Molecular biomarkers for ionizing radiation exposure (gene expression changes, blood proteins) can be measured in real time using such diagnostic detection technologies as miniaturized nucleic acid sequences and antigen-based biosensors, but they require validation of dose-dependent targets and development of optimized protocols and analysis systems. The Biodosimetry Assessment Tool, a software application, calculates radiation dose based on a patient's physical signs and symptoms and blood cell count analysis. It also annotates location of personnel dosimeters, displays a summary of a patient's dosimetric information to healthcare professionals, and archives the data for further use. These radiation assessment diagnostic technologies can have dual-use applications supporting general medical-related care.

A conceptual review on systems biology in health and diseases: from biological networks to modern therapeutics.

PubMed

Somvanshi, Pramod Rajaram; Venkatesh, K V

2014-03-01

Human physiology is an ensemble of various biological processes spanning from intracellular molecular interactions to the whole body phenotypic response. Systems biology endures to decipher these multi-scale biological networks and bridge the link between genotype to phenotype. The structure and dynamic properties of these networks are responsible for controlling and deciding the phenotypic state of a cell. Several cells and various tissues coordinate together to generate an organ level response which further regulates the ultimate physiological state. The overall network embeds a hierarchical regulatory structure, which when unusually perturbed can lead to undesirable physiological state termed as disease. Here, we treat a disease diagnosis problem analogous to a fault diagnosis problem in engineering systems. Accordingly we review the application of engineering methodologies to address human diseases from systems biological perspective. The review highlights potential networks and modeling approaches used for analyzing human diseases. The application of such analysis is illustrated in the case of cancer and diabetes. We put forth a concept of cell-to-human framework comprising of five modules (data mining, networking, modeling, experimental and validation) for addressing human physiology and diseases based on a paradigm of system level analysis. The review overtly emphasizes on the importance of multi-scale biological networks and subsequent modeling and analysis for drug target identification and designing efficient therapies.

Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

PubMed Central

Liu, Bing; Gao, Yankun; Ruan, Hai-Bin; Chen, Yue

2016-01-01

Proline hydroxylation is a critical cellular mechanism regulating oxygen-response pathways in tumor initiation and progression. Yet, its substrate diversity and functions remain largely unknown. Here, we report a system-wide analysis to characterize proline hydroxylation substrates in cancer cells using an immunoaffinity-purification assisted proteomics strategy. We identified 562 sites from 272 proteins in HeLa cells. Bioinformatic analysis revealed that proline hydroxylation substrates are significantly enriched with mRNA processing and stress-response cellular pathways with canonical and diverse flanking sequence motifs. Structural analysis indicates a significant enrichment of proline hydroxylation participating in the secondary structure of substrate proteins. Our study identified and validated Brd4, a key transcription factor, as a novel proline hydroxylation substrate. Functional analysis showed that the inhibition of proline hydroxylation pathway significantly reduced the proline hydroxylation abundance on Brd4 and affected Brd4-mediated transcriptional activity as well as cell proliferation in AML leukemia cells. Taken together, our study identified a broad regulatory role of proline hydroxylation in cellular oxygen-sensing pathways and revealed potentially new targets that dynamically respond to hypoxia microenvironment in tumor cells. PMID:27764789

Analysis of Average Telomere Length in Human Telomeric Protein Knockout Cells Generated by CRISPR/Cas9.

PubMed

Xu, Jun; Songyang, Zhou; Liu, Dan; Kim, Hyeung

2017-01-01

Telomeres play an important role in ensuring the integrity of the genome. Telomere shortening can lead to loss of genetic information and trigger DNA damage responses. Cultured mammalian cells have served as critical model systems for studying the function of telomere binding proteins and telomerase. Tremendous heterogeneity can be observed both between species and within a single cell population. Recent advances in genome editing (such as the development of the CRISPR/Cas9 platform) have further enabled researchers to carry out loss-of-function analysis of how disrupting key players in telomere maintenance affects telomere length regulation. Here we describe the steps to be carried out in order to analyze the average length of telomeres in CRISPR-engineered human knockout (KO) cells (TRF analysis).

Contact-independent cell death of human microglial cells due to pathogenic Naegleria fowleri trophozoites.

PubMed

Kim, Jong-Hyun; Kim, Daesik; Shin, Ho-Joon

2008-12-01

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increase of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.

Contact-Independent Cell Death of Human Microglial Cells due to Pathogenic Naegleria fowleri Trophozoites

PubMed Central

Kim, Jong-Hyun

2008-01-01

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increasse of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death. PMID:19127326

Techno-economic analysis of fuel cell auxiliary power units as alternative to idling

NASA Astrophysics Data System (ADS)

Jain, Semant; Chen, Hsieh-Yeh; Schwank, Johannes

This paper presents a techno-economic analysis of fuel-cell-based auxiliary power units (APUs), with emphasis on applications in the trucking industry and the military. The APU system is intended to reduce the need for discretionary idling of diesel engines or gas turbines. The analysis considers the options for on-board fuel processing of diesel and compares the two leading fuel cell contenders for automotive APU applications: proton exchange membrane fuel cell and solid oxide fuel cell. As options for on-board diesel reforming, partial oxidation and auto-thermal reforming are considered. Finally, using estimated and projected efficiency data, fuel consumption patterns, capital investment, and operating costs of fuel-cell APUs, an economic evaluation of diesel-based APUs is presented, with emphasis on break-even periods as a function of fuel cost, investment cost, idling time, and idling efficiency. The analysis shows that within the range of parameters studied, there are many conditions where deployment of an SOFC-based APU is economically viable. Our analysis indicates that at an APU system cost of 100 kW -1, the economic break-even period is within 1 year for almost the entire range of conditions. At 500 kW -1 investment cost, a 2-year break-even period is possible except for the lowest end of the fuel consumption range considered. However, if the APU investment cost is 3000 kW -1, break-even would only be possible at the highest fuel consumption scenarios. For Abram tanks, even at typical land delivered fuel costs, a 2-year break-even period is possible for APU investment costs as high as 1100 kW -1.

Functional Module Search in Protein Networks based on Semantic Similarity Improves the Analysis of Proteomics Data*

PubMed Central

Boyanova, Desislava; Nilla, Santosh; Klau, Gunnar W.; Dandekar, Thomas; Müller, Tobias; Dittrich, Marcus

2014-01-01

The continuously evolving field of proteomics produces increasing amounts of data while improving the quality of protein identifications. Albeit quantitative measurements are becoming more popular, many proteomic studies are still based on non-quantitative methods for protein identification. These studies result in potentially large sets of identified proteins, where the biological interpretation of proteins can be challenging. Systems biology develops innovative network-based methods, which allow an integrated analysis of these data. Here we present a novel approach, which combines prior knowledge of protein-protein interactions (PPI) with proteomics data using functional similarity measurements of interacting proteins. This integrated network analysis exactly identifies network modules with a maximal consistent functional similarity reflecting biological processes of the investigated cells. We validated our approach on small (H9N2 virus-infected gastric cells) and large (blood constituents) proteomic data sets. Using this novel algorithm, we identified characteristic functional modules in virus-infected cells, comprising key signaling proteins (e.g. the stress-related kinase RAF1) and demonstrate that this method allows a module-based functional characterization of cell types. Analysis of a large proteome data set of blood constituents resulted in clear separation of blood cells according to their developmental origin. A detailed investigation of the T-cell proteome further illustrates how the algorithm partitions large networks into functional subnetworks each representing specific cellular functions. These results demonstrate that the integrated network approach not only allows a detailed analysis of proteome networks but also yields a functional decomposition of complex proteomic data sets and thereby provides deeper insights into the underlying cellular processes of the investigated system. PMID:24807868

Polarized targeting of a shaker-like (A-type) K(+)-channel in the polarized epithelial cell line MDCK.

PubMed

Le Maout, S; Sewing, S; Coudrier, E; Elalouf, J M; Pongs, O; Merot, J

1996-01-01

Functional Kv 1-4 channels were stably expressed in filter-grown MDCK cells which form a polarized epithelium with two distinct plasma membrane domains: a basolateral and an apical cell surface. The Shaker-related Kv 1-4 channels mediated in MDCK cells fast transient (A-type) voltage-activated outward currents having similar properties to the ones reported for Kv 1-4 in the Xenopus oocytes expression system. Immunoblot analysis with specific anti-Kv 1-4 antibodies showed that two Kv 1-4 protein forms are expressed in MDCK cells which most likely represent the glycosylated and non-glycosylated Kv 1-4 protein, respectively. Using immunocytochemistry and confocal microscopy we showed that the Kv 1-4 channels are specifically localized in the basolateral membranes of MDCK cells. Thus, the MDCK cells may provide an important model system to analyse the polarized transport of ion channels such as Kv 1-4, which are distinctly expressed in the mammalian central nervous system.

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

PubMed Central

Macdonald, Patrick J.; Chen, Yan; Mueller, Joachim D.

2012-01-01

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction. PMID:22093611

Inhibitory effect of hot-water extract from dried fruit of Crataegus pinnatifida on low-density lipoprotein (LDL) oxidation in cell and cell-free systems.

PubMed

Chu, Chia-Yih; Lee, Miao-Jane; Liao, Chuen-Lan; Lin, Wea-Lung; Yin, Yu-Fang; Tseng, Tsui-Hwa

2003-12-17

The dried fruit of Crataegus pinnatifida, a local soft drink material and medical herb, was found to possess potential against oxidative stress. In the preliminary study, the antioxidant potential of a hot-water extract obtained from the dried fruit of C. pinnatifida (CF-H) was evaluated in terms of its capacity of quenching 1,1-diphenyl-2-picrylhydrazyl free radicals (EC(50) = 0.118 mg/mL). After content analysis, it was found that CF-H is mainly composed of polyphenols including flavonoids (6.9%), procyanidins (2.2%), (+)-catechin (0.5%), and (-)-epicatechin (0.2%). The antioxidative bioactivity of CF-H had been assess previously using the models of CuSO(4) as cell-free system and sodium nitroprusside (SNP) plus macrophage RAW 264.7 cells as cell system to induce human low-density lipoprotein oxidation. CF-H was found to inhibit relative electrophoretic mobility and thiobarbituric acid reactive substances at the concentration of 0.5-1.0 mg/mL in the cell-free system and at 0.01-0.10 mg/mL in the cell system. Furthermore, it was found that CF-H decreased the SNP-induced cell lipid peroxidation and reduced glutathione depletion.

Tomographic flow cytometry assisted by intelligent wavefronts analysis

NASA Astrophysics Data System (ADS)

Merola, F.; Memmolo, P.; Miccio, L.; Mugnano, M.; Ferraro, P.

2017-06-01

High-throughput single-cell analysis is a challenging target for implementing advanced biomedical applications. An excellent candidate for this aim is label-free tomographic phase microscopy. However, in-line tomography is very difficult to be implemented in practice, as it requires complex setup for rotating the sample and/or illuminate the cell along numerous directions [1]. We exploit random rolling of cells while they are flowing along a microfluidic channel demonstrating that it is possible to obtain in-line phase-contrast tomography by adopting strategies for intelligent wavefronts analysis thus obtaining complete retrieval of both 3D-position and orientation of rotating cells [2]. Thus, by numerical wavefront analysis a-priori knowledge of such information is no longer needed. This approach makes continuos-flow cyto-tomography suitable for practical operation in real-world, single-cell analysis and with substantial simplification of the optical system avoiding any mechanical/optical scanning of light source. Demonstration is given for different classes of biosamples, red-blood-cells (RBCs), diatom algae and fibroblast cells [3]. Accurate characterization of each type of cells is reported despite their very different nature and materials content, thus showing the proposed method can be extended, by adopting two alternate strategies of wavefront-analysis, to many classes of cells. In particular, for RBCs we furnish important parameters as 3D morphology, Corpuscular Hemoglobin (CH), Volume (V), and refractive index (RI) for each single cell in the total population [3]. This could open a new route in blood disease diagnosis, for example for the isolation and characterization of "foreign" cells in the blood stream, the so called Circulating Tumor Cells (CTC), early manifestation of metastasis.

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT: UTC FUEL CELLS' PC25C POWER PLANT - GAS PROCESSING UNIT PERFORMANCE FOR ANAEROBIC DIGESTER GAS

EPA Science Inventory

Under EPA’s Environmental Technology Verification program, which provides objective and scientific third party analysis of new technology that can benefit the environment, a combined heat and power system based on the UTC Fuel Cell's PC25C Fuel Cell Power Plant was evaluated. The...

2016 Annual Progress Report: DOE Hydrogen and Fuel Cells Program

DOE Office of Scientific and Technical Information (OSTI.GOV)

None, None

The 2016 Annual Progress Report summarizes fiscal year 2016 activities and accomplishments by projects funded by the DOE Hydrogen and Fuel Cells Program. It covers the program areas of hydrogen production; hydrogen delivery; hydrogen storage; fuel cells; manufacturing R&D; technology validation; safety, codes and standards; systems analysis; market transformation; and Small Business Innovation Research projects.

Peroxisome Proliferator-Activated Receptor-γ Ligands Alter Breast Cancer Cell Motility through Modulation of the Plasminogen Activator System

PubMed Central

Carter, Jennifer C.; Church, Frank C.

2011-01-01

We investigated peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands effect on cell motility and the plasminogen activator system using normal MCF-10A and malignant MCF-10CA1 cell lines. Ciglitazone reduced both wound-induced migration and chemotaxis. However, the effect was not reversed with pretreatment of cells with the PPAR-γ-specific antagonist GW9662. Immunoblot analysis of conditioned media showed ciglitazone decreased plasminogen activator inhibitor-1 (PAI-1) in both cell lines; this effect was also unaltered by PPAR-γ antagonism. Alternatively, treatment with the ω-6 fatty acid arachidonic acid (ArA), but not the ω-3 fatty acid docosahexanoic acid, increased both MCF-10A cell migration and cell surface uPA activity. Pretreatment with a PPAR-γ antagonist reversed these effects, suggesting that ArA mediates its effect on cell motility and uPA activity through PPAR-γ activation. Collectively, the data suggest PPAR-γ ligands have a differential effect on normal and malignant cell migration and the plasminogen activation system, resulting from PPAR-γ-dependent and PPAR-γ-independent effects. PMID:22131991

Individually programmable cell stretching microwell arrays actuated by a Braille display.

PubMed

Kamotani, Yoko; Bersano-Begey, Tommaso; Kato, Nobuhiro; Tung, Yi-Chung; Huh, Dongeun; Song, Jonathan W; Takayama, Shuichi

2008-06-01

Cell culture systems are often static and are therefore nonphysiological. In vivo, many cells are exposed to dynamic surroundings that stimulate cellular responses in a process known as mechanotransduction. To recreate this environment, stretchable cell culture substrate systems have been developed, however, these systems are limited by being macroscopic and low throughput. We have developed a device consisting of 24 miniature cell stretching chambers with flexible bottom membranes that are deformed using the computer-controlled, piezoelectrically actuated pins of a Braille display. We have also developed efficient image capture and analysis protocols to quantify morphological responses of the cells to applied strain. Human dermal microvascular endothelial cells (HDMECs) were found to show increasing degrees of alignment and elongation perpendicular to the radial strain in response to cyclic stretch at increasing frequencies of 0.2, 1, and 5 Hz, after 2, 4, and 12h. Mouse myogenic C2C12 cells were also found to align in response to the stretch, while A549 human lung adenocarcinoma epithelial cells did not respond to stretch.

Individually Programmable Cell Stretching Microwell Arrays Actuated by a Braille Display

PubMed Central

Kamotani, Yoko; Bersano-Begey, Tommaso; Kato, Nobuhiro; Tung, Yi-chung; Huh, Dongeun; Song, Jonathan W.; Takayama, Shuichi

2008-01-01

Cell culture systems are often static and are therefore nonphysiological. In vivo, many cells are exposed to dynamic surroundings that stimulate cellular responses in a process known as mechanotransduction. To recreate this environment, stretchable cell culture substrate systems have been developed, however these systems are limited by being macroscopic and low throughput. We have developed a device consisting of 24 miniature cell stretching chambers with flexible bottom membranes that are deformed using the computer-controlled, piezoelectrically actuated pins of a Braille display. We have also developed efficient image capture and analysis protocols to quantify morphological responses of the cells to applied strain. Human dermal microvascular endothelial cells (HDMECs) were found to show increasing degrees of alignment and elongation perpendicular to the radial strain in response to cyclic stretch at increasing frequencies of 0.2, 1, and 5 Hz, after 2, 4, and 12 hours. Mouse myogenic C2C12 cells were also found to align in response to the stretch, while A549 human lung adenocarcinoma epithelial cells did not respond to stretch. PMID:18342367

Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads

PubMed Central

Wang, Lin; Acosta, Miguel A.; Leach, Jennie B.; Carrier, Rebecca L.

2013-01-01

Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems. PMID:23443975

Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads.

PubMed

Wang, Lin; Acosta, Miguel A; Leach, Jennie B; Carrier, Rebecca L

2013-04-21

Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems.

Tissue-resident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells

PubMed Central

Sojka, Dorothy K; Plougastel-Douglas, Beatrice; Yang, Liping; Pak-Wittel, Melissa A; Artyomov, Maxim N; Ivanova, Yulia; Zhong, Chao; Chase, Julie M; Rothman, Paul B; Yu, Jenny; Riley, Joan K; Zhu, Jinfang; Tian, Zhigang; Yokoyama, Wayne M

2014-01-01

Natural killer (NK) cells belong to the innate immune system; they can control virus infections and developing tumors by cytotoxicity and producing inflammatory cytokines. Most studies of mouse NK cells, however, have focused on conventional NK (cNK) cells in the spleen. Recently, we described two populations of liver NK cells, tissue-resident NK (trNK) cells and those resembling splenic cNK cells. However, their lineage relationship was unclear; trNK cells could be developing cNK cells, related to thymic NK cells, or a lineage distinct from both cNK and thymic NK cells. Herein we used detailed transcriptomic, flow cytometric, and functional analysis and transcription factor-deficient mice to determine that liver trNK cells form a distinct lineage from cNK and thymic NK cells. Taken together with analysis of trNK cells in other tissues, there are at least four distinct lineages of NK cells: cNK, thymic, liver (and skin) trNK, and uterine trNK cells. DOI: http://dx.doi.org/10.7554/eLife.01659.001 PMID:24714492

Construction of Gallium Point at NMIJ

NASA Astrophysics Data System (ADS)

Widiatmo, J. V.; Saito, I.; Yamazawa, K.

2017-03-01

Two open-type gallium point cells were fabricated using ingots whose nominal purities are 7N. Measurement systems for the realization of the melting point of gallium using these cells were built. The melting point of gallium is repeatedly realized by means of the measurement systems for evaluating the repeatability. Measurements for evaluating the effect of hydrostatic pressure coming from the molten gallium existing during the melting process and the effect of gas pressure that fills the cell were also performed. Direct cell comparisons between those cells were conducted. This comparison was aimed to evaluate the consistency of each cell, especially related to the nominal purity. Direct cell comparison between the open-type and the sealed-type gallium point cell was also conducted. Chemical analysis was conducted using samples extracted from ingots used in both the newly built open-type gallium point cells, from which the effect of impurities in the ingot was evaluated.

Measurement and Characterization of Concentrator Solar Cells II

NASA Technical Reports Server (NTRS)

Scheiman, Dave; Sater, Bernard L.; Chubb, Donald; Jenkins, Phillip; Snyder, Dave

2005-01-01

Concentrator solar cells are continuing to get more consideration for use in power systems. This interest is because concentrator systems can have a net lower cost per watt in solar cell materials plus ongoing improvements in sun-tracking technology. Quantitatively measuring the efficiency of solar cells under concentration is difficult. Traditionally, the light concentration on solar cells has been determined by using a ratio of the measured solar cell s short circuit current to that at one sun, this assumes that current changes proportionally with light intensity. This works well with low to moderate (<20 suns) concentration levels on "well-behaved" linear cells but does not apply when cells respond superlinearly, current increases faster than intensity, or sublinearly, current increases more slowly than intensity. This paper continues work on using view factors to determine the concentration level and linearity of the solar cell with mathematical view factor analysis and experimental results [1].

Zoledronate Triggers Vδ2 T Cells to Destroy and Kill Spheroids of Colon Carcinoma: Quantitative Image Analysis of Three-Dimensional Cultures.

PubMed

Varesano, Serena; Zocchi, Maria Raffaella; Poggi, Alessandro

2018-01-01

New successful anti-cancer strategies are based on the stimulation of immune reaction against tumors: however, preclinical testing of such treatments is still a challenge. To improve the screening of anti-cancer drugs, three-dimensional (3D) culture systems, including spheroids, have been validated as preclinical models. We propose the spheroid 3D system to test anti-tumor drug-induced immune responses. We show that colorectal carcinoma (CRC) spheroids, generated with the epithelial growth factor (EGF), can be co-cultured with Vδ2 T cells to evaluate the anti-tumor activity of these effector lymphocytes. By computerized image analysis, the precise and unbiased measure of perimeters and areas of tumor spheroids is achievable, beside the calculation of their volume. CRC spheroid size is related to ATP content and cell number, as parameters for cell metabolism and proliferation; in turn, crystal violet staining can check the viability of cells inside the spheroids to detect tumor killing by Vδ2 T cells. In this 3D cultures, we tested (a) zoledronate that is known to activate Vδ2 T cells and (b) the therapeutic anti-EGF receptor humanized antibody cetuximab that can elicit the antibody-dependent cytotoxicity of tumor cells by effector lymphocytes. Zoledronate triggers Vδ2 T cells to kill and degrade CRC spheroids; we detected the T-cell receptor dependency of zoledronate effect, conceivably due to the recognition of phosphoantigens produced as a drug effect on target cell metabolism. In addition, cetuximab triggered Vδ2 T lymphocytes to exert the antibody-dependent cellular cytotoxicity of CRC spheroids. Finally, the system reveals differences in the sensitivity of CRC cell lines to the action of Vδ2 T lymphocytes and in the efficiency of anti-tumor effectors from distinct donors. A limitation of this model is the absence of cells, including fibroblasts, that compose tumor microenvironment and influence drug response. Nevertheless, the system can be improved by setting mixed spheroids, made of stromal and cancer cells. We conclude that this type of spheroid 3D culture is a feasible and reliable system to evaluate and measure anti-tumor drug-induced immune responses beside direct anti-cancer drug effect.

Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.

PubMed

Bae, Seunghee; An, In-Sook; An, Sungkwan

2015-09-01

Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts.

Using low-risk factors to generate non-integrated human induced pluripotent stem cells from urine-derived cells.

PubMed

Wang, Linli; Chen, Yuehua; Guan, Chunyan; Zhao, Zhiju; Li, Qiang; Yang, Jianguo; Mo, Jian; Wang, Bin; Wu, Wei; Yang, Xiaohui; Song, Libing; Li, Jun

2017-11-02

Because the lack of an induced pluripotent stem cell (iPSC) induction system with optimal safety and efficiency limits the application of these cells, development of such a system is important. To create such an induction system, we screened a variety of reprogrammed plasmid combinations and multiple compounds and then verified the system's feasibility using urine cells from different individuals. We also compared large-scale iPSC chromosomal variations and expression of genes associated with genomic stability between this system and the traditional episomal system using karyotype and quantitative reverse transcription polymerase chain reaction analyses. We developed a high-efficiency episomal system, the 6F/BM1-4C system, lacking tumorigenic factors for human urine-derived cell (hUC) reprogramming. This system includes six low-risk factors (6F), Oct4, Glis1, Klf4, Sox2, L-Myc, and the miR-302 cluster. Transfected hUCs were treated with four compounds (4C), inhibitor of lysine-demethylase1, methyl ethyl ketone, glycogen synthase kinase 3 beta, and histone deacetylase, within a short time period. Comparative analysis revealed significantly decreased chromosomal variation in iPSCs and significantly increased Sirt1 expression compared with iPSCs induced using the traditional episomal system. The 6F/BM1-4C system effectively induces reprogramming of urine cells in samples obtained from different individuals. iPSCs induced using the 6F/BM1-4C system are more stable at the cytogenetic level and have potential value for clinical application.

Differential diagnosis of lung carcinoma with three-dimensional quantitative molecular vibrational imaging

NASA Astrophysics Data System (ADS)

Gao, Liang; Hammoudi, Ahmad A.; Li, Fuhai; Thrall, Michael J.; Cagle, Philip T.; Chen, Yuanxin; Yang, Jian; Xia, Xiaofeng; Fan, Yubo; Massoud, Yehia; Wang, Zhiyong; Wong, Stephen T. C.

2012-06-01

The advent of molecularly targeted therapies requires effective identification of the various cell types of non-small cell lung carcinomas (NSCLC). Currently, cell type diagnosis is performed using small biopsies or cytology specimens that are often insufficient for molecular testing after morphologic analysis. Thus, the ability to rapidly recognize different cancer cell types, with minimal tissue consumption, would accelerate diagnosis and preserve tissue samples for subsequent molecular testing in targeted therapy. We report a label-free molecular vibrational imaging framework enabling three-dimensional (3-D) image acquisition and quantitative analysis of cellular structures for identification of NSCLC cell types. This diagnostic imaging system employs superpixel-based 3-D nuclear segmentation for extracting such disease-related features as nuclear shape, volume, and cell-cell distance. These features are used to characterize cancer cell types using machine learning. Using fresh unstained tissue samples derived from cell lines grown in a mouse model, the platform showed greater than 97% accuracy for diagnosis of NSCLC cell types within a few minutes. As an adjunct to subsequent histology tests, our novel system would allow fast delineation of cancer cell types with minimum tissue consumption, potentially facilitating on-the-spot diagnosis, while preserving specimens for additional tests. Furthermore, 3-D measurements of cellular structure permit evaluation closer to the native state of cells, creating an alternative to traditional 2-D histology specimen evaluation, potentially increasing accuracy in diagnosing cell type of lung carcinomas.

A broad-host range dual-fluorescence reporter system for gene expression analysis in Gram-negative bacteria.

PubMed

Hennessy, Rosanna C; Christiansen, Line; Olsson, Stefan; Stougaard, Peter

2018-01-01

Fluorescence-based reporter systems are valuable tools for studying gene expression dynamics in living cells. Here we describe a dual-fluorescence reporter system carrying the red fluorescent marker mCherry and the blue fluorescent protein EBFP2 enabling the simultaneous analysis of two promoters in broad-host range autofluorescent Gram-negative bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

An ultrastructural analysis of platelets, erythrocytes, white blood cells, and fibrin network in systemic lupus erythematosus.

PubMed

Pretorius, Etheresia; du Plooy, Jenny; Soma, Prashilla; Gasparyan, Armen Yuri

2014-07-01

The study suggests that patients with systemic lupus erythematosus (SLE) present with distinct inflammatory ultrastructural changes such as platelets blebbing, generation of platelet-derived microparticles, spontaneous formation of massive fibrin network and fusion of the erythrocytes membranes. Lupoid platelets actively interact with other inflammatory cells, particularly with white blood cells (WBCs), and the massive fibrin network facilitates such an interaction. It is possible that the concerted actions of platelets, erythrocytes and WBC, caught in the inflammatory fibrin network, predispose to pro-thrombotic states in patients with SLE.

Untargeted Metabolomic Analysis of Rat Neuroblastoma Cells as a Model System to Study the Biochemical Effects of the Acute Administration of Methamphetamine.

PubMed

Maker, Garth L; Green, Tobias; Mullaney, Ian; Trengove, Robert D

2018-06-07

Methamphetamine is an illicit psychostimulant drug that is linked to a number of diseases of the nervous system. The downstream biochemical effects of its primary mechanisms are not well understood, and the objective of this study was to investigate whether untargeted metabolomic analysis of an in vitro model could generate data relevant to what is already known about this drug. Rat B50 neuroblastoma cells were treated with 1 mM methamphetamine for 48 h, and both intracellular and extracellular metabolites were profiled using gas chromatography⁻mass spectrometry. Principal component analysis of the data identified 35 metabolites that contributed most to the difference in metabolite profiles. Of these metabolites, the most notable changes were in amino acids, with significant increases observed in glutamate, aspartate and methionine, and decreases in phenylalanine and serine. The data demonstrated that glutamate release and, subsequently, excitotoxicity and oxidative stress were important in the response of the neuronal cell to methamphetamine. Following this, the cells appeared to engage amino acid-based mechanisms to reduce glutamate levels. The potential of untargeted metabolomic analysis has been highlighted, as it has generated biochemically relevant data and identified pathways significantly affected by methamphetamine. This combination of technologies has clear uses as a model for the study of neuronal toxicology.

Performance evaluation of thermophotovoltaic GaSb cell technology in high temperature waste heat

NASA Astrophysics Data System (ADS)

Utlu, Z.; Önal, B. S.

2018-02-01

In this study, waste heat was evaluated and examined by means of thermophotovoltaic systems with the application of energy production potential GaSb cells. The aim of our study is to examine GaSb cell technology at high temperature waste heat. The evaluation of the waste heat to be used in the system is designed to be used in the electricity, industry and iron and steel industry. Our work is research. Graphic analysis is done with Matlab program. The high temperature waste heat graphs applied on the GaSb cell are in the results section. Our study aims to provide a source for future studies.

Dissection of Drosophila Visual Circuits Implicative in Figure Motion

NASA Astrophysics Data System (ADS)

Kelley, Ross G.

The Drosophila visual system offers a model to study the foundations of how motion signals are computed from raw visual input and transformed into behavioral output. My studies focus on how specific cells in the Drosophila nervous system implement this input-output transformation. The individual cell types are known from classical studies using Golgi impregnations, but the assembly of motion processing circuits and the behavioral outputs remain poorly understood. Using an electronic flight simulator for flies and a white-noise analysis developed by Aptekar et al., I screen specific neurons in the optic lobes for behavioral ramifications. This approach produces wing responses to both the spatial and temporal dynamics of motion signals. The results of these experiments give Spatiotemporal Action Fields (STAFs) across the entire visual panorama. Genetically inactivating a distinct grouping of cells in the third optic ganglion, the Lobula Plate, the Horizontal System (HS) cell group, produced a robust phenotype through STAF analysis. Using the Gal4-UAS transgene expression system, we selectively inactivated the HS cells by expressing in their membrane inward rectifying potassium channels (Kir2.1) to hyperpolarize these cells, preventing their role in synaptic signaling. The results of the experiments show mutants lose steering responses to several distinct categories of figure motion and reduced behavioral responses to figure motion set against a contrasting moving background, highlighting their role in figure tracking behavior. Finally, a synapse inactivating protein, tetanus toxin (TNT), expressed in the HS cell group, produces a different behavioral phenotype than overexpressing inward rectifier. TNT, a bacterial neurotoxin, cleaves SNARE proteins resulting in loss of synaptic output of the cell, but the dendrites are intact and signal normally, preserving dendro-dendritic interactions known to sculpt the visual receptive fields of these cells. The two distinct phenotypes to each genetically targeted silencer differentiate the functional role of dendritic integration versus axonal output in this important cell group.

Mutations in spalt cause a severe but reversible neurodegenerative phenotype in the embryonic central nervous system of Drosophila melanogaster.

PubMed

Cantera, Rafael; Lüer, Karin; Rusten, Tor Erik; Barrio, Rosa; Kafatos, Fotis C; Technau, Gerhard M

2002-12-01

The gene spalt is expressed in the embryonic central nervous system of Drosophila melanogaster but its function in this tissue is still unknown. To investigate this question, we used a combination of techniques to analyse spalt mutant embryos. Electron microscopy showed that in the absence of spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway which influences the expression of the beta-catenin Armadillo, its ligand N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organisation of the central nervous system.

Thermodynamic analysis of a combined gas turbine power plant with a solid oxide fuel cell for marine applications

NASA Astrophysics Data System (ADS)

Welaya, Yousri M. A.; Mosleh, M.; Ammar, Nader R.

2013-12-01

Strong restrictions on emissions from marine power plants (particularly SOx, NOx) will probably be adopted in the near future. In this paper, a combined solid oxide fuel cell (SOFC) and gas turbine fuelled by natural gas is proposed as an attractive option to limit the environmental impact of the marine sector. It includes a study of a heatrecovery system for 18 MW SOFC fuelled by natural gas, to provide the electric power demand onboard commercial vessels. Feasible heat-recovery systems are investigated, taking into account different operating conditions of the combined system. Two types of SOFC are considered, tubular and planar SOFCs, operated with either natural gas or hydrogen fuels. This paper includes a detailed thermodynamic analysis for the combined system. Mass and energy balances are performed, not only for the whole plant but also for each individual component, in order to evaluate the thermal efficiency of the combined cycle. In addition, the effect of using natural gas as a fuel on the fuel cell voltage and performance is investigated. It is found that a high overall efficiency approaching 70% may be achieved with an optimum configuration using SOFC system under pressure. The hybrid system would also reduce emissions, fuel consumption, and improve the total system efficiency.

In silico lineage tracing through single cell transcriptomics identifies a neural stem cell population in planarians.

PubMed

Molinaro, Alyssa M; Pearson, Bret J

2016-04-27

The planarian Schmidtea mediterranea is a master regenerator with a large adult stem cell compartment. The lack of transgenic labeling techniques in this animal has hindered the study of lineage progression and has made understanding the mechanisms of tissue regeneration a challenge. However, recent advances in single-cell transcriptomics and analysis methods allow for the discovery of novel cell lineages as differentiation progresses from stem cell to terminally differentiated cell. Here we apply pseudotime analysis and single-cell transcriptomics to identify adult stem cells belonging to specific cellular lineages and identify novel candidate genes for future in vivo lineage studies. We purify 168 single stem and progeny cells from the planarian head, which were subjected to single-cell RNA sequencing (scRNAseq). Pseudotime analysis with Waterfall and gene set enrichment analysis predicts a molecularly distinct neoblast sub-population with neural character (νNeoblasts) as well as a novel alternative lineage. Using the predicted νNeoblast markers, we demonstrate that a novel proliferative stem cell population exists adjacent to the brain. scRNAseq coupled with in silico lineage analysis offers a new approach for studying lineage progression in planarians. The lineages identified here are extracted from a highly heterogeneous dataset with minimal prior knowledge of planarian lineages, demonstrating that lineage purification by transgenic labeling is not a prerequisite for this approach. The identification of the νNeoblast lineage demonstrates the usefulness of the planarian system for computationally predicting cellular lineages in an adult context coupled with in vivo verification.

Optical spectrum measurement of a cell-adhered microcavity for the cell-cycle analysis applications

NASA Astrophysics Data System (ADS)

Saito, Ryusuke; Terakawa, Mitsuhiro; Tanabe, Takasumi

2015-03-01

We build a setup and demonstrate successful measurement of the transmittance spectrum of a whispering gallery mode silica optical microcavity in which NIH 3T3 cells adhered on the top surface to achieve real-time and label-free measurement of the cell cycle. Label-free measurement is expected to prevent the cells to exhibit secondary effect. We build a system that enables the control of the gap distance between the microcavity and the tapered fiber, both of which are placed in the cell culture medium. The optimization of the tapered fiber diameter is the key to measure the spectrum of a microcavity in liquid. A swept wavelength laser light at a wavelength of 766 to 780 nm is used for the measurement. The cavity exhibit a Q of 1 . 0 ×106 in air, where the value is 1 . 0 ×105 in the medium and drops to 3 . 1 ×104 after the cell-adhesion. Still the Q of the microcavity is sufficiently high to detect the change at the cavity surface. Indeed we observe slight spectrum shift toward a longer wavelength, which we believe is due to the adherence of NIH 3T3 cells on the silica microcavity.The successful measurement of the transmittance spectrum of a microcavity in cell culture medium is the first step to realize the analysis of the cell-cycle based on microcavity system.

Measurement of marine picoplankton cell size by using a cooled, charge-coupled device camera with image-analyzed fluorescence microscopy.

PubMed Central

Viles, C L; Sieracki, M E

1992-01-01

Accurate measurement of the biomass and size distribution of picoplankton cells (0.2 to 2.0 microns) is paramount in characterizing their contribution to the oceanic food web and global biogeochemical cycling. Image-analyzed fluorescence microscopy, usually based on video camera technology, allows detailed measurements of individual cells to be taken. The application of an imaging system employing a cooled, slow-scan charge-coupled device (CCD) camera to automated counting and sizing of individual picoplankton cells from natural marine samples is described. A slow-scan CCD-based camera was compared to a video camera and was superior for detecting and sizing very small, dim particles such as fluorochrome-stained bacteria. Several edge detection methods for accurately measuring picoplankton cells were evaluated. Standard fluorescent microspheres and a Sargasso Sea surface water picoplankton population were used in the evaluation. Global thresholding was inappropriate for these samples. Methods used previously in image analysis of nanoplankton cells (2 to 20 microns) also did not work well with the smaller picoplankton cells. A method combining an edge detector and an adaptive edge strength operator worked best for rapidly generating accurate cell sizes. A complete sample analysis of more than 1,000 cells averages about 50 min and yields size, shape, and fluorescence data for each cell. With this system, the entire size range of picoplankton can be counted and measured. Images PMID:1610183

Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis.

PubMed

Chang, Chia-Hao; Mau-Hsu, Daxen; Chen, Ke-Cheng; Wei, Cheng-Wey; Chiu, Chiung-Ying; Young, Tai-Horng

2018-02-21

In a single-cell study, isolating and identifying single cells are essential, but these processes often require a large investment of time or money. The aim of this study was to isolate and analyse single cells using a novel platform, the PanelChip™ Analysis System, which includes 2500 microwells chip and a digital real-time polymerase chain reaction (dqPCR) assay, in comparison with a standard PCR (qPCR) assay. Through the serial dilution of a known concentration standard, namely pUC19, the accuracy and sensitivity levels of two methodologies were compared. The two systems were tested on the basis of expression levels of the genetic markers vimentin, E-cadherin, N-cadherin and GAPDH in A549 lung carcinoma cells at two known concentrations. Furthermore, the influence of a known PCR inhibitor commonly found in blood samples, heparin, was evaluated in both methodologies. Finally, mathematical models were proposed and separation method of single cells was verified; moreover, gene expression levels during epithelial-mesenchymal transition in single cells under TGFβ1 treatment were measured. The drawn conclusion is that dqPCR performed using PanelChip™ is superior to the standard qPCR in terms of sensitivity, precision, and heparin tolerance. The dqPCR assay is a potential tool for clinical diagnosis and single-cell applications.

Microchimerism in a female patient with systemic lupus erythematosus.

PubMed

Johnson, K L; McAlindon, T E; Mulcahy, E; Bianchi, D W

2001-09-01

Systemic lupus erythematosus (SLE) is a serious multisystem disease that has a striking propensity to affect women. The cause of SLE remains elusive. Fetomaternal cell trafficking, or the passage of fetal cells into the maternal circulation, is now a well-established phenomenon. In addition, fetal cells have been implicated in the development of preeclampsia and in the pathogenesis of scleroderma. We undertook this study to determine whether fetomaternal cell trafficking might also be involved in pathogenic processes in SLE. Fluorescence in situ hybridization analysis was performed using X and Y chromosome-specific probes on affected and unaffected tissue obtained at autopsy from a woman who had previously given birth to 2 males and who had died of complications of SLE. The goal of the analysis was to detect the presence of male cells of putative fetal origin. Male cells were found in every histologically abnormal tissue type that was examined, but were not found in histologically normal tissue. These data suggest that fetal cells may be associated with SLE. It is unclear whether their presence may be related to disease causation, an effect of disease progression, or unrelated to disease pathology. However, this case study is an important step toward understanding the potential relationship between fetomaternal cell trafficking and SLE pathology.

Microgravity effects during fertilization, cell division, development, and calcium metabolism in sea urchins

NASA Technical Reports Server (NTRS)

Schatten, Heide

1996-01-01

The overall objectives of this project are to explore the role of microgravity during fertilization, early development, cytoskeletal organization, and skeletal calcium deposition in a model development system: the sea urchin eggs and embryos. While pursuing these objectives, we have also helped to develop, test, and fly the Aquatic Research Facility (ARF) system. Cells were fixed at preselected time points to preserve the structures and organelles of interest with regards to cell biology events during development. The protocols used for the analysis of the results had been developed during the earlier part of this research and were applied for post-flight analysis using light and (immuno)fluorescence microscopy, scanning electron microscopy, and transmission electron microscopy. The structures of interest are: microtubules during fertilization, cell division, and cilia movement; microfilaments during cell surface restructuring and cell division; centrosomes and centrioles during cell division, cell differentiation, and cilia formation and movement; membranes, Golgi, endoplasmic reticulum, mitochondria, and chromosomes at all stages of development; and calcium deposits during spicule formation in late-stage embryos. In addition to further explore aspects important or living in space, several aspects of this research are also aimed at understanding diseases that affect humans on Earth which may be accelerated in space.

Evaluation of the Effects of Airborne Particulate Matter on Bone Marrow-Mesenchymal Stem Cells (BM-MSCs): Cellular, Molecular and Systems Biological Approaches

PubMed Central

Abu-Elmagd, Muhammad; Alghamdi, Mansour A.; Shamy, Magdy; Khoder, Mamdouh I.; Costa, Max; Assidi, Mourad; Kadam, Roaa; Alsehli, Haneen; Gari, Mamdooh; Pushparaj, Peter Natesan; Kalamegam, Gauthaman; Al-Qahtani, Mohammed H.

2017-01-01

Particulate matter (PM) contains heavy metals that affect various cellular functions and gene expression associated with a range of acute and chronic diseases in humans. However, the specific effects they exert on the stem cells remain unclear. Here, we report the effects of PM collected from the city of Jeddah on proliferation, cell death, related gene expression and systems of biological analysis in bone marrow mesenchymal stem cells (BM-MSCs), with the aim of understanding the underlying mechanisms. PM2.5 and PM10 were tested in vitro at various concentrations (15 to 300 µg/mL) and durations (24 to 72 h). PMs induced cellular stress including membrane damage, shrinkage and death. Lower concentrations of PM2.5 increased proliferation of BM-MSCs, while higher concentrations served to decrease it. PM10 decreased BM-MSCs proliferation in a concentration-dependent manner. The X-ray fluorescence spectrometric analysis showed that PM contains high levels of heavy metals. Ingenuity Pathway Analysis (IPA) and hierarchical clustering analyses demonstrated that heavy metals were associated with signaling pathways involving cell stress/death, cancer and chronic diseases. qRT-PCR results showed differential expression of the apoptosis genes (BCL2, BAX); inflammation associated genes (TNF-α and IL-6) and the cell cycle regulation gene (p53). We conclude that PM causes inflammation and cell death, and thereby predisposes to chronic debilitating diseases. PMID:28425934

PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing

PubMed Central

Dasa, Siva Sai Krishna; Kelly, Kimberly A.

2016-01-01

Next-generation sequencing has enhanced the phage display process, allowing for the quantification of millions of sequences resulting from the biopanning process. In response, many valuable analysis programs focused on specificity and finding targeted motifs or consensus sequences were developed. For targeted drug delivery and molecular imaging, it is also necessary to find peptides that are selective—targeting only the cell type or tissue of interest. We present a new analysis strategy and accompanying software, PHage Analysis for Selective Targeted PEPtides (PHASTpep), which identifies highly specific and selective peptides. Using this process, we discovered and validated, both in vitro and in vivo in mice, two sequences (HTTIPKV and APPIMSV) targeted to pancreatic cancer-associated fibroblasts that escaped identification using previously existing software. Our selectivity analysis makes it possible to discover peptides that target a specific cell type and avoid other cell types, enhancing clinical translatability by circumventing complications with systemic use. PMID:27186887

Modeling and analysis of cell membrane systems with probabilistic model checking

PubMed Central

2011-01-01

Background Recently there has been a growing interest in the application of Probabilistic Model Checking (PMC) for the formal specification of biological systems. PMC is able to exhaustively explore all states of a stochastic model and can provide valuable insight into its behavior which are more difficult to see using only traditional methods for system analysis such as deterministic and stochastic simulation. In this work we propose a stochastic modeling for the description and analysis of sodium-potassium exchange pump. The sodium-potassium pump is a membrane transport system presents in all animal cell and capable of moving sodium and potassium ions against their concentration gradient. Results We present a quantitative formal specification of the pump mechanism in the PRISM language, taking into consideration a discrete chemistry approach and the Law of Mass Action aspects. We also present an analysis of the system using quantitative properties in order to verify the pump reversibility and understand the pump behavior using trend labels for the transition rates of the pump reactions. Conclusions Probabilistic model checking can be used along with other well established approaches such as simulation and differential equations to better understand pump behavior. Using PMC we can determine if specific events happen such as the potassium outside the cell ends in all model traces. We can also have a more detailed perspective on its behavior such as determining its reversibility and why its normal operation becomes slow over time. This knowledge can be used to direct experimental research and make it more efficient, leading to faster and more accurate scientific discoveries. PMID:22369714

Design of a large-scale femtoliter droplet array for single-cell analysis of drug-tolerant and drug-resistant bacteria.

PubMed

Iino, Ryota; Matsumoto, Yoshimi; Nishino, Kunihiko; Yamaguchi, Akihito; Noji, Hiroyuki

2013-01-01

Single-cell analysis is a powerful method to assess the heterogeneity among individual cells, enabling the identification of very rare cells with properties that differ from those of the majority. In this Methods Article, we describe the use of a large-scale femtoliter droplet array to enclose, isolate, and analyze individual bacterial cells. As a first example, we describe the single-cell detection of drug-tolerant persisters of Pseudomonas aeruginosa treated with the antibiotic carbenicillin. As a second example, this method was applied to the single-cell evaluation of drug efflux activity, which causes acquired antibiotic resistance of bacteria. The activity of the MexAB-OprM multidrug efflux pump system from Pseudomonas aeruginosa was expressed in Escherichia coli and the effect of an inhibitor D13-9001 were assessed at the single cell level.

Single Cell Mass Cytometry for Analysis of Immune System Functional States

PubMed Central

Bjornson, Zach B.; Nolan, Garry P.; Fantl, Wendy J.

2013-01-01

Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with antibody panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the periodic table. Isotope labelled antibodies recognize surface markers to delineate cell types and intracellular signaling molecules to provide a measure of the network state—and thereby demarcating multiple cell state functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at its network state. This review will cover the basics of mass cytometry as well as outline steps already taken to allow it to stand aside traditional fluorescence based cytometry in the immunologist’s analytical arsenal in their study of immune states during infection. PMID:23999316

Developing multi-cellular tumor spheroid model (MCTS) in the chitosan/collagen/alginate (CCA) fibrous scaffold for anticancer drug screening.

PubMed

Wang, Jian-Zheng; Zhu, Yu-Xia; Ma, Hui-Chao; Chen, Si-Nan; Chao, Ji-Ye; Ruan, Wen-Ding; Wang, Duo; Du, Feng-guang; Meng, Yue-Zhong

2016-05-01

In this work, a 3D MCTS-CCA system was constructed by culturing multi-cellular tumor spheroid (MCTS) in the chitosan/collagen/alginate (CCA) fibrous scaffold for anticancer drug screening. The CCA scaffolds were fabricated by spray-spinning. The interactions between the components of the spray-spun fibers were evidenced by methods of Coomassie Blue stain, X-ray diffraction (XRD) and Fourier transform-infrared spectroscopy (FTIR). Co-culture indicated that MCF-7 cells showed a spatial growth pattern of multi-cellular tumor spheroid (MCTS) in the CCA fibrous scaffold with increased proliferation rate and drug-resistance to MMC, ADM and 5-Aza comparing with the 2D culture cells. Significant increases of total viable cells were found in 3D MCTS groups after drug administration by method of apoptotic analysis. Glucose-lactate analysis indicated that the metabolism of MCTS in CCA scaffold was closer to the tumor issue in vivo than the monolayer cells. In addition, MCTS showed the characteristic of epithelial mesenchymal transition (EMT) which is subverted by carcinoma cells to facilitate metastatic spread. These results demonstrated that MCTS in CCA scaffold possessed a more conservative phenotype of tumor than monolayer cells, and anticancer drug screening in 3D MCTS-CCA system might be superior to the 2D culture system. Copyright © 2016 Elsevier B.V. All rights reserved.

Computational fluid modeling and performance analysis of a bidirectional rotating perfusion culture system.

PubMed

Kang, Chang-Wei; Wang, Yan; Tania, Marshella; Zhou, Huancheng; Gao, Yi; Ba, Te; Tan, Guo-Dong Sean; Kim, Sangho; Leo, Hwa Liang

2013-01-01

A myriad of bioreactor configurations have been investigated as extracorporeal medical support systems for temporary replacement of vital organ functions. In recent years, studies have demonstrated that the rotating bioreactors have the potential to be utilized as bioartificial liver assist devices (BLADs) owing to their advantage of ease of scalability of cell-culture volume. However, the fluid movement in the rotating chamber will expose the suspended cells to unwanted flow structures with abnormally high shear conditions that may result in poor cell stability and in turn lower the efficacy of the bioreactor system. In this study, we compared the hydrodynamic performance of our modified rotating bioreactor design with that of an existing rotating bioreactor design. Computational fluid dynamic analysis coupled with experimental results were employed in the optimization process for the development of the modified bioreactor design. Our simulation results showed that the modified bioreactor had lower fluid induced shear stresses and more uniform flow conditions within its rotating chamber than the conventional design. Experimental results revealed that the cells within the modified bioreactor also exhibited better cell-carrier attachment, higher metabolic activity, and cell viability compared to those in the conventional design. In conclusion, this study was able to provide important insights into the flow physics within the rotating bioreactors, and help enhanced the hydrodynamic performance of an existing rotating bioreactor for BLAD applications. © 2013 American Institute of Chemical Engineers.

Understanding Regulation of Metabolism through Feasibility Analysis

PubMed Central

Nikerel, Emrah; Berkhout, Jan; Hu, Fengyuan; Teusink, Bas; Reinders, Marcel J. T.; de Ridder, Dick

2012-01-01

Understanding cellular regulation of metabolism is a major challenge in systems biology. Thus far, the main assumption was that enzyme levels are key regulators in metabolic networks. However, regulation analysis recently showed that metabolism is rarely controlled via enzyme levels only, but through non-obvious combinations of hierarchical (gene and enzyme levels) and metabolic regulation (mass action and allosteric interaction). Quantitative analyses relating changes in metabolic fluxes to changes in transcript or protein levels have revealed a remarkable lack of understanding of the regulation of these networks. We study metabolic regulation via feasibility analysis (FA). Inspired by the constraint-based approach of Flux Balance Analysis, FA incorporates a model describing kinetic interactions between molecules. We enlarge the portfolio of objectives for the cell by defining three main physiologically relevant objectives for the cell: function, robustness and temporal responsiveness. We postulate that the cell assumes one or a combination of these objectives and search for enzyme levels necessary to achieve this. We call the subspace of feasible enzyme levels the feasible enzyme space. Once this space is constructed, we can study how different objectives may (if possible) be combined, or evaluate the conditions at which the cells are faced with a trade-off among those. We apply FA to the experimental scenario of long-term carbon limited chemostat cultivation of yeast cells, studying how metabolism evolves optimally. Cells employ a mixed strategy composed of increasing enzyme levels for glucose uptake and hexokinase and decreasing levels of the remaining enzymes. This trade-off renders the cells specialized in this low-carbon flux state to compete for the available glucose and get rid of over-overcapacity. Overall, we show that FA is a powerful tool for systems biologists to study regulation of metabolism, interpret experimental data and evaluate hypotheses. PMID:22808034

Systems Imaging of the Immune Synapse.

PubMed

Ambler, Rachel; Ruan, Xiangtao; Murphy, Robert F; Wülfing, Christoph

2017-01-01

Three-dimensional live cell imaging of the interaction of T cells with antigen-presenting cells (APCs) visualizes the subcellular distributions of signaling intermediates during T cell activation at thousands of resolved positions within a cell. These information-rich maps of local protein concentrations are a valuable resource in understanding T cell signaling. Here, we describe a protocol for the efficient acquisition of such imaging data and their computational processing to create four-dimensional maps of local concentrations. This protocol allows quantitative analysis of T cell signaling as it occurs inside live cells with resolution in time and space across thousands of cells.

Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.

PubMed

Bleckmann, Maren; Fritz, Markus H-Y; Bhuju, Sabin; Jarek, Michael; Schürig, Margitta; Geffers, Robert; Benes, Vladimir; Besir, Hüseyin; van den Heuvel, Joop

2015-01-01

The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.

Flow-injection analysis of catecholamine secretion from bovine adrenal medulla cells on microbeads.

PubMed

Herrera, M; Kao, L S; Curran, D J; Westhead, E W

1985-01-01

Bovine adrenal medullary cells have been cultured on microbeads which are placed in a low-volume flow system for measurements of stimulation-response parameters. Electronically controlled stream switching allows stimulation of cells with pulse lengths from 1 s to many minutes; pulses may be repeated indefinitely. Catecholamines secreted are detected by an electrochemical detector downstream from the cells. This flow-injection analysis technique provides a new level of sensitivity and precision for measurement of kinetic parameters of secretion. A manual injection valve allows stimulation by higher levels of stimulant in the presence of constant low levels of stimulant. Such experiments show interesting differences between the effects of K+ and acetylcholine on cells partially desensitized to acetylcholine.

Sampling-based Bayesian approaches reveal the importance of quasi-bistable behavior in cellular decision processes on the example of the MAPK signaling pathway in PC-12 cell lines.

PubMed

Jensch, Antje; Thomaseth, Caterina; Radde, Nicole E

2017-01-25

Positive and negative feedback loops are ubiquitous motifs in biochemical signaling pathways. The mitogen-activated protein kinase (MAPK) pathway module is part of many distinct signaling networks and comprises several of these motifs, whose functioning depends on the cell line at hand and on the particular context. The maintainance of specificity of the response of the MAPK module to distinct stimuli has become a key paradigm especially in PC-12 cells, where the same module leads to different cell fates, depending on the stimulating growth factor. This cell fate is regulated by differences in the ERK (MAPK) activation profile, which shows a transient response upon stimulation with EGF, while the response is sustained in case of NGF. This behavior was explained by different effective network topologies. It is widely believed that this sustained response requires a bistable system. In this study we present a sampling-based Bayesian model analysis on a dataset, in which PC-12 cells have been stimulated with different growth factors. This is combined with novel analysis methods to investigate the role of feedback interconnections to shape ERK response. Results strongly suggest that, besides bistability, an additional effect called quasi-bistability can contribute to explain the observed responses of the system to different stimuli. Quasi-bistability is the ability of a monostable system to maintain two distinct states over a long time period upon a transient signal, which is also related to positive feedback, but cannot be detected by standard steady state analysis methods. Although applied on a specific example, our framework is generic enough to be also relevant for other regulatory network modeling studies that comprise positive feedback to explain cellular decision making processes. Overall, this study advices to focus not only on steady states, but also to take transient behavior into account in the analysis.

Architecture and inherent robustness of a bacterial cell-cycle control system.

PubMed

Shen, Xiling; Collier, Justine; Dill, David; Shapiro, Lucy; Horowitz, Mark; McAdams, Harley H

2008-08-12

A closed-loop control system drives progression of the coupled stalked and swarmer cell cycles of the bacterium Caulobacter crescentus in a near-mechanical step-like fashion. The cell-cycle control has a cyclical genetic circuit composed of four regulatory proteins with tight coupling to processive chromosome replication and cell division subsystems. We report a hybrid simulation of the coupled cell-cycle control system, including asymmetric cell division and responses to external starvation signals, that replicates mRNA and protein concentration patterns and is consistent with observed mutant phenotypes. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Formal model-checking analysis of the digital circuit showed that the cell-cycle control is robust to intrinsic stochastic variations in reaction rates and nutrient supply, and that it reliably stops and restarts to accommodate nutrient starvation. Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. The hybrid cell-cycle simulation implementation is inherently extensible and provides a promising approach for development of whole-cell behavioral models that can replicate the observed functionality of the cell and its responses to changing environmental conditions.

3.0-3.7μm infrared sensor system for cell analysis

NASA Astrophysics Data System (ADS)

van den Driesche, Sander; Witarski, Wojciech; Vellekoop, Michael J.

2009-05-01

In this contribution we present a novel LED-photodiode based infrared absorbance sensor in the wavelength range of 3.0 - 3.7 μm for cell analysis. Instead of using time consuming and expensive labelling and staining techniques to distinguish healthy from malignant cell types, this IR sensor system can perform faster, cheaper and without the need of additional chemicals. Depending on the used narrow bandpass filters, absorbance due to specific molecular vibration can be measured, such as the functional absorbance peaks at 3.38 μm (CH3-antisymmetric stretch), 3.42 μm (CH2- antisymmetric stretch), 3.48 μm (CH3-symmetric stretch) and 3.51 μm (CH2-symmetric stretch). For normalization and baseline correction the absorbance at wavelengths 3.33 and 3.57 μm are used. By recording the IR absorbance spectra of healthy and malignant epithelial kidney cell lines with an IR spectroscope, we found significant differences in the absorbance ratio 3.51 μm / 3.42 μm (CH2-symmetric/antisymmetric stretch). This result has led us to a sensor concept where only four wavelengths are being measured. In the 3.0 - 3.7 μm wavelength region a low cost LED-photodiode system can be used instead of a spectroscope. Yeast cells, which also contain the CH2 symmetric and antisymmetric stretch bands, are used to validate this sensor system and to make a first comparison of the system to spectroscopic recordings. Sensor experiments on dried spots of baker's yeast on calcium-fluoride slides yielded a comparable CH2 stretch ratio with the IR spectroscope measurement. This confirms the usability of the sensor to measure the CH2 stretch ratio and its potential for fast, label-free and low cost screening of cell samples.

Spatial Distribution and Receptor Specificity of Zebrafish Kit System - Evidence for a Kit-Mediated Bi-Directional Communication System in the Preovulatory Ovarian Follicle

PubMed Central

Yao, Kai; Ge, Wei

2013-01-01

Consisting of Kit ligand and receptor Kit, the Kit system is involved in regulating many ovarian functions such as follicle activation, granulosa cell proliferation, and oocyte growth and maturation. In mammals, Kit ligand is derived from the granulosa cells and Kit receptor is expressed in the oocyte and theca cells. In the zebrafish, the Kit system contains two ligands (Kitlga and Kitlgb) and two receptors (Kita and Kitb). Interestingly, Kitlga and Kitb are localized in the somatic follicle cells, but Kitlgb and Kita are expressed in the oocyte. Using recombinant zebrafish Kitlga and Kitlgb, we demonstrated that Kitlga preferentially activated Kita whereas Kitlgb specifically activated Kitb by Western analysis for receptor phosphorylation. In support of this, Kitlgb triggered a stronger and longer MAPK phosphorylation in follicle cells than Kitlga, whereas Kitlga but not Kitlgb activated MAPK in the denuded oocytes, in agreement with the distribution of Kita and Kitb in the follicle and their specificity for Kitlga and Kitlgb. Further analysis of the interaction between Kit ligands and receptors by homology modeling showed that Kitlga-Kita and Kitlgb-Kitb both have more stable electrostatic interaction than Kitlgb-Kita or Kitlga-Kitb. A functional study of Kit involvement in final oocyte maturation showed that Kitlga and Kitlgb both suppressed the spontaneous maturation significantly; in contrast, Kitlgb but not Kitlga significantly promoted 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) -induced oocyte maturation. Our results provided strong evidence for a Kit-mediated bi-directional communication system in the zebrafish ovarian follicle, which could be part of the complex interplay between the oocyte and the follicle cells in the development of follicles. PMID:23409152

Selective Heterogeneity in Exoprotease Production by Bacillus subtilis

PubMed Central

Davidson, Fordyce A.; Seon-Yi, Chung; Stanley-Wall, Nicola R.

2012-01-01

Bacteria have elaborate signalling mechanisms to ensure a behavioural response that is most likely to enhance survival in a changing environment. It is becoming increasingly apparent that as part of this response, bacteria are capable of cell differentiation and can generate multiple, mutually exclusive co-existing cell states. These cell states are often associated with multicellular processes that bring benefit to the community as a whole but which may be, paradoxically, disadvantageous to an individual subpopulation. How this process of cell differentiation is controlled is intriguing and remains a largely open question. In this paper, we consider an important aspect of cell differentiation that is known to occur in the Gram-positive bacterium Bacillus subtilis: we investigate the role of two master regulators DegU and Spo0A in the control of extra-cellular protease production. Recent work in this area focussed the on role of DegU in this process and suggested that transient effects in protein production were the drivers of cell-response heterogeneity. Here, using a combination of mathematical modelling, analysis and stochastic simulations, we provide a complementary analysis of this regulatory system that investigates the roles of both DegU and Spo0A in extra-cellular protease production. In doing so, we present a mechanism for bimodality, or system heterogeneity, without the need for a bistable switch in the underlying regulatory network. Moreover, our analysis leads us to conclude that this heterogeneity is in fact a persistent, stable feature. Our results suggest that system response is divided into three zones: low and high signal levels induce a unimodal or undifferentiated response from the cell population with all cells OFF and ON, respectively for exoprotease production. However, for intermediate levels of signal, a heterogeneous response is predicted with a spread of activity levels, representing typical “bet-hedging” behaviour. PMID:22745669

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

PubMed

Shiba, Tomonori; Mii, Masahiro

2005-12-01

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

Note: An automated image analysis method for high-throughput classification of surface-bound bacterial cell motions.

PubMed

Shen, Simon; Syal, Karan; Tao, Nongjian; Wang, Shaopeng

2015-12-01

We present a Single-Cell Motion Characterization System (SiCMoCS) to automatically extract bacterial cell morphological features from microscope images and use those features to automatically classify cell motion for rod shaped motile bacterial cells. In some imaging based studies, bacteria cells need to be attached to the surface for time-lapse observation of cellular processes such as cell membrane-protein interactions and membrane elasticity. These studies often generate large volumes of images. Extracting accurate bacterial cell morphology features from these images is critical for quantitative assessment. Using SiCMoCS, we demonstrated simultaneous and automated motion tracking and classification of hundreds of individual cells in an image sequence of several hundred frames. This is a significant improvement from traditional manual and semi-automated approaches to segmenting bacterial cells based on empirical thresholds, and a first attempt to automatically classify bacterial motion types for motile rod shaped bacterial cells, which enables rapid and quantitative analysis of various types of bacterial motion.

Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis

PubMed Central

1993-01-01

We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn2+, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis. PMID:8408207

Droplet-based gene expression analysis using a device with magnetic force-based-droplet-handling system.

PubMed

Okochi, Mina; Tsuchiya, Hiroyoshi; Kumazawa, Fumitaka; Shikida, Mitsuhiro; Honda, Hiroyuki

2010-02-01

A droplet-based cell lysis and reverse transcription-polymerase chain reaction (PCR) were performed on-chip employing magnetic force-based-droplet-handling system. The actuation with a magnet offers a simple system for droplet manipulation; it does not need mechanical fluidic systems such as pumps and valves for handling solutions. It can be used as a powerful tool for various biochemical applications by moving and coalescing sample droplets using magnetic beads immersed in mineral oil. The droplet containing magnetic beads and the cells were manipulated with the magnet located underneath the channel, and coalesced with a droplet of lysis buffer. Using K562 cells as the leukemia model, the cell lysis, cDNA synthesis, and amplification of WT1 gene that is known as the prognostic factor for acute leukemia were successfully performed from a single cell. Copyright (c) 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Fast and Label-Free Isolation of Circulating Tumor Cells from Blood: From a Research Microfluidic Platform to an Automated Fluidic Instrument, VTX-1 Liquid Biopsy System.

PubMed

Lemaire, Clementine A; Liu, Sean Z; Wilkerson, Charles L; Ramani, Vishnu C; Barzanian, Nasim A; Huang, Kuo-Wei; Che, James; Chiu, Michael W; Vuppalapaty, Meghah; Dimmick, Adam M; Carlo, Dino Di; Kochersperger, Michael L; Crouse, Steve C; Jeffrey, Stefanie S; Englert, Robert F; Hengstler, Stephan; Renier, Corinne; Sollier-Christen, Elodie

2018-02-01

Tumor tissue biopsies are invasive, costly, and collect a limited cell population not completely reflective of patient cancer cell diversity. Circulating tumor cells (CTCs) can be isolated from a simple blood draw and may be representative of the diverse biology from multiple tumor sites. The VTX-1 Liquid Biopsy System was designed to automate the isolation of clinically relevant CTC populations, making the CTCs available for easy analysis. We present here the transition from a cutting-edge microfluidic innovation in the lab to a commercial, automated system for isolating CTCs directly from whole blood. As the technology evolved into a commercial system, flexible polydimethylsiloxane microfluidic chips were replaced by rigid poly(methyl methacrylate) chips for a 2.2-fold increase in cell recovery. Automating the fluidic processing with the VTX-1 further improved cancer cell recovery by nearly 1.4-fold, with a 2.8-fold decrease in contaminating white blood cells and overall improved reproducibility. Two isolation protocols were optimized that favor either the cancer cell recovery (up to 71.6% recovery) or sample purity (≤100 white blood cells/mL). The VTX-1's performance was further tested with three different spiked breast or lung cancer cell lines, with 69.0% to 79.5% cell recovery. Finally, several cancer research applications are presented using the commercial VTX-1 system.

Efficacy of rituximab (anti‐CD20) for refractory systemic lupus erythematosus involving the central nervous system

PubMed Central

Tokunaga, Mikiko; Saito, Kazuyoshi; Kawabata, Daisuke; Imura, Yoshitaka; Fujii, Takao; Nakayamada, Shingo; Tsujimura, Shizuyo; Nawata, Masao; Iwata, Shigeru; Azuma, Taeko; Mimori, Tsuneyo; Tanaka, Yoshiya

2007-01-01

Aim Neuropsychiatric systemic lupus erythematosus (NPSLE) is a serious treatment‐resistant phenotype of systemic lupus erythematosus. A standard treatment for NPSLE is not available. This report describes the clinical and laboratory tests of 10 patients with NPSLE before and after rituximab treatment, including changes in lymphocyte phenotypes. Methods Rituximab was administered at different doses in 10 patients with refractory NPSLE, despite intensive treatment. Results Treatment with rituximab resulted in rapid improvement of central nervous system‐related manifestations, particularly acute confusional state. Rituximab also improved cognitive dysfunction, psychosis and seizure, and reduced the SLE Disease Activity Index Score at day 28 in all 10 patients. These effects lasted for >1 year in five patients. Flow cytometric analysis showed that rituximab down regulated CD40 and CD80 on B cells and CD40L, CD69 and inducible costimulator on CD4+ T cells. Conclusions Rituximab rapidly improved refractory NPSLE, as evident by resolution of various clinical signs and symptoms and improvement of radiographic findings. The down regulation of functional molecules on B and T cells suggests that rituximab modulates the interaction of activated B and T cells through costimulatory molecules. These results warrant further analysis of rituximab as treatment for NPSLE. PMID:17107983

Power System Trade Studies for the Lunar Surface Access Module

NASA Technical Reports Server (NTRS)

Kohout, Lisa, L.

2008-01-01

A Lunar Lander Preparatory Study (LLPS) was undertaken for NASA's Lunar Lander Pre-Project in 2006 to explore a wide breadth of conceptual lunar lander designs. Civil servant teams from nearly every NASA center responded with dozens of innovative designs that addressed one or more specific lander technical challenges. Although none of the conceptual lander designs sought to solve every technical design issue, each added significantly to the technical database available to the Lunar Lander Project Office as it began operations in 2007. As part of the LLPS, a first order analysis was performed to identify candidate power systems for the ascent and descent stages of the Lunar Surface Access Module (LSAM). A power profile by mission phase was established based on LSAM subsystem power requirements. Using this power profile, battery and fuel cell systems were modeled to determine overall mass and volume. Fuel cell systems were chosen for both the descent and ascent stages due to their low mass. While fuel cells looked promising based on these initial results, several areas have been identified for further investigation in subsequent studies, including the identification and incorporation of peak power requirements into the analysis, refinement of the fuel cell models to improve fidelity and incorporate ongoing technology developments, and broadening the study to include solar power.

An improved Red/ET recombineering system and mouse ES cells culture conditions for the generation of targeted mutant mice.

PubMed

Kumagai, Katsuyoshi; Takanashi, Masakatsu; Ohno, Shin-Ichiro; Kuroda, Masahiko; Sudo, Katsuko

2017-05-03

Targeted mutant mice generated on a C57BL/6 background are powerful tools for analysis of the biological functions of genes, and gene targeting technologies using mouse embryonic stem (ES) cells have been used to generate such mice. Recently, a bacterial artificial chromosome (BAC) recombineering system was established for the construction of targeting vectors. However, gene retrieval from BACs for the generation of gene targeting vectors using this system remains difficult. Even when construction of a gene targeting vector is successful, the efficiency of production of targeted mutant mice from ES cells derived from C57BL/6 mice are poor. Therefore, in this study, we first improved the strategy for the retrieval of genes from BACs and their transfer into a DT-A plasmid, for the generation of gene targeting vectors using the BAC recombineering system. Then, we attempted to generate targeted mutant mice from ES cell lines derived from C57BL/6 mice, by culturing in serum-free medium. In conclusion, we established an improved strategy for the efficient generation of targeted mutant mice on a C57BL/6 background, which are useful for the in vivo analysis of gene functions and regulation.

Investigation of type-I interferon dysregulation by arenaviruses : a multidisciplinary approach.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kozina, Carol L.; Moorman, Matthew Wallace; Branda, Catherine

2011-09-01

This report provides a detailed overview of the work performed for project number 130781, 'A Systems Biology Approach to Understanding Viral Hemorrhagic Fever Pathogenesis.' We report progress in five key areas: single cell isolation devices and control systems, fluorescent cytokine and transcription factor reporters, on-chip viral infection assays, molecular virology analysis of Arenavirus nucleoprotein structure-function, and development of computational tools to predict virus-host protein interactions. Although a great deal of work remains from that begun here, we have developed several novel single cell analysis tools and knowledge of Arenavirus biology that will facilitate and inform future publications and funding proposals.

[Quantitative analysis of the structure of neuronal dendritic spines in the striatum using the Leitz-ASM system].

PubMed

Leontovich, T A; Zvegintseva, E G

1985-10-01

Two principal classes of striatum long axonal neurons (sparsely ramified reticular cells and densely ramified dendritic cells) were analyzed quantitatively in four animal species: hedgehog, rabbit, dog and monkey. The cross section area, total dendritic length and the area of dendritic field were measured using "LEITZ-ASM" system. Classes of neurons studied were significantly different in dogs and monkeys, while no differences were noted between hedgehog and rabbit. Reticular neurons of different species varied much more than dendritic ones. Quantitative analysis has revealed the progressive increase in the complexity of dendritic tree in mammals from rabbit to monkey.

Automated Static Culture System Cell Module Mixing Protocol and Computational Fluid Dynamics Analysis

NASA Technical Reports Server (NTRS)

Kleis, Stanley J.; Truong, Tuan; Goodwin, Thomas J,

2004-01-01

This report is a documentation of a fluid dynamic analysis of the proposed Automated Static Culture System (ASCS) cell module mixing protocol. The report consists of a review of some basic fluid dynamics principles appropriate for the mixing of a patch of high oxygen content media into the surrounding media which is initially depleted of oxygen, followed by a computational fluid dynamics (CFD) study of this process for the proposed protocol over a range of the governing parameters. The time histories of oxygen concentration distributions and mechanical shear levels generated are used to characterize the mixing process for different parameter values.

Compartmentalized microchannel array for high-throughput analysis of single cell polarized growth and dynamics

DOE PAGES

Geng, Tao; Bredeweg, Erin L.; Szymanski, Craig J.; ...

2015-11-04

Here, interrogating polarized growth is technologically challenging due to extensive cellular branching and uncontrollable environmental conditions in conventional assays. Here we present a robust and high-performance microfluidic system that enables observations of polarized growth with enhanced temporal and spatial control over prolonged periods. The system has built-in tunability and versatility to accommodate a variety of science applications requiring precisely controlled environments. Using the model filamentous fungus, Neurospora crassa, this microfluidic system enabled direct visualization and analysis of cellular heterogeneity in a clonal fungal cell population, nuclear distribution and dynamics at the subhyphal level, and quantitative dynamics of gene expression withmore » single hyphal compartment resolution in response to carbon source starvation and exchange experiments. Although the microfluidic device is demonstrated on filamentous fungi, our technology is immediately extensible to a wide array of other biosystems that exhibit similar polarized cell growth with applications ranging from bioenergy production to human health.« less

CFD analysis of laboratory scale phase equilibrium cell operation

NASA Astrophysics Data System (ADS)

Jama, Mohamed Ali; Nikiforow, Kaj; Qureshi, Muhammad Saad; Alopaeus, Ville

2017-10-01

For the modeling of multiphase chemical reactors or separation processes, it is essential to predict accurately chemical equilibrium data, such as vapor-liquid or liquid-liquid equilibria [M. Šoóš et al., Chem. Eng. Process.: Process Intensif. 42(4), 273-284 (2003)]. The instruments used in these experiments are typically designed based on previous experiences, and their operation verified based on known equilibria of standard components. However, mass transfer limitations with different chemical systems may be very different, potentially falsifying the measured equilibrium compositions. In this work, computational fluid dynamics is utilized to design and analyze laboratory scale experimental gas-liquid equilibrium cell for the first time to augment the traditional analysis based on plug flow assumption. Two-phase dilutor cell, used for measuring limiting activity coefficients at infinite dilution, is used as a test case for the analysis. The Lagrangian discrete model is used to track each bubble and to study the residence time distribution of the carrier gas bubbles in the dilutor cell. This analysis is necessary to assess whether the gas leaving the cell is in equilibrium with the liquid, as required in traditional analysis of such apparatus. Mass transfer for six different bio-oil compounds is calculated to determine the approach equilibrium concentration. Also, residence times assuming plug flow and ideal mixing are used as reference cases to evaluate the influence of mixing on the approach to equilibrium in the dilutor. Results show that the model can be used to predict the dilutor operating conditions for which each of the studied gas-liquid systems reaches equilibrium.

CFD analysis of laboratory scale phase equilibrium cell operation.

PubMed

Jama, Mohamed Ali; Nikiforow, Kaj; Qureshi, Muhammad Saad; Alopaeus, Ville

2017-10-01

For the modeling of multiphase chemical reactors or separation processes, it is essential to predict accurately chemical equilibrium data, such as vapor-liquid or liquid-liquid equilibria [M. Šoóš et al., Chem. Eng. Process Intensif. 42(4), 273-284 (2003)]. The instruments used in these experiments are typically designed based on previous experiences, and their operation verified based on known equilibria of standard components. However, mass transfer limitations with different chemical systems may be very different, potentially falsifying the measured equilibrium compositions. In this work, computational fluid dynamics is utilized to design and analyze laboratory scale experimental gas-liquid equilibrium cell for the first time to augment the traditional analysis based on plug flow assumption. Two-phase dilutor cell, used for measuring limiting activity coefficients at infinite dilution, is used as a test case for the analysis. The Lagrangian discrete model is used to track each bubble and to study the residence time distribution of the carrier gas bubbles in the dilutor cell. This analysis is necessary to assess whether the gas leaving the cell is in equilibrium with the liquid, as required in traditional analysis of such apparatus. Mass transfer for six different bio-oil compounds is calculated to determine the approach equilibrium concentration. Also, residence times assuming plug flow and ideal mixing are used as reference cases to evaluate the influence of mixing on the approach to equilibrium in the dilutor. Results show that the model can be used to predict the dilutor operating conditions for which each of the studied gas-liquid systems reaches equilibrium.

Automated microbeam observation environment for biological analysis—Custom portable environmental control applied to a vertical microbeam system

PubMed Central

England, Matthew J.; Bigelow, Alan W.; Merchant, Michael J.; Velliou, Eirini; Welch, David; Brenner, David J.; Kirkby, Karen J.

2018-01-01

Vertical Microbeams (VMB) are used to irradiate individual cells with low MeV energy ions. The irradiation of cells using VMBs requires cells to be removed from an incubator; this can cause physiological changes to cells because of the lower CO2 concentration, temperature and relative humidity outside of the incubator. Consequently, for experiments where cells require irradiation and observation for extended time periods, it is important to provide a controlled environment. The highly customised nature of the microscopes used on VMB systems means that there are no commercially available environmentally controlled microscope systems for VMB systems. The Automated Microbeam Observation Environment for Biological Analysis (AMOEBA) is a highly flexible modular environmental control system used to create incubator conditions on the end of a VMB. The AMOEBA takes advantage of the recent “maker” movement to create an open source control system that can be easily configured by the user to fit their control needs even beyond VMB applications. When applied to the task of controlling cell medium temperature, CO2 concentration and relative humidity on VMBs it creates a stable environment that allows cells to multiply on the end of a VMB over a period of 36 h, providing a low-cost (costing less than $2700 to build), customisable alternative to commercial time-lapse microscopy systems. AMOEBA adds the potential of VMBs to explore the long-term effects of radiation on single cells opening up new research areas for VMBs. PMID:29515291

Dynamical analysis of uterine cell electrical activity model.

PubMed

Rihana, S; Santos, J; Mondie, S; Marque, C

2006-01-01

The uterus is a physiological system consisting of a large number of interacting smooth muscle cells. The uterine excitability changes remarkably with time, generally quiescent during pregnancy, the uterus exhibits forceful synchronized contractions at term leading to fetus expulsion. These changes characterize thus a dynamical system susceptible of being studied through formal mathematical tools. Multiple physiological factors are involved in the regulation process of this complex system. Our aim is to relate the physiological factors to the uterine cell dynamic behaviors. Taking into account a previous work presented, in which the electrical activity of a uterine cell is described by a set of ordinary differential equations, we analyze the impact of physiological parameters on the response of the model, and identify the main subsystems generating the complex uterine electrical activity, with respect to physiological data.

Ethylene Inhibits Cell Proliferation of the Arabidopsis Root Meristem1[OPEN

PubMed Central

Street, Ian H.; Aman, Sitwat; Zubo, Yan; Ramzan, Aleena; Wang, Xiaomin; Shakeel, Samina N.; Kieber, Joseph J.; Schaller, G. Eric

2015-01-01

The root system of plants plays a critical role in plant growth and survival, with root growth being dependent on both cell proliferation and cell elongation. Multiple phytohormones interact to control root growth, including ethylene, which is primarily known for its role in controlling root cell elongation. We find that ethylene also negatively regulates cell proliferation at the root meristem of Arabidopsis (Arabidopsis thaliana). Genetic analysis indicates that the inhibition of cell proliferation involves two pathways operating downstream of the ethylene receptors. The major pathway is the canonical ethylene signal transduction pathway that incorporates CONSTITUTIVE TRIPLE RESPONSE1, ETHYLENE INSENSITIVE2, and the ETHYLENE INSENSITIVE3 family of transcription factors. The secondary pathway is a phosphorelay based on genetic analysis of receptor histidine kinase activity and mutants involving the type B response regulators. Analysis of ethylene-dependent gene expression and genetic analysis supports SHORT HYPOCOTYL2, a repressor of auxin signaling, as one mediator of the ethylene response and furthermore, indicates that SHORT HYPOCOTYL2 is a point of convergence for both ethylene and cytokinin in negatively regulating cell proliferation. Additional analysis indicates that ethylene signaling contributes but is not required for cytokinin to inhibit activity of the root meristem. These results identify key elements, along with points of cross talk with cytokinin and auxin, by which ethylene negatively regulates cell proliferation at the root apical meristem. PMID:26149574