WorldWideScience

Sample records for viable cell mass

  1. Viable Cell Culture Banking for Biodiversity Characterization and Conservation.

    Science.gov (United States)

    Ryder, Oliver A; Onuma, Manabu

    2018-02-15

    Because living cells can be saved for indefinite periods, unprecedented opportunities for characterizing, cataloging, and conserving biological diversity have emerged as advanced cellular and genetic technologies portend new options for preventing species extinction. Crucial to realizing the potential impacts of stem cells and assisted reproductive technologies on biodiversity conservation is the cryobanking of viable cell cultures from diverse species, especially those identified as vulnerable to extinction in the near future. The advent of in vitro cell culture and cryobanking is reviewed here in the context of biodiversity collections of viable cell cultures that represent the progress and limitations of current efforts. The prospects for incorporating collections of frozen viable cell cultures into efforts to characterize the genetic changes that have produced the diversity of species on Earth and contribute to new initiatives in conservation argue strongly for a global network of facilities for establishing and cryobanking collections of viable cells.

  2. High speed flow cytometric separation of viable cells

    Science.gov (United States)

    Sasaki, Dennis T.; Van den Engh, Gerrit J.; Buckie, Anne-Marie

    1995-01-01

    Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

  3. Surface Charge Visualization at Viable Living Cells.

    Science.gov (United States)

    Perry, David; Paulose Nadappuram, Binoy; Momotenko, Dmitry; Voyias, Philip D; Page, Ashley; Tripathi, Gyanendra; Frenguelli, Bruno G; Unwin, Patrick R

    2016-03-09

    Scanning ion conductance microscopy (SICM) is demonstrated to be a powerful technique for quantitative nanoscale surface charge mapping of living cells. Utilizing a bias modulated (BM) scheme, in which the potential between a quasi-reference counter electrode (QRCE) in an electrolyte-filled nanopipette and a QRCE in bulk solution is modulated, it is shown that both the cell topography and the surface charge present at cellular interfaces can be measured simultaneously at high spatial resolution with dynamic potential measurements. Surface charge is elucidated by probing the properties of the diffuse double layer (DDL) at the cellular interface, and the technique is sensitive at both low-ionic strength and under typical physiological (high-ionic strength) conditions. The combination of experiments that incorporate pixel-level self-referencing (calibration) with a robust theoretical model allows for the analysis of local surface charge variations across cellular interfaces, as demonstrated on two important living systems. First, charge mapping at Zea mays root hairs shows that there is a high negative surface charge at the tip of the cell. Second, it is shown that there are distinct surface charge distributions across the surface of human adipocyte cells, whose role is the storage and regulation of lipids in mammalian systems. These are new features, not previously recognized, and their implications for the functioning of these cells are highlighted.

  4. Inkjet printing of viable human dental follicle stem cells

    Directory of Open Access Journals (Sweden)

    Mau Robert

    2015-09-01

    Full Text Available Inkjet printing technology has the potential to be used for seeding of viable cells for tissue engineering approaches. For this reason, a piezoelectrically actuated, drop-on-demand inkjet printing system was applied to deliver viable human dental follicle stem cells (hDFSC of sizes of about 15 μm up to 20 μm in diameter. The purpose of these investigations was to verify the stability of the printing process and to evaluate cell viability post printing. Using a Nanoplotter 2.1 (Gesim, Germany equipped with the piezoelectric printhead NanoTip HV (Gesim, Germany, a concentration of 6.6 ×106 cells ml−1 in DMEM with 10% fetal calf serum (FCS could be dispensed. The piezoelectric printhead has a nominal droplet volume of ~ 400 pl and was set to a voltage of 75 V and a pulse of 50 μs while dosing 50 000 droplets over a time of 100 seconds. The volume and trajectory of the droplet were checked by a stroboscope test right before and after the printing process. It was found that the droplet volume decreases significantly by 35% during printing process, while the trajectory of the droplets remains stable with only an insignificant number of degrees deviation from the vertical line. It is highly probable that some cell sedimentations or agglomerations affect the printing performance. The cell viability post printing was assessed by using the Trypan Blue dye exclusion test. The printing process was found to have no significant influence on cell survival. In conclusion, drop-on-demand inkjet printing can be a potent tool for the seeding of viable cells.

  5. The molecularly crowded cytoplasm of bacterialcCells : Dividing cells contrasted with viable but non-culturable (VBNC) bacterial cells

    NARCIS (Netherlands)

    Trevors, J. T.; van Elsas, J. D.; Bej, A. K.

    2013-01-01

    In this perspective, we discuss the cytoplasm in actively growing bacterial cells contrasted with viable but non-culturable (VBNC) cells. Actively growing bacterial cells contain a more molecularly crowded and organized cytoplasm, and are capable of completing their cell cycle resulting in cell

  6. Non-viable antagonist cells are associated with reduced biocontrol performance by viable cells of the yeast Papiliotrema flavescens against Fusarium head blight of wheat.

    Science.gov (United States)

    Microbially-based plant disease control products have achieved commercial market success, but the efficacy of such biocontrol products is sometimes deemed inconsistent. Improper processing of harvested microbial biomass or long-term storage can reduce the proportion of viable cells and necessitate t...

  7. Modelling the number of viable vegetative cells of Bacillus cereus passing through the stomach

    NARCIS (Netherlands)

    Wijnands, L.M.; Pielaat, A.; Dufrenne, J.B.; Zwietering, M.H.; Leusden, van F.M.

    2009-01-01

    Aims: Model the number of viable vegetative cells of B. cereus surviving the gastric passage after experiments in simulated gastric conditions. Materials and Methods: The inactivation of stationary and exponential phase vegetative cells of twelve different strains of Bacillus cereus, both mesophilic

  8. Combining ethidium monoazide treatment with real-time PCR selectively quantifies viable Batrachochytrium dendrobatidis cells.

    Science.gov (United States)

    Blooi, Mark; Martel, An; Vercammen, Francis; Pasmans, Frank

    2013-02-01

    Detection of the lethal amphibian fungus Batrachochytrium dendrobatidis relies on PCR-based techniques. Although highly accurate and sensitive, these methods fail to distinguish between viable and dead cells. In this study a novel approach combining the DNA intercalating dye ethidium monoazide (EMA) and real-time PCR is presented that allows quantification of viable B. dendrobatidis cells without the need for culturing. The developed method is able to suppress real-time PCR signals of heat-killed B. dendrobatidis zoospores by 99.9 % and is able to discriminate viable from heat-killed B. dendrobatidis zoospores in mixed samples. Furthermore, the novel approach was applied to assess the antifungal activity of the veterinary antiseptic F10(®) Antiseptic Solution. This disinfectant killed B. dendrobatidis zoospores effectively within 1 min at concentrations as low as 1:6400. Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  9. A simple way to identify non-viable cells within living plant tissue using confocal microscopy

    Directory of Open Access Journals (Sweden)

    Truernit Elisabeth

    2008-06-01

    Full Text Available Abstract Background Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. Results Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy. The dyes were selective for non-viable cells and showed very little background staining in living cells. Simultaneous detection of SYTOX dye and fluorescent protein (e.g. GFP fluorescence was possible. Conclusion The fluorescent SYTOX dyes are useful for an easy and quick first assay of plant cell viability in living plant samples using fluorescence and confocal laser-scanning microscopy.

  10. Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

    Science.gov (United States)

    Zhou, Ming-Da; Hao, Sijie; Williams, Anthony J.; Harouaka, Ramdane A.; Schrand, Brett; Rawal, Siddarth; Ao, Zheng; Brennaman, Randall; Gilboa, Eli; Lu, Bo; Wang, Shuwen; Zhu, Jiyue; Datar, Ram; Cote, Richard; Tai, Yu-Chong; Zheng, Si-Yang

    2014-12-01

    The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 103), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

  11. Use of propidium monoazide for selective profiling of viable microbial cells during Gouda cheese ripening

    NARCIS (Netherlands)

    Erkus, O.; Jager, V.C. de; Geene, R.T.; Alen-Boerrigter, I.J. van; Hazelwood, L.; Hijum, S.A.F.T. van; Kleerebezem, M; Smid, E.J.

    2016-01-01

    DNA based microbial community profiling of food samples is confounded by the presence of DNA derived from membrane compromised (dead or injured) cells. Selective amplification of DNA from viable (intact) fraction of the community by propidium monoazide (PMA) treatment could circumvent this problem.

  12. In vitro and in vivo bioluminescent quantification of viable stem cells in engineered constructs.

    Science.gov (United States)

    Logeart-Avramoglou, Delphine; Oudina, Karim; Bourguignon, Marianne; Delpierre, Laetitia; Nicola, Marie-Anne; Bensidhoum, Morad; Arnaud, Eric; Petite, Herve

    2010-06-01

    Bioluminescent quantification of viable cells inside three-dimensional porous scaffolds was performed in vitro and in vivo. The assay quantified the bioluminescence of murine stem (C3H10T1/2) cells tagged with the luciferase gene reporter and distributed inside scaffolds of either soft, translucent, AN69 polymeric hydrogel or hard, opaque, coral ceramic materials. Quantitative evaluation of bioluminescence emitted from tagged cells adhering to these scaffolds was performed in situ using either cell lysates and a luminometer or intact cells and a bioluminescence imaging system. Despite attenuation of the signal when compared to cells alone, the bioluminescence correlated with the number of cells (up to 1.5 x 10(5)) present on each material scaffold tested, both in vitro and noninvasively in vivo (subcutaneous implants in the mouse model). The noninvasive bioluminescence measurement technique proved to be comparable to the cell-destructive bioluminescence measurement technique. Monitoring the kinetics of luciferase expression via bioluminescence enabled real-time assessment of cell survival and proliferation on the scaffolds tested over prolonged (up to 59 days) periods of time. This novel, sensitive, easy, fast-to-implement, quantitative bioluminescence assay has great, though untapped, potential for screening and determining noninvasively the presence of viable cells on biomaterial constructs in the tissue engineering and tissue regeneration fields.

  13. Systematic study of one-loop Dirac neutrino masses and viable dark matter candidates

    Science.gov (United States)

    Yao, Chang-Yuan; Ding, Gui-Jun

    2017-11-01

    We study the generation of small neutrino masses from the dimension six effective operator at tree level and one-loop level, the neutrinos are assumed to be Dirac particles. We find out all possible tree and one-loop diagrams which lead to finite neutrino masses. The relevant mediator fields and the predictions for neutrino masses are presented. The lower order contributions to neutrino masses can be forbidden by introducing soft breaking Abelian symmetry or finite non-Abelian symmetry. The messengers inside the loop can be dark matter particles. The possible dark matter candidates and the corresponding constraints on the parameter α are analyzed for each model.

  14. Mobilization of Viable Tumor Cells Into the Circulation During Radiation Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Olga A. [Division of Radiation Oncology and Cancer Imaging, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Molecular Radiation Biology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC (Australia); Anderson, Robin L. [The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC (Australia); Metastasis Research Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Russell, Prudence A. [Department of Anatomical Pathology, St. Vincent Hospital, Fitzroy, VIC (Australia); Ashley Cox, R. [Division of Radiation Oncology and Cancer Imaging, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Ivashkevich, Alesia [Molecular Radiation Biology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Laboratory of DNA Repair and Genomics, Centre for Innate Immunity and Infectious Disease, Monash Institute for Medical Research, Monash University, Clayton, VIC (Australia); Swierczak, Agnieszka; Doherty, Judy P. [Metastasis Research Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Jacobs, Daphne H.M. [Division of Radiation Oncology and Cancer Imaging, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Smith, Jai [Molecular Radiation Biology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Siva, Shankar; Daly, Patricia E. [Division of Radiation Oncology and Cancer Imaging, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); Ball, David L. [Division of Radiation Oncology and Cancer Imaging, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia); The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC (Australia); and others

    2014-02-01

    Purpose: To determine whether radiation therapy (RT) could mobilize viable tumor cells into the circulation of non-small cell lung cancer (NSCLC) patients. Methods and Materials: We enumerated circulating tumor cells (CTCs) by fluorescence microscopy of blood samples immunostained with conventional CTC markers. We measured their DNA damage levels using γ-H2AX, a biomarker for radiation-induced DNA double-strand breaks, either by fluorescence-activated cell sorting or by immunofluorescence microscopy. Results: Twenty-seven RT-treated NSCLC patients had blood samples analyzed by 1 or more methods. We identified increased CTC numbers after commencement of RT in 7 of 9 patients treated with palliative RT, and in 4 of 8 patients treated with curative-intent RT. Circulating tumor cells were also identified, singly and in clumps in large numbers, during RT by cytopathologic examination (in all 5 cases studied). Elevated γ-H2AX signal in post-RT blood samples signified the presence of CTCs derived from irradiated tumors. Blood taken after the commencement of RT contained tumor cells that proliferated extensively in vitro (in all 6 cases studied). Circulating tumor cells formed γ-H2AX foci in response to ex vivo irradiation, providing further evidence of their viability. Conclusions: Our findings provide a rationale for the development of strategies to reduce the concentration of viable CTCs by modulating RT fractionation or by coadministering systemic therapies.

  15. Desiccation induces viable but Non-Culturable cells in Sinorhizobium meliloti 1021.

    Science.gov (United States)

    Vriezen, Jan Ac; de Bruijn, Frans J; Nüsslein, Klaus R

    2012-01-20

    Sinorhizobium meliloti is a microorganism commercially used in the production of e.g. Medicago sativa seed inocula. Many inocula are powder-based and production includes a drying step. Although S. meliloti survives drying well, the quality of the inocula is reduced during this process. In this study we determined survival during desiccation of the commercial strains 102F84 and 102F85 as well as the model strain USDA1021.The survival of S. meliloti 1021 was estimated during nine weeks at 22% relative humidity. We found that after an initial rapid decline of colony forming units, the decline slowed to a steady 10-fold reduction in colony forming units every 22 days. In spite of the reduction in colony forming units, the fraction of the population identified as viable (42-54%) based on the Baclight live/dead stain did not change significantly over time. This change in the ability of viable cells to form colonies shows (i) an underestimation of the survival of rhizobial cells using plating methods, and that (ii) in a part of the population desiccation induces a Viable But Non Culturable (VBNC)-like state, which has not been reported before. Resuscitation attempts did not lead to a higher recovery of colony forming units indicating the VBNC state is stable under the conditions tested. This observation has important consequences for the use of rhizobia. Finding methods to resuscitate this fraction may increase the quality of powder-based seed inocula.

  16. Mathematical modelling of the viable epidermis: impact of cell shape and vertical arrangement

    KAUST Repository

    Wittum, Rebecca

    2017-12-07

    In-silico methods are valuable tools for understanding the barrier function of the skin. The key benefit is that mathematical modelling allows the interplay between cell shape and function to be elucidated. This study focuses on the viable (living) epidermis. For this region, previous works suggested a diffusion model and an approximation of the cells by hexagonal prisms. The work at hand extends this in three ways. First, the extracellular space is treated with full spatial resolution. This induces a decrease of permeability by about 10%. Second, cells of tetrakaidecahedral shape are considered, in addition to the original hexagonal prisms. For both cell types, the resulting membrane permeabilities are compared. Third, for the first time, the influence of cell stacking in the vertical direction is considered. This is particularly important for the stratum granulosum, where tight junctions are present.

  17. Progress in biocatalysis with immobilized viable whole cells: systems development, reaction engineering and applications.

    Science.gov (United States)

    Polakovič, Milan; Švitel, Juraj; Bučko, Marek; Filip, Jaroslav; Neděla, Vilém; Ansorge-Schumacher, Marion B; Gemeiner, Peter

    2017-05-01

    Viable microbial cells are important biocatalysts in the production of fine chemicals and biofuels, in environmental applications and also in emerging applications such as biosensors or medicine. Their increasing significance is driven mainly by the intensive development of high performance recombinant strains supplying multienzyme cascade reaction pathways, and by advances in preservation of the native state and stability of whole-cell biocatalysts throughout their application. In many cases, the stability and performance of whole-cell biocatalysts can be highly improved by controlled immobilization techniques. This review summarizes the current progress in the development of immobilized whole-cell biocatalysts, the immobilization methods as well as in the bioreaction engineering aspects and economical aspects of their biocatalytic applications.

  18. Trypan blue dye enters viable cells incubated with the pore-forming toxin HlyII of Bacillus cereus.

    Directory of Open Access Journals (Sweden)

    Seav-Ly Tran

    Full Text Available Trypan blue is a dye that has been widely used for selective staining of dead tissues or cells. Here, we show that the pore-forming toxin HlyII of Bacillus cereus allows trypan blue staining of macrophage cells, despite the cells remaining viable and metabolically active. These findings suggest that the dye enters viable cells through the pores. To our knowledge, this is the first demonstration that trypan blue may enter viable cells. Consequently, the use of trypan blue staining as a marker of vital status should be interpreted with caution. The blue coloration does not necessarily indicate cell lysis, but may rather indicate pore formation in the cell membranes and more generally increased membrane permeability.

  19. Can Mass Trapping Reduce Thrips Damage and Is It Economically Viable? Management of the Western Flower Thrips in Strawberry

    Science.gov (United States)

    Sampson, Clare; Kirk, William D. J.

    2013-01-01

    The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) is a cosmopolitan, polyphagous insect pest that causes bronzing to fruit of strawberry (Fragaria x ananassa). The main aim of this study was to test whether mass trapping could reduce damage and to predict whether this approach would be economically viable. In semi-protected strawberry crops, mass trapping of F. occidentalis using blue sticky roller traps reduced adult thrips numbers per flower by 61% and fruit bronzing by 55%. The addition of the F. occidentalis aggregation pheromone, neryl (S)-2-methylbutanoate, to the traps doubled the trap catch, reduced adult thrips numbers per flower by 73% and fruit bronzing by 68%. The factors affecting trapping efficiency through the season are discussed. Damage that would result in downgrading of fruit to a cheaper price occurred when bronzing affected about 10% of the red fruit surface. Cost-benefit analysis using this threshold showed that mass trapping of thrips using blue sticky roller traps can be cost-effective in high-value crops. The addition of blue sticky roller traps to an integrated pest management programme maintained thrips numbers below the damage threshold and increased grower returns by a conservative estimate of £2.2k per hectare. Further work is required to develop the F. occidentalis aggregation pheromone for mass trapping and to determine the best timing for trap deployment. Mass trapping of thrips is likely to be cost-effective in other countries and other high-value crops affected by F. occidentalis damage, such as cucumber and cut flowers. PMID:24282554

  20. Can mass trapping reduce thrips damage and is it economically viable? Management of the Western flower thrips in strawberry.

    Directory of Open Access Journals (Sweden)

    Clare Sampson

    Full Text Available The western flower thrips Frankliniella occidentalis (Pergande (Thysanoptera: Thripidae is a cosmopolitan, polyphagous insect pest that causes bronzing to fruit of strawberry (Fragaria x ananassa. The main aim of this study was to test whether mass trapping could reduce damage and to predict whether this approach would be economically viable. In semi-protected strawberry crops, mass trapping of F. occidentalis using blue sticky roller traps reduced adult thrips numbers per flower by 61% and fruit bronzing by 55%. The addition of the F. occidentalis aggregation pheromone, neryl (S-2-methylbutanoate, to the traps doubled the trap catch, reduced adult thrips numbers per flower by 73% and fruit bronzing by 68%. The factors affecting trapping efficiency through the season are discussed. Damage that would result in downgrading of fruit to a cheaper price occurred when bronzing affected about 10% of the red fruit surface. Cost-benefit analysis using this threshold showed that mass trapping of thrips using blue sticky roller traps can be cost-effective in high-value crops. The addition of blue sticky roller traps to an integrated pest management programme maintained thrips numbers below the damage threshold and increased grower returns by a conservative estimate of £2.2k per hectare. Further work is required to develop the F. occidentalis aggregation pheromone for mass trapping and to determine the best timing for trap deployment. Mass trapping of thrips is likely to be cost-effective in other countries and other high-value crops affected by F. occidentalis damage, such as cucumber and cut flowers.

  1. IN-VITRO BIOREDUCTION OF HEXAVALENT CHROMIUM BY VIABLE WHOLE CELLS OF Arthrobacter sp. SUK 1201

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    Satarupa Dey

    2014-08-01

    Full Text Available A chromium resistant and reducing bacterium Arthrobacter sp. SUK 1201 was isolated from chromite mine overburden dumps of Orissa, India. Viable whole cells of this isolate was capable of completely reducing 100 µM Cr(VI in chemically defined MS medium within 28 h of incubation under batch cultivation. Reduction of chromate increased with increased cell density and was maximum at a density of 1010 cells/ml, but the reduction potential of the suspended cells decreased with increase in Cr(VI concentration in the medium. Chromate reducing efficiency was promoted when glycerol and glucose was used as electron donors, while the optimum pH and temperature of Cr(VI reduction was found to be 7.0 and 35°C respectively. The reduction process was inhibited by divalent cations Ni, Co and Cd, but not by Cu and Fe. Similarly, carbonyl cyanide m-chlorophenylhydrazone (CCCP, N,N,-Di cyclohexyl carboiimide (DCC, sodium azide and sodium fluoride were inhibitory to chromate reduction, while in presence of 2,4 dinitrophenol (2,4 DNP chromate reduction by SUK 1201 cells remained unaffected.

  2. Assessing prognosis and optimizing treatment in patients with postchemotherapy viable nonseminomatous germ-cell tumors (NSGCT): results of the sCR2 international study

    DEFF Research Database (Denmark)

    Fizazi, K.; Oldenburg, J.; Dunant, A.

    2008-01-01

    malignant cells, and a good International Germ Cell Consensus Classification group at presentation. Patients were assigned to one of three risk groups defined in sCR1: no risk factor (good risk), one risk factor (intermediate risk) and two to three risk factors (poor risk group). The 5-year PFS rate was 92...... with surveillance and treatment only at relapse. CONCLUSION: In patients with postchemotherapy viable NSGCT, a complete resection of residual masses should be rigorously pursued. These data validate the sCR1 prognostic index. Given their excellent outcome, patients in the favorable group may not require......BACKGROUND: The purpose of this study was to validate a prognostic index [surgical complete response 1 (sCR1)] in patients with postchemotherapy viable nonseminomatous germ-cell tumors (NSGCT). PATIENTS AND METHODS: Data and specimens from 61 patients with normalized tumor markers...

  3. Viable bacteria associated with red blood cells and plasma in freshly drawn blood donations.

    Directory of Open Access Journals (Sweden)

    Christian Damgaard

    Full Text Available Infection remains a leading cause of post-transfusion mortality and morbidity. Bacterial contamination is, however, detected in less than 0.1% of blood units tested. The aim of the study was to identify viable bacteria in standard blood-pack units, with particular focus on bacteria from the oral cavity, and to determine the distribution of bacteria revealed in plasma and in the red blood cell (RBC-fraction.Cross-sectional study. Blood were separated into plasma and RBC-suspensions, which were incubated anaerobically or aerobically for 7 days on trypticase soy blood agar (TSA or blue lactose plates. For identification colony PCR was performed using primers targeting 16S rDNA.Blood donors attending Capital Region Blood Bank, Copenhagen University Hospital, Rigshospitalet, Hvidovre, Denmark, October 29th to December 10th 2013.60 donors (≥50 years old, self-reported medically healthy.Bacterial growth was observed on plates inoculated with plasma or RBCs from 62% of the blood donations. Growth was evident in 21 (35% of 60 RBC-fractions and in 32 (53% of 60 plasma-fractions versus 8 of 60 negative controls (p = 0.005 and p = 2.6x10-6, respectively. Propionibacterium acnes was found in 23% of the donations, and Staphylococcus epidermidis in 38%. The majority of bacteria identified in the present study were either facultative anaerobic (59.5% or anaerobic (27.8% species, which are not likely to be detected during current routine screening.Viable bacteria are present in blood from donors self-reported as medically healthy, indicating that conventional test systems employed by blood banks insufficiently detect bacteria in plasma. Further investigation is needed to determine whether routine testing for anaerobic bacteria and testing of RBC-fractions for adherent bacteria should be recommended.

  4. Clinical validation of an ultra high-throughput spiral microfluidics for the detection and enrichment of viable circulating tumor cells.

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    Bee Luan Khoo

    Full Text Available Circulating tumor cells (CTCs are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation.Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56 (Breast cancer samples: 12-1275 CTCs/ml; Lung cancer samples: 10-1535 CTCs/ml rapidly from clinically relevant blood volumes (7.5 ml under 5 min. Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM, fluorescence in-situ hybridization (FISH (EML4-ALK or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA, and demonstrate concordance with the original tumor-biopsy samples.We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS or proteomic analysis.

  5. Increasing Vero viable cell densities for yellow fever virus production in stirred-tank bioreactors using serum-free medium.

    Science.gov (United States)

    Mattos, Diogo A; Silva, Marlon V; Gaspar, Luciane P; Castilho, Leda R

    2015-08-20

    In this work, changes in Vero cell cultivation methods have been employed in order to improve cell growth conditions to obtain higher viable cell densities and to increase viral titers. The propagation of the 17DD yellow fever virus (YFV) in Vero cells grown on Cytodex I microcarriers was evaluated in 3-L bioreactor vessels. Prior to the current changes, Vero cells were repeatedly displaying insufficient microcarrier colonization. A modified cultivation process with four changes has resulted in higher cell densities and higher virus titers than previously observed for 17DD YFV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Cellular bone matrices: viable stem cell-containing bone graft substitutes.

    Science.gov (United States)

    Skovrlj, Branko; Guzman, Javier Z; Al Maaieh, Motasem; Cho, Samuel K; Iatridis, James C; Qureshi, Sheeraz A

    2014-11-01

    Advances in the field of stem cell technology have stimulated the development and increased use of allogenic bone grafts containing live mesenchymal stem cells (MSCs), also known as cellular bone matrices (CBMs). It is estimated that CBMs comprise greater than 17% of all bone grafts and bone graft substitutes used. To critically evaluate CBMs, specifically their technical specifications, existing published data supporting their use, US Food and Drug Administration (FDA) regulation, cost, potential pitfalls, and other aspects pertaining to their use. Areview of literature. A series of Ovid, Medline, and Pubmed-National Library of Medicine/National Institutes of Health (www.ncbi.nlm.nih.gov) searches were performed. Only articles in English journals or published with English language translations were included. Level of evidence of the selected articles was assessed. Specific technical information on each CBM was obtained by direct communication from the companies marketing the individual products. Five different CBMs are currently available for use in spinal fusion surgery. There is a wide variation between the products with regard to the average donor age at harvest, total cellular concentration, percentage of MSCs, shelf life, and cell viability after defrosting. Three retrospective studies evaluating CBMs and fusion have shown fusion rates ranging from 90.2% to 92.3%, and multiple industry-sponsored trials are underway. No independent studies evaluating spinal fusion rates with the use of CBMs exist. All the commercially available CBMs claim to meet the FDA criteria under Section 361, 21 CFR Part 1271, and are not undergoing FDA premarket review. The CBMs claim to provide viable MSCs and are offered at a premium cost. Numerous challenges exist in regard to MSCs' survival, function, osteoblastic potential, and cytokine production once implanted into the intended host. Cellular bone matrices may be a promising bone augmentation technology in spinal fusion surgery

  7. Combined bromodeoxyuridine immunohistochemistry and Masson trichrome staining: facilitated detection of cell proliferation in viable vs. infarcted myocardium.

    Science.gov (United States)

    Lazarous, D F; Shou, M; Unger, E F

    1992-09-01

    Cells in the S-phase of the cell cycle can be identified in tissue sections by immunohistochemical localization of the thymidine analogue bromodeoxyuridine (BrdU). Generally, a single counterstain is used to visualize the underlying tissue; however, interpretation of morphologic detail is often difficult. We have utilized BrdU to localize proliferating cells in myocardium exposed to angiogenic mitogens. To facilitate identification of labelled nuclei in the context of infarcted vs. viable myocardium, BrdU immunohistochemistry was followed by a modified Masson trichrome stain. The time of exposure to the counterstains and the wash protocol were re-revised, permitting clear identification of the labelled brown nuclei against a background of red viable myocardium vs. blue infarct. The combined technique also provides color contrast suitable for computer-based image analysis.

  8. Quantitative assessment of viable cells of Lactobacillus plantarum strains in single, dual and multi-strain biofilms.

    Science.gov (United States)

    Fernández Ramírez, Mónica D; Kostopoulos, Ioannis; Smid, Eddy J; Nierop Groot, Masja N; Abee, Tjakko

    2017-03-06

    Biofilms of Lactobacillus plantarum are a potential source for contamination and recontamination of food products. Although biofilms have been mostly studied using single species or even single strains, it is conceivable that in a range of environmental settings including food processing areas, biofilms are composed of multiple species with each species represented by multiple strains. In this study six spoilage related L. plantarum strains FBR1-FBR6 and the model strain L. plantarum WCFS1 were characterised in single, dual and multiple strain competition models. A quantitative PCR approach was used with added propidium monoazide (PMA) enabling quantification of intact cells in the biofilm, representing the viable cell fraction that determines the food spoilage risk. Our results show that the performance of individual strains in multi-strain cultures generally correlates with their performance in pure culture, and relative strain abundance in multi-strain biofilms positively correlated with the relative strain abundance in suspended (planktonic) cultures. Performance of individual strains in dual-strain biofilms was highly influenced by the presence of the secondary strain, and in most cases no correlation between the relative contributions of viable planktonic cells and viable cells in the biofilm was noted. The total biofilm quantified by CV staining of the dual and multi-strain biofilms formed was mainly correlated to CV values of the dominant strain obtained in single strain studies. However, the combination of strain FBR5 and strain WCFS1 showed significantly higher CV values compared to the individual performances of both strains indicating that total biofilm formation was higher in this specific condition. Notably, L. plantarum FBR5 was able to outgrow all other strains and showed the highest relative abundance in dual and multi-strain biofilms. All the dual and multi-strain biofilms contained a considerable number of viable cells, representing a potential

  9. A multimodality imaging model to track viable breast cancer cells from single arrest to metastasis in the mouse brain

    Science.gov (United States)

    Parkins, Katie M.; Hamilton, Amanda M.; Makela, Ashley V.; Chen, Yuanxin; Foster, Paula J.; Ronald, John A.

    2016-10-01

    Cellular MRI involves sensitive visualization of iron-labeled cells in vivo but cannot differentiate between dead and viable cells. Bioluminescence imaging (BLI) measures cellular viability, and thus we explored combining these tools to provide a more holistic view of metastatic cancer cell fate in mice. Human breast carcinoma cells stably expressing Firefly luciferase were loaded with iron particles, injected into the left ventricle, and BLI and MRI were performed on days 0, 8, 21 and 28. The number of brain MR signal voids (i.e., iron-loaded cells) on day 0 significantly correlated with BLI signal. Both BLI and MRI signals decreased from day 0 to day 8, indicating a loss of viable cells rather than a loss of iron label. Total brain MR tumour volume on day 28 also correlated with BLI signal. Overall, BLI complemented our sensitive cellular MRI technologies well, allowing us for the first time to screen animals for successful injections, and, in addition to MR measures of cell arrest and tumor burden, provided longitudinal measures of cancer cell viability in individual animals. We predict this novel multimodality molecular imaging framework will be useful for evaluating the efficacy of emerging anti-cancer drugs at different stages of the metastatic cascade.

  10. Viable cell yield from active dry yeast products and effects of storage temperature and diluent on yeast cell viability.

    Science.gov (United States)

    Sullivan, M L; Bradford, B J

    2011-01-01

    Active dry yeast (ADY) products are commonly fed in the dairy industry, but research regarding quality control for such products is limited. The objectives of this study were to determine yeast viability in field samples relative to manufacturers' guarantees (experiment 1), measure the effects of high-temperature storage on yeast viability (experiment 1), and determine the effect of vitamin-trace mineral (VTM) premix on yeast viability (experiment 2). Commercially available ADY products were acquired in triplicate through normal distribution channels and stored at 4°C upon receipt. Initial samples were evaluated for colony-forming units and compared with product label guarantees. Only 1 of the 6 products sampled in experiment 1 met product guarantees for all 3 samples. To determine effects of storage temperature and duration on viability, ADY samples were stored in an incubator at 40°C with ambient humidity for 1, 2, and 3 mo. High-temperature storage significantly decreased viability over the 3-mo period; approximately 90% of viable cells were lost each month. Three of the 5 products sampled in experiment 2 met product guarantees. Fresh samples of 4 of these 5 ADY products were mixed in duplicate with ground corn (GC) or a VTM premix to achieve a target concentration of 2.2×10(8) cfu/g. For each product, GC and VTM samples were stored at ambient temperature (22°C) and at an elevated temperature (40°C) for 2 wk. No differences in viable yeast count were observed between GC and VTM samples immediately after mixing or after storage at ambient temperature. Yeast viability in GC and VTM samples decreased during storage at an elevated temperature. There also was a significant interaction of diluent and storage temperature; VTM samples had higher cell viability than GC samples when subjected to high-temperature storage. Results suggest that (1) ADY products failed to consistently meet product guarantees; (2) viability of ADY products was greatly diminished during

  11. Sensitive and Specific Biomimetic Lipid Coated Microfluidics to Isolate Viable Circulating Tumor Cells and Microemboli for Cancer Detection.

    Directory of Open Access Journals (Sweden)

    Jia-Yang Chen

    Full Text Available Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB "smart coating" to capture viable circulating tumor cells (CTCs and circulating tumor microemboli (CTM directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.

  12. Viable cell sorting of dinoflagellates by multi-parametric flow cytometry.

    Science.gov (United States)

    Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking of cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor as fragile dinoflagellates, such a...

  13. Fuel cells are a commercially viable alternative for the production of "clean" energy.

    Science.gov (United States)

    Niakolas, Dimitris K; Daletou, Maria; Neophytides, Stylianos G; Vayenas, Constantinos G

    2016-01-01

    Fuel cells present a highly efficient and environmentally friendly alternative technology for decentralized energy production. The scope of the present study is to provide an overview of the technological and commercialization readiness level of fuel cells. Specifically, there is a brief description of their general advantages and weaknesses in correlation with various technological actions and political strategies, which are adopted towards their proper positioning in the global market. Some of the most important key performance indicators are also discussed, alongside with a few examples of broad commercialization. It is concluded that the increasing number of companies which utilize and invest on this technology, in combination with the supply chain improvements and the concomitant technological maturity and recognition, reinforce the fuel cell industry so as to become well-aligned for global success.

  14. A viable electrode material for use in microbial fuel cells for tropical regions

    DEFF Research Database (Denmark)

    Offei, Felix; Thygesen, Anders; Mensah, Moses

    2016-01-01

    Electrode materials are critical for microbial fuel cells (MFC) since they influence the construction and operational costs. This study introduces a simple and efficient electrode material in the form of palm kernel shell activated carbon (AC) obtained in tropical regions. The novel introduction...

  15. Progress in emerging techniques for characterization of immobilized viable whole-cell biocatalysts

    Czech Academy of Sciences Publication Activity Database

    Bučko, M.; Vikartovská, A.; Schenkmayerová, A.; Tkáč, J.; Filip, J.; Chorvát Jr., D.; Neděla, Vilém; Ansorge-Schumacher, M.B.; Gemeiner, P.

    2017-01-01

    Roč. 71, č. 11 (2017), s. 2309-2324 ISSN 0366-6352 Institutional support: RVO:68081731 Keywords : bioelectrocatalysis * imaging techniques * immobilized whole- cell biocatalyst * multienzyme cascade reactions * online kinetics Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 1.258, year: 2016

  16. Progress in biocatalysis with immobilized viable whole cells: systems development, reaction engineering and applications

    Czech Academy of Sciences Publication Activity Database

    Polakovič, M.; Švitel, J.; Bučko, M.; Filip, J.; Neděla, Vilém; Ansorge-Schumacher, M.B.; Gemeiner, P.

    2017-01-01

    Roč. 39, č. 5 (2017), s. 667-683 ISSN 0141-5492 Institutional support: RVO:68081731 Keywords : biocatalysis * immobilization methods * immobilized whole-cell biocatalyst * multienzyme cascade reactions * process economics * reaction engineering Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 1.730, year: 2016

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

    Science.gov (United States)

    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.

  18. Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157:H7 Cells by Targeting Z3276 as a Genetic Marker

    Science.gov (United States)

    Chen, Jin-Qiang

    2012-01-01

    The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)–real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 107 dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA–real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources. PMID:22635992

  19. Viable Bacteria Associated with Red Blood Cells and Plasma in Freshly Drawn Blood Donations

    DEFF Research Database (Denmark)

    Damgaard, Christian; Magnussen, Karin; Enevold, Christian

    2015-01-01

    the oral cavity, and to determine the distribution of bacteria revealed in plasma and in the red blood cell (RBC)-fraction. DESIGN: Cross-sectional study. Blood were separated into plasma and RBC-suspensions, which were incubated anaerobically or aerobically for 7 days on trypticase soy blood agar (TSA......), self-reported medically healthy. RESULTS: Bacterial growth was observed on plates inoculated with plasma or RBCs from 62% of the blood donations. Growth was evident in 21 (35%) of 60 RBC-fractions and in 32 (53%) of 60 plasma-fractions versus 8 of 60 negative controls (p = 0.005 and p = 2.6x10...... of RBC-fractions for adherent bacteria should be recommended....

  20. A Viable Electrode Material for Use in Microbial Fuel Cells for Tropical Regions

    Directory of Open Access Journals (Sweden)

    Felix Offei

    2016-01-01

    Full Text Available Electrode materials are critical for microbial fuel cells (MFC since they influence the construction and operational costs. This study introduces a simple and efficient electrode material in the form of palm kernel shell activated carbon (AC obtained in tropical regions. The novel introduction of this material is also targeted at introducing an inexpensive and durable electrode material, which can be produced in rural communities to improve the viability of MFCs. The maximum voltage and power density obtained (under 1000 Ω load using an H-shaped MFC with AC as both anode and cathode electrode material was 0.66 V and 1.74 W/m3, respectively. The power generated by AC was as high as 86% of the value obtained with the extensively used carbon paper. Scanning electron microscopy and Denaturing Gradient Gel Electrophoresis (DGGE analysis of AC anode biofilms confirmed that electrogenic bacteria were present on the electrode surface for substrate oxidation and the formation of nanowires.

  1. Response of Listeria monocytogenes to disinfection stress at the single-cell and population levels as monitored by intracellular pH measurements and viable-cell counts

    DEFF Research Database (Denmark)

    Kastbjerg, Vicky Gaedt; Nielsen, Dennis S.; Arneborg, Nils

    2009-01-01

    .05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (P ... that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present....

  2. A factor converting viable but nonculturable Vibrio cholerae to a culturable state in eukaryotic cells is a human catalase.

    Science.gov (United States)

    Senoh, Mitsutoshi; Hamabata, Takashi; Takeda, Yoshifumi

    2015-08-01

    In our previous work, we demonstrated that viable but nonculturable (VBNC) Vibrio cholerae O1 and O139 were converted to culturable by coculture with eukaryotic cells. Furthermore, we isolated a factor converting VBNC V. cholerae to culturable (FCVC) from a eukaryotic cell line, HT-29. In this study, we purified FCVC by successive column chromatographies comprising UNO Q-6 anion exchange, Bio-Scale CHT2-1 hydroxyapatite, and Superdex 200 10/300 GL. Homogeneity of the purified FCVC was demonstrated by SDS-PAGE. Nano-LC MS/MS analysis showed that the purified FCVC was a human catalase. An experiment of RNAi knockdown of catalase mRNA from HT-29 cells and treatment of the purified FCVC with a catalase inhibitor, 3-amino-1,2,4-triazole confirmed that the FCVC was a catalase. A possible role of the catalase in converting a VBNC V. cholerae to a culturable state in the human intestine is discussed. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  3. Drugs with anti-oxidant properties can interfere with cell viability measurements by assays that rely on the reducing property of viable cells.

    Science.gov (United States)

    Shenoy, Niraj; Stenson, Mary; Lawson, Joshua; Abeykoon, Jithma; Patnaik, Mrinal; Wu, Xiaosheng; Witzig, Thomas

    2017-02-27

    Cell viability assays such as Cell Titer Blue and Alamar Blue rely on the reducing property of viable cells to reduce the reagent dye to a product which gives a fluorescent signal. The current manufacture-recommended protocols do not take into account the possibility of the reagent substrate being reduced directly to the fluorescent product by drugs with an anti-oxidant property. After suspecting spurious results while determining the cytotoxic potential of a drug of interest (DOI) with known anti-oxidant property against a renal cell cancer (RCC) cell line, we aimed to establish that drugs with anti-oxidant property can indeed cause false-negative results with the current protocols of these assays by direct reduction of the reagent substrate. We also aimed to counter the same with a simple modification added to the protocol. Through our experiments, we conclusively demonstrate that drugs with anti-oxidant properties can indeed interfere with cell viability measurements by assays that rely on the reducing property of viable cells. A simple modification in the protocol, as elaborated in the manuscript, can prevent spurious results with these otherwise convenient assays.Laboratory Investigation advance online publication, 27 February 2017; doi:10.1038/labinvest.2017.18.

  4. A Novel Application for Low Frequency Electrochemical Impedance Spectroscopy as an Online Process Monitoring Tool for Viable Cell Concentrations

    Directory of Open Access Journals (Sweden)

    Christoph Slouka

    2016-11-01

    Full Text Available New approaches in process monitoring during industrial fermentations are not only limited to classical pH, dO2 and offgas analysis, but use different in situ and online sensors based on different physical principles to determine biomass, product quality, lysis and far more. One of the very important approaches is the in situ accessibility of viable cell concentration (VCC. This knowledge provides increased efficiency in monitoring and controlling strategies during cultivations. Electrochemical impedance spectroscopy—EIS—is used to monitor biomass in a fermentation of E. coli BL21(DE3, producing a recombinant protein using a fed batch-based approach. Increases in the double layer capacitance (Cdl, determined at frequencies below 1 kHz, are proportional to the increase of biomass in the batch and fed batch phase, monitored in offline and online modes for different cultivations. A good correlation of Cdl with cell density is found and in order to get an appropriate verification of this method, different state-of-the-art biomass measurements are performed and compared. Since measurements in this frequency range are largely determined by the double layer region between the electrode and media, rather minor interferences with process parameters (aeration, stirring are to be expected. It is shown that impedance spectroscopy at low frequencies is a powerful tool for cultivation monitoring.

  5. A viable foal obtained by equine somatic cell nuclear transfer using oocytes recovered from immature follicles of live mares.

    Science.gov (United States)

    Choi, Young-Ho; Norris, Jody D; Velez, Isabel C; Jacobson, Candace C; Hartman, David L; Hinrichs, Katrin

    2013-03-15

    The presence of heterogenous mitochondria from the host ooplast affects the acceptance of offspring obtained by somatic cell nuclear transfer. This might be avoided by obtaining oocytes from selected females, but is then complicated by low numbers of available oocytes. We examined the efficiency of equine somatic cell nuclear transfer using oocytes recovered by transvaginal aspiration of immature follicles from 11 mares. Use of metaphase I oocytes as cytoplasts and of scriptaid (a histone deacetylase inhibitor) treatment during oocyte activation were evaluated to determine if these approaches would increase blastocyst production. In experiment 1, blastocyst development was 0/14 for metaphase I oocytes and 4/103 (4%) for metaphase II oocytes. Three blastocysts were transferred to recipient mares, resulting in two pregnancies and one live foal, which died shortly after birth. In experiment 2, blastocyst development was 2/47 (4%) for control oocytes and 1/83 (1%) for scriptaid-treated oocytes. No foals were born from two blastocysts transferred in the control group. The blastocyst from the scriptaid treatment resulted in birth of a live foal. In conclusion, this is apparently the first report of production of a viable cloned foal from oocytes collected from immature follicles of live mares, supporting the possibility of cloning using oocytes from selected mares. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Quantitation of viable Coxiella burnetii in milk using an integrated cell culture-polymerase chain reaction (ICC-PCR) assay.

    Science.gov (United States)

    Stewart, Diana; Shieh, Y-Carol; Tortorello, Mary; Kukreja, Ankush; Shazer, Arlette; Schlesser, Joseph

    2015-11-01

    The obligate intracellular pathogen Coxiella burnetii has long been considered the most heat resistant pathogen in raw milk, making it the reference pathogen for determining pasteurisation conditions for milk products. New milk formulations and novel non-thermal processes require validation of effectiveness which requires a more practical method for analysis than using the currently used animal model for assessing Coxiella survival. Also, there is an interest in better characterising thermal inactivation of Coxiella in various milk formulations. To avoid the use of the guinea pig model for evaluating Coxiella survival, an Integrated Cell Culture-PCR (ICC-PCR) method was developed for determining Coxiella viability in milk. Vero cell cultures were directly infected from Coxiella-contaminated milk in duplicate 24-well plates. Viability of the Coxiella in milk was shown by a ≥ 0.5 log genome equivalent (ge)/ml increase in the quantity of IS111a gene from the baseline post-infection (day 0) level after 9-11 d propagation. Coxiella in skim, 2%, and whole milk, and half and half successfully infected Vero cells and increased in number by at least 2 logs using a 48-h infection period followed by 9-d propagation time. As few as 125 Coxiella ge/ml in whole milk was shown to infect and propagate at least 2 logs in the optimised ICC-PCR assay, though variable confirmation of propagation was shown for as low as 25 Coxiella ge/ml. Applicability of the ICC-PCR method was further proven in an MPN format to quantitate the number of viable Coxiella remaining in whole milk after 60 °C thermal treatment at 0, 20, 40, 60 and 90 min.

  7. Polyelectrolyte Complex Beads by Novel Two-Step Process for Improved Performance of Viable Whole-Cell Baeyer-Villiger Monoxygenase by Immobilization

    Directory of Open Access Journals (Sweden)

    Tomáš Krajčovič

    2017-11-01

    Full Text Available A novel immobilization matrix for the entrapment of viable whole-cell Baeyer–Villiger monooxygenase was developed. Viable recombinant Escherichia coli cells overexpressing cyclohexanone monooxygenase were entrapped in polyelectrolyte complex beads prepared by a two-step reaction of oppositely-charged polymers including highly defined cellulose sulphate. Immobilized cells exhibited higher operational stability than free cells during 10 repeated cycles of Baeyer–Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to the corresponding lactones (1R,5S-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R-2-oxabicyclo-[3.3.0]oct-6-en-3-one. The morphology of polyelectrolyte complex beads was characterised by environmental scanning electron microscopy; the spatial distribution of polymers in the beads and cell viability were examined using confocal laser scanning microscopy, and the texture was characterised by the mechanical resistance measurements.

  8. Enhanced inhibition of murine prostatic carcinoma growth by immunization with or administration of viable human umbilical vein endothelial cells and CRM197

    Directory of Open Access Journals (Sweden)

    Zhang Huiyong

    2011-02-01

    Full Text Available Vaccination with xenogeneic and syngeneic endothelial cells is effective for inhibiting tumor growth. Nontoxic diphtheria toxin (CRM197, as an immunogen or as a specific inhibitor of heparin-binding EGF-like growth factor, has shown promising antitumor activity. Therefore, immunization with or administration of viable human umbilical vein endothelial cells (HUVECs combined with CRM197 could have an enhanced antitumor effect. Six-week-old C57BL/6J male mice were vaccinated with viable HUVECs, 1 x 10(6 viable HUVECs combined with 100 μg CRM197, or 100 μg CRM197 alone by ip injections once a week for 4 consecutive weeks. RM-1 cells (5 x 10(5 were inoculated by sc injection as a preventive procedure. During the therapeutic procedure, 6-week-old male C57BL/6J mice were challenged with 1 x 10(5 RM-1 cells, then injected sc with 1 x 10(6 viable HUVECs, 1 x 10(6 viable HUVECs + 100 μg CRM197, and 100 μg CRM197 alone twice a week for 4 consecutive weeks. Tumor volume and life span were monitored. We also investigated the effects of immunization with HUVECs on the aortic arch wall and on wound healing. Vaccination with or administration of viable HUVECs+CRM197 enhanced the inhibition of RM-1 prostatic carcinoma by 24 and 29%, respectively, and prolonged the life span for 3 and 4 days, respectively, compared with those of only vaccination or administration with viable HUVECs of tumor-bearing C57BL/6J mice. Furthermore, HUVEC immunization caused some damage to the aortic arch wall but did not have remarkable effects on the rate of wound healing; the wounds healed in approximately 13 days. Treatment with CRM197 in combination with viable HUVECs resulted in a marked enhancement of the antitumor effect in the preventive or therapeutic treatment for prostatic carcinoma in vivo, suggesting a novel combination for anti-cancer therapy.

  9. Mass transfer in fuel cells

    Science.gov (United States)

    Walker, R. D., Jr.

    1973-01-01

    Developments in the following areas are reported: surface area and pore size distribution in electrolyte matrices, electron microscopy of electrolyte matrices, surface tension of KOH solutions, water transport in fuel cells, and effectiveness factors for fuel cell components.

  10. Viable Cancer Cells in the Remnant Stomach are a Potential Source of Peritoneal Metastasis after Curative Distal Gastrectomy for Gastric Cancer.

    Science.gov (United States)

    Murata, Satoshi; Yamamoto, Hiroshi; Yamaguchi, Tsuyoshi; Kaida, Sachiko; Ishida, Mitsuaki; Kodama, Hirokazu; Takebayashi, Katsushi; Shimizu, Tomoharu; Miyake, Toru; Tani, Tohru; Kushima, Ryoji; Tani, Masaji

    2016-09-01

    The mechanisms underlying peritoneal metastasis (PM) after curative gastrectomy for gastric cancer (GC) are not well elucidated. This study assessed whether viable cancer cells, including cancer stemlike cells (CSCs), were present in the remnant stomach immediately before gastrointestinal (GI) tract reconstruction because these could be a source of PM after gastrectomy. Saline fluid used for remnant stomach lumen irrigation before GI reconstruction was prospectively collected from 142 consecutive patients undergoing distal gastrectomy for GC and cytologically examined. Proliferative activity (Ki67 staining) and stemness (expression of the CSC surface markers CD44s or CD44v6) were evaluated in detected cancer cells. Viable cancer cells were detected in 33 (23.2 %) of the 142 remnant stomachs. These cells formed clusters and stained positively for Ki67, indicating proliferation. Cancer cells in remnant stomachs and surface cancer cells in primary GCs from 10 (30.3 %) of these 33 cases also stained positively for CD44s or CD44v6. In a multiple logistic regression analysis, advanced cancer (odds ratio [OR], 4.65; 95 % confidence interval [CI], 1.32-16.4; P = 0.017), tumor size of 40 mm or larger (OR, 3.78; 95 % CI, 1.12-12.8; P = 0.033), and histologic differentiation (OR, 3.10; 95 % CI, 1.30-7.40; P = 0.011) were associated independently with the presence of cancer cells in the remnant stomach. Viable, proliferative, and clustered cancer cells, including CSCs, were found in remnant gastric lumens immediately before GI reconstruction, indicating a possible cellular source of PM after curative gastrectomy for GC. Dissemination of gastric contents into the peritoneal cavity should be avoided during GI reconstruction.

  11. Designing primers and evaluation of the efficiency of propidium monoazide – Quantitative polymerase chain reaction for counting the viable cells of Lactobacillus gasseri and Lactobacillus salivarius

    Directory of Open Access Journals (Sweden)

    Chieh-Hsien Lai

    2017-07-01

    Full Text Available The purpose of this study is to evaluate the efficiency of using propidium monoazide (PMA real-time quantitative polymerase chain reaction (qPCR to count the viable cells of Lactobacillus gasseri and Lactobacillus salivarius in probiotic products. Based on the internal transcription spacer and 23S rRNA genes, two primer sets specific for these two Lactobacillus species were designed. For a probiotic product, the total deMan Rogosa Sharpe plate count was 8.65±0.69 log CFU/g, while for qPCR, the cell counts of L. gasseri and L. salivarius were 8.39±0.14 log CFU/g and 8.57±0.24 log CFU/g, respectively. Under the same conditions, for its heat-killed product, qPCR counts for L. gasseri and L. salivarius were 6.70±0.16 log cells/g and 7.67±0.20 log cells/g, while PMA-qPCR counts were 5.33±0.18 log cells/g and 5.05±0.23 log cells/g, respectively. For cell dilutions with a viable cell count of 8.5 log CFU/mL for L. gasseri and L. salivarius, after heat killing, the PMA-qPCR count for both Lactobacillus species was near 5.5 log cells/mL. When the PMA-qPCR counts of these cell dilutions were compared before and after heat killing, although some DNA might be lost during the heat killing, significant qPCR signals from dead cells, i.e., about 4–5 log cells/mL, could not be reduced by PMA treatment. Increasing PMA concentrations from 100 μM to 200 μM or light exposure time from 5 minutes to 15 minutes had no or, if any, only minor effect on the reduction of qPCR signals from their dead cells. Thus, to differentiate viable lactic acid bacterial cells from dead cells using the PMA-qPCR method, the efficiency of PMA to reduce the qPCR signals from dead cells should be notable.

  12. Designing primers and evaluation of the efficiency of propidium monoazide - Quantitative polymerase chain reaction for counting the viable cells of Lactobacillus gasseri and Lactobacillus salivarius.

    Science.gov (United States)

    Lai, Chieh-Hsien; Wu, Sih-Rong; Pang, Jen-Chieh; Ramireddy, Latha; Chiang, Yu-Cheng; Lin, Chien-Ku; Tsen, Hau-Yang

    2017-07-01

    The purpose of this study is to evaluate the efficiency of using propidium monoazide (PMA) real-time quantitative polymerase chain reaction (qPCR) to count the viable cells of Lactobacillus gasseri and Lactobacillus salivarius in probiotic products. Based on the internal transcription spacer and 23S rRNA genes, two primer sets specific for these two Lactobacillus species were designed. For a probiotic product, the total deMan Rogosa Sharpe plate count was 8.65±0.69 log CFU/g, while for qPCR, the cell counts of L. gasseri and L. salivarius were 8.39±0.14 log CFU/g and 8.57±0.24 log CFU/g, respectively. Under the same conditions, for its heat-killed product, qPCR counts for L. gasseri and L. salivarius were 6.70±0.16 log cells/g and 7.67±0.20 log cells/g, while PMA-qPCR counts were 5.33±0.18 log cells/g and 5.05±0.23 log cells/g, respectively. For cell dilutions with a viable cell count of 8.5 log CFU/mL for L. gasseri and L. salivarius, after heat killing, the PMA-qPCR count for both Lactobacillus species was near 5.5 log cells/mL. When the PMA-qPCR counts of these cell dilutions were compared before and after heat killing, although some DNA might be lost during the heat killing, significant qPCR signals from dead cells, i.e., about 4-5 log cells/mL, could not be reduced by PMA treatment. Increasing PMA concentrations from 100 μM to 200 μM or light exposure time from 5 minutes to 15 minutes had no or, if any, only minor effect on the reduction of qPCR signals from their dead cells. Thus, to differentiate viable lactic acid bacterial cells from dead cells using the PMA-qPCR method, the efficiency of PMA to reduce the qPCR signals from dead cells should be notable. Copyright © 2016. Published by Elsevier B.V.

  13. Pancreatic α-cell mass in obesity.

    Science.gov (United States)

    Ellenbroek, Johanne H; Töns, Hendrica A M; Hanegraaf, Maaike A J; Rabelink, Ton J; Engelse, Marten A; Carlotti, Françoise; de Koning, Eelco J P

    2017-12-01

    While it is well recognized that obesity is associated with an increased β-cell mass, the association with α-cell mass is less clear. Type 2 diabetes (T2DM) associated with obesity is a bihormonal disease characterized by inadequate insulin secretion and hyperglucagonaemia. We examined β- and α-cell mass throughout the pancreas in obese and lean subjects. Pancreatic tissue of the head, body and tail region of the pancreas was examined from 15 obese subjects (body mass index [BMI] ≥ 27 kg/m2 ) and 15 age-matched lean subjects (BMI ≤ 25 kg/m2 ) without diabetes. In obese subjects both β- and α-cell mass were proportionally higher compared with lean subjects, thereby maintaining the α- to β-cell ratio. The adaptation to obesity occurred preferentially in the head of the pancreas. As data so far have been derived from histological studies of β- and α-cell adaptation, in which the head region of the human pancreas was not included, the adaptive capacity of humans to obesity has previously been underestimated. Obesity is associated with an increased α-cell mass, which could contribute to the hyperglucagonaemia observed in people with T2DM. © 2017 John Wiley & Sons Ltd.

  14. Not single but periodic injections of synovial mesenchymal stem cells maintain viable cells in knees and inhibit osteoarthritis progression in rats.

    Science.gov (United States)

    Ozeki, N; Muneta, T; Koga, H; Nakagawa, Y; Mizuno, M; Tsuji, K; Mabuchi, Y; Akazawa, C; Kobayashi, E; Matsumoto, K; Futamura, K; Saito, T; Sekiya, I

    2016-06-01

    We investigated the effects of single or repetitive intra-articular injections of synovial mesenchymal stem cells (MSCs) on a rat osteoarthritis (OA) model, and elucidated the behaviors and underlying mechanisms of the stem cells after the injection. One week after the transection of the anterior cruciate ligament (ACL) of wild type Lewis rats, one million synovial MSCs were injected into the knee joint every week. Cartilage degeneration was evaluated with safranin-o staining after the first injection. To analyze cell kinetics or MSC properties, luciferase, LacZ, and GFP expressing synovial MSCs were used. To confirm the role of MSCs, species-specific microarray and PCR analyses were performed using human synovial MSCs. Histological analysis for femoral and tibial cartilage showed that a single injection was ineffective but weekly injections had significant chondroprotective effects for 12 weeks. Histological and flow-cytometric analyses of LacZ and GFP expressing synovial MSCs revealed that injected MSCs migrated mainly into the synovium and most of them retained their undifferentiated MSC properties though the migrated cells rapidly decreased. In vivo imaging analysis revealed that MSCs maintained in knees while weekly injection. Species-specific microarray and PCR analyses showed that the human mRNAs on day 1 for 21 genes increased over 50-fold, and increased the expressions of PRG-4, BMP-2, and BMP-6 genes encoding chondroprotective proteins, and TSG-6 encoding an anti-inflammatory one. Not single but periodic injections of synovial MSCs maintained viable cells without losing their MSC properties in knees and inhibited osteoarthritis (OA) progression by secretion of trophic factors. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments

    Energy Technology Data Exchange (ETDEWEB)

    Patheja, Pooja, E-mail: pooja.patheja8@gmail.com [Laser Biomedical Applications Section, Raja Ramanna Centre for Advanced Technology, Indore 452013, Madhya Pradesh (India); Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, Maharashtra (India); Sahu, Khageswar [Laser Biomedical Applications Section, Raja Ramanna Centre for Advanced Technology, Indore 452013, Madhya Pradesh (India)

    2017-06-15

    Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MφCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MφCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.

  16. Bacillus subtilis mutants with knockouts of the genes encoding ribonucleases RNase Y and RNase J1 are viable, with major defects in cell morphology, sporulation, and competence.

    Science.gov (United States)

    Figaro, Sabine; Durand, Sylvain; Gilet, Laetitia; Cayet, Nadège; Sachse, Martin; Condon, Ciarán

    2013-05-01

    The genes encoding the ribonucleases RNase J1 and RNase Y have long been considered essential for Bacillus subtilis cell viability, even before there was concrete knowledge of their function as two of the most important enzymes for RNA turnover in this organism. Here we show that this characterization is incorrect and that ΔrnjA and Δrny mutants are both viable. As expected, both strains grow relatively slowly, with doubling times in the hour range in rich medium. Knockout mutants have major defects in their sporulation and competence development programs. Both mutants are hypersensitive to a wide range of antibiotics and have dramatic alterations to their cell morphologies, suggestive of cell envelope defects. Indeed, RNase Y mutants are significantly smaller in diameter than wild-type strains and have a very disordered peptidoglycan layer. Strains lacking RNase J1 form long filaments in tight spirals, reminiscent of mutants of the actin-like proteins (Mre) involved in cell shape determination. Finally, we combined the rnjA and rny mutations with mutations in other components of the degradation machinery and show that many of these strains are also viable. The implications for the two known RNA degradation pathways of B. subtilis are discussed.

  17. Effects of the oral administration of viable and heat-killed Streptococcus bovis HC5 cells to pre-sensitized BALB/c mice.

    Directory of Open Access Journals (Sweden)

    Aline D Paiva

    Full Text Available Antimicrobial peptides have been suggested as an alternative to classical antibiotics in livestock production and bacteriocin-producing bacteria could be added to animal feeds to deliver bacteriocins in the gastrointestinal (GI tract of ruminant and monogastric animals. In this study, viable (V and heat-killed (HK Streptococcus bovis HC5 cells were orally administered to pre-sensitized mice in order to assess the effects of a bacteriocin-producing bacteria on histological parameters and the immune response of the GI tract of monogastric animals. The administration of V and HK S. bovis HC5 cells during 58 days to BALB/c mice did not affect weight gain, but an increase in gut permeability was detected in animals receiving the HK cells. Viable and heat killed cells caused similar morphological alterations in the GI tract of the animals, but the most prominent effects were detected in the small intestine. The oral administration of S. bovis HC5 also influenced cytokine production in the small intestine, and the immune-mediated activity differed between V and HK cells. The relative expression of IL-12 and INF-γ was significantly higher in the small intestine of mice treated with V cells, while an increase in IL-5, IL-13 and TNF-α expression was only detected in mice treated with HK cells. Considering that even under a condition of severe challenge (pre-sensitization followed by daily exposure to the same bacterial immunogen the general health of the animals was maintained, it appears that oral administration of S. bovis HC5 cells could be a useful route to deliver bacteriocin in the GI tract of livestock animals.

  18. A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells.

    Science.gov (United States)

    Volovitz, Ilan; Shapira, Netanel; Ezer, Haim; Gafni, Aviv; Lustgarten, Merav; Alter, Tal; Ben-Horin, Idan; Barzilai, Ori; Shahar, Tal; Kanner, Andrew; Fried, Itzhak; Veshchev, Igor; Grossman, Rachel; Ram, Zvi

    2016-06-01

    Conducting research on the molecular biology, immunology, and physiology of brain tumors (BTs) and primary brain tissues requires the use of viably dissociated single cells. Inadequate methods for tissue dissociation generate considerable loss in the quantity of single cells produced and in the produced cells' viability. Improper dissociation may also demote the quality of data attained in functional and molecular assays due to the presence of large quantities cellular debris containing immune-activatory danger associated molecular patterns, and due to the increased quantities of degraded proteins and RNA. Over 40 resected BTs and non-tumorous brain tissue samples were dissociated into single cells by mechanical dissociation or by mechanical and enzymatic dissociation. The quality of dissociation was compared for all frequently used dissociation enzymes (collagenase, DNase, hyaluronidase, papain, dispase) and for neutral protease (NP) from Clostridium histolyticum. Single-cell-dissociated cell mixtures were evaluated for cellular viability and for the cell-mixture dissociation quality. Dissociation quality was graded by the quantity of subcellular debris, non-dissociated cell clumps, and DNA released from dead cells. Of all enzymes or enzyme combinations examined, NP (an enzyme previously not evaluated on brain tissues) produced dissociated cell mixtures with the highest mean cellular viability: 93 % in gliomas, 85 % in brain metastases, and 89 % in non-tumorous brain tissue. NP also produced cell mixtures with significantly less cellular debris than other enzymes tested. Dissociation using NP was non-aggressive over time-no changes in cell viability or dissociation quality were found when comparing 2-h dissociation at 37 °C to overnight dissociation at ambient temperature. The use of NP allows for the most effective dissociation of viable single cells from human BTs or brain tissue. Its non-aggressive dissociative capacity may enable ambient

  19. Prognostic impact of the number of viable circulating cells with high telomerase activity in gastric cancer patients: a prospective study.

    Science.gov (United States)

    Ito, Hiroaki; Inoue, Haruhiro; Kimura, Satoshi; Ohmori, Tohru; Ishikawa, Fumihiro; Gohda, Keigo; Sato, Jun

    2014-07-01

    The identification of circulating tumor cells (CTCs) in peripheral blood is a useful approach to estimate prognosis, monitor disease progression and measure treatment effects in several types of malignancies. We have previously used OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene. GFP-positive cells (GFP+ cells) were counted under a fluorescence microscope. Our results showed that the number of at least 7.735 µm in diameter GFP+ cells (L-GFP+ cells) in the peripheral blood was a significant marker of prognosis in gastric cancer patients. However, tumor cells undergoing epithelial-mesenchymal transition (EMT) have been reported to be smaller in size than cells without EMT features; thus, CTCs undergoing EMT may escape detection with this technique. Therefore, in this study, we analyzed the relationship between patient outcome and the number of GFP+ cells of any size. We obtained peripheral blood samples from 65 patients with gastric cancer. After infection of OBP-401, GFP+ cells were counted and measured. The relationship between the number of GFP+ cells and surgical outcome was analyzed. The median follow-up period of the surviving patients was 36 months. A significant difference in overall survival was found between patients with 0-5 and patients with ≥6 L-GFP+ cells. No clear relationship was established between the number of small-sized GFP+ cells and patient prognosis. The number of L-GFP+ cells was significantly related to overall survival in patients with gastric cancer. The detection of L-GFP+ cells using OBP-401 may be a useful prognostic marker in gastric cancer.

  20. Binding of CLL subset 4 B-cell receptor immunoglobulins to viable human memory B lymphocytes requires a distinctive IGKV somatic mutation.

    Science.gov (United States)

    Catera, Rosa; Liu, Yun; Gao, Chao; Yan, Xiao-Jie; Magli, Amanda; Allen, Steven L; Kolitz, Jonathan E; Rai, Kanti R; Chu, Charles C; Feizi, Ten; Stamatopoulos, Kostas; Chiorazzi, Nicholas

    2017-01-12

    Amino acid replacement mutations in certain CLL stereotyped B-cell receptor (BCR) immunoglobulins (IGs) at defined positions within antigen-binding sites strongly imply antigen selection. Prime examples of this are CLL subset 4 BCR IGs using IGHV4-34/IGHD5-18/IGHJ6 and IGKV2-30/IGKJ2 rearrangements. Conspicuously and unlike most CLL IGs, subset 4 IGs do not bind apoptotic cells. By testing the (auto)antigenic reactivities of subset 4 IGs toward viable lymphoid-lineage cells and specific autoantigens typically bound by IGHV4-34+ IGs, we found IGs from both subset 4 and non-subset 4 IGHV4-34-expressing CLL cases bind naïve B cells. However, only subset 4 IGs react with memory B cells. Furthermore, subset 4 IGs do not bind DNA nor i or I carbohydrate antigens, common targets of IGHV4-34-utilizing antibodies in systemic lupus erythematosus and cold agglutinin disease, respectively. Notably, we found that subset 4 IG binding to memory B lymphocytes depends on an aspartic acid at position 66 of FR3 in the rearranged IGKV2-30 gene; this amino acid residue is acquired by somatic mutation. Our findings illustrate the importance of positive and negative selection criteria for structural elements in CLL IGs and suggest that autoantigens driving normal B cells to become subset 4 CLL cells differ from those driving IGHV4-34+ B cells in other diseases.

  1. Waste association in mass for coating formulations: a viable alternative to dispose; Associacao de residuos em formulacoes de massas para revestimentos: uma alternativa viavel ao descarte

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, E.A.C.; Soares Filho, J.E.; Souza, F.J.P.; Almeida, V.S. de; Oliveira, T.M. de, E-mail: erikcferreira@gmail.com [Universidade Federal do Rio Grande do Norte (UFRN), Natal RN (Brazil)

    2016-07-01

    The ceramic coatings industries are able to use in their formulations whose waste Eco disposal make the costly disposal, being able to reduce production costs by replacing traditional inputs for mining and industrial waste. Their raw materials are classified as plasticizers, fluxes and structural according to their physicochemical characteristics. Since waste falls within these classifications, their use in formulations becomes a viable and attractive alternative from an ecological point of view and marketing. Several studies have attested to waste incorporating viability porcelains formulations, however, is not common to find studies evaluating the addition of more than one simultaneously in formulations. It is the objective of the study, to examine whether fine waste rock and kaolin together with traditional raw materials are able to produce porcelain wet as technological properties defined by the NBR-13818. (author)

  2. Fluorescence Quenching Property of C-Phycocyanin from Spirulina platensis and its Binding Efficacy with Viable Cell Components.

    Science.gov (United States)

    Paswan, Meenakshi B; Chudasama, Meghna M; Mitra, Madhusree; Bhayani, Khushbu; George, Basil; Chatterjee, Shruti; Mishra, Sandhya

    2016-03-01

    Phycocyanin is a natural brilliant blue colored, fluorescent protein, which is commonly present in cyanobacteria. In this study, C-phycocyanin was extracted and purified from Spirulina platensis, which are multicellular and filamentous cyanobacteria of greater importance because of its various biological and pharmacological potential. It was analyzed for its binding affinity towards blood cells, algal cells, genomic DNA of microalgae, and bacteria at different temperature and incubation time. It showed good binding affinity with these components even at low concentration of 2.5 μM. The purpose of this study was to evaluate the applicability of C-phycocyanin as a green fluorescent dye substituting carcinogenic chemical dyes.

  3. Single cell analysis of human tissues and solid tumors with mass cytometry.

    Science.gov (United States)

    Leelatian, Nalin; Doxie, Deon B; Greenplate, Allison R; Mobley, Bret C; Lehman, Jonathan M; Sinnaeve, Justine; Kauffmann, Rondi M; Werkhaven, Jay A; Mistry, Akshitkumar M; Weaver, Kyle D; Thompson, Reid C; Massion, Pierre P; Hooks, Mary A; Kelley, Mark C; Chambless, Lola B; Ihrie, Rebecca A; Irish, Jonathan M

    2017-01-01

    Mass cytometry measures 36 or more markers per cell and is an appealing platform for comprehensive phenotyping of cells in human tissue and tumor biopsies. While tissue disaggregation and fluorescence cytometry protocols were pioneered decades ago, it is not known whether established protocols will be effective for mass cytometry and maintain cancer and stromal cell diversity. Tissue preparation techniques were systematically compared for gliomas and melanomas, patient derived xenografts of small cell lung cancer, and tonsil tissue as a control. Enzymes assessed included DNase, HyQTase, TrypLE, collagenase (Col) II, Col IV, Col V, and Col XI. Fluorescence and mass cytometry were used to track cell subset abundance following different enzyme combinations and treatment times. Mechanical disaggregation paired with enzymatic dissociation by Col II, Col IV, Col V, or Col XI plus DNase for 1 h produced the highest yield of viable cells per gram of tissue. Longer dissociation times led to increasing cell death and disproportionate loss of cell subsets. Key markers for establishing cell identity included CD45, CD3, CD4, CD8, CD19, CD64, HLA-DR, CD11c, CD56, CD44, GFAP, S100B, SOX2, nestin, vimentin, cytokeratin, and CD31. Mass and fluorescence cytometry identified comparable frequencies of cancer cell subsets, leukocytes, and endothelial cells in glioma (R = 0.97), and tonsil (R = 0.98). This investigation establishes standard procedures for preparing viable single cell suspensions that preserve the cellular diversity of human tissue microenvironments. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  4. Managing Viable Knowledge

    NARCIS (Netherlands)

    Achterbergh, J.M.I.M.; Vriens, D.J.

    2002-01-01

    In this paper, Beer's Viable System Model (VSM) is applied to knowledge management. Based on the VSM, domains of knowledge are identified that an organization should possess to maintain its viability. The logic of the VSM is also used to support the diagnosis, design and implementation of the

  5. Determination of Viable Salmonella Typhimurium Cells in Heat Treated Milk By PMA/Real-Time PCR Method

    Directory of Open Access Journals (Sweden)

    Zülal Kesmen

    2017-06-01

    Full Text Available Applying different technological processes during the production of food has a lethal effect on the bacteria but DNA of these bacterial strains may cause false positive results when detected by real time PCR technique because they preserve their existence for a certain period of time. To overcome this shortcoming of the real time PCR technique, a new method has been developed in recent years, based on the removal of dead cell DNA from the medium by treatment with Propodium Monoazide (PMA before DNA extraction. In this study, real-time PCR method was combined with PMA application for the detection of live cells of Salmonella Typhimurium in heat treated milk samples. For this purpose, milk samples inoculated with S. Tyhimurium were heat treated at different temperatures (60, 65, 70 and 75°C and times (15, 60, 300, 900 sec and number of live bacteria was determined comparatively by direct real-time PCR, PMA/real-time PCR and conventional cultural method. As a result, unlike the direct real time PCR technique, PMA/real-time PCR method prevents to a certain extent of false positive results from dead cells at all tested temperatures and times but higher results were obtained from PMA/real-time PCR method when compared to conventional cultural results. Therefore, further studies should be carried out to optimize the conditions of the PMA application in order to eliminate the high positive results detected by the PMA / real-time PCR method

  6. Advances toward regenerative medicine in the central nervous system: challenges in making stem cell therapy a viable clinical strategy.

    Science.gov (United States)

    Stoll, Elizabeth A

    2014-01-01

    Over recent years, there has been a great deal of interest in the prospects of stem cell-based therapies for the treatment of nervous system disorders. The eagerness of scientists, clinicians, and spin-out companies to develop new therapies led to premature clinical trials in human patients, and now the initial excitement has largely turned to skepticism. Rather than embracing a defeatist attitude or pressing blindly ahead, I argue it is time to evaluate the challenges encountered by regenerative medicine in the central nervous system and the progress that is being made to solve these problems. In the twenty years since the adult brain was discovered to have an endogenous regenerative capacity, much basic research has been done to elucidate mechanisms controlling proliferation and cellular identity; how stem cells may be directed into neuronal lineages; genetic, pharmacological, and behavioral interventions that modulate neurogenic activity; and the exact nature of limitations to regeneration in the adult, aged, diseased and injured CNS. These findings should prove valuable in designing realistic clinical strategies to improve the prospects of stem cell-based therapies. In this review, I discuss how basic research continues to play a critical role in identifying both barriers and potential routes to regenerative therapy in the CNS.

  7. Induction of Viable but Nonculturable Salmonella in Exponentially Grown Cells by Exposure to a Low-Humidity Environment and Their Resuscitation by Catalase.

    Science.gov (United States)

    Morishige, Yuta; Koike, Atsushi; Tamura-Ueyama, Ai; Amano, Fumio

    2017-02-01

    Salmonella is a major cause of foodborne disease that sometimes occurs in massive outbreaks around the world. This pathogen is tolerant of low-humidity conditions. We previously described a method for induction of viable but nonculturable (VBNC) Salmonella enterica serovar Enteritidis by treatment with hydrogen peroxide (H2O2) and subsequent resuscitation with 0.3 mM sodium pyruvate. Here, we report a new method for the induction of the VBNC state in Salmonella Enteritidis cells, one involving dehydration. Exposure of Salmonella Enteritidis cells to dehydration stress under poor nutritional conditions (0.9% [wt/vol] NaCl) and 10 to 20% relative humidity at room temperature decreased the presence of culturable population to 0.0067%, but respiratory and glucose uptake active populations were maintained at 0.46 and 1.12%, respectively, meaning that approximately 1% may have entered the VBNC state. Furthermore, these VBNC cells could be resuscitated to acquire culturability by incubation with catalase in M9 minimal medium without glucose in a manner dependent on the dose of catalase but not sodium pyruvate. These results suggest that a low-humidity environment could cause Salmonella Enteritidis cells to enter the VBNC state and the cells could then be resuscitated for growth by treatment with catalase, suggesting a potential risk of Salmonella Enteritidis to survive in low water activity foods in the VBNC state and to start regrowth for foodborne illness.

  8. Freezing Nitrogen Ethanol Composite May be a Viable Approach for Cryotherapy of Human Giant Cell Tumor of Bone.

    Science.gov (United States)

    Wu, Po-Kuei; Chen, Cheng-Fong; Wang, Jir-You; Chen, Paul Chih-Hsueh; Chang, Ming-Chau; Hung, Shih-Chieh; Chen, Wei-Ming

    2017-06-01

    Liquid nitrogen has been used as adjuvant cryotherapy for treating giant cell tumor (GCT) of bone. However, the liquid phase and ultrafreezing (-196° C) properties increase the risk of damage to the adjacent tissues and may lead to perioperative complications. A novel semisolid cryogen, freezing nitrogen ethanol composite, might mitigate these shortcomings because of less-extreme freezing. We therefore wished to evaluate freezing nitrogen ethanol composite as a coolant to determine its properties in tumor cryoablation. (1) Is freezing nitrogen ethanol composite-mediated freezing effective for tumor cryoablation in an ex vivo model, and if yes, is apoptosis involved in the tumor-killing mechanism? (2) Does freezing nitrogen ethanol composite treatment block neovascularization and neoplastic progression of the grafted GCTs and is it comparable to that of liquid nitrogen in an in vivo chicken model? (3) Can use of freezing nitrogen ethanol composite as an adjuvant to curettage result in successful short-term treatment, defined as absence of GCT recurrence at a minimum of 1 year in a small proof-of-concept clinical series? The cryogenic effect on bone tissue mediated by freezing nitrogen ethanol composite and liquid nitrogen was verified by thermal measurement in a time-course manner. Cryoablation on human GCT tissue was examined ex vivo for effect on morphologic features (cell shrinkage) and DNA fragmentation (apoptosis). The presumed mechanism was investigated by molecular analysis of apoptosis regulatory proteins including caspases 3, 8, and 9 and Bax/Bcl-2. Chicken chorioallantoic membrane was used as an in vivo model to evaluate the effects of freezing nitrogen ethanol composite and liquid nitrogen treatment on GCT-derived neovascularization and tumor neoplasm. A small group of patients with GCT of bone was treated by curettage and adjuvant freezing nitrogen ethanol composite cryotherapy in a proof-of-concept study. Tumor recurrence and perioperative

  9. Quality of raw cow milk in Republic of Macedonia determined through the testing of somatic cell count and total viable count

    Directory of Open Access Journals (Sweden)

    Angelovski Ljupco

    2008-11-01

    Full Text Available Somatic cells count and total viable count are criteria used to estimate the compliance of raw cow milk with the Book of rules for demands for safety and hygiene and procedures for official controls of milk and milk products, Official Gazette of RM 157/2007. According to the given demands, raw milk operators are obliged to conduct all procedures and to guarantee that milk is in compliance with the criteria laid down in Book of rules. At the same time, Republic of Macedonia have to fulfill EU criteria laid down in Directive 92/46 (Council directive 92/46/EEC laying down the health rules for the production and placing on the market of raw milk, heat-treated milk and milkbased products for quality of raw milk as part of implementation of community legislation and milk production. The independent laboratory for milk quality control at FVM-Skopje, in frame of its activities in the period February- August 2008 has conducted a study for obtaining preliminary results for the situation with raw milk quality produced in R. of Macedonia for somatic cells counts and total viable count. In the study we analyzed 2065 samples for TVC and 1625 samples for SCC of raw milk samples produced in different parts of the country. From the tested samples only 41,8% fulfill criteria for SCC and 41,45% criteria for TVC lay down in Book of rules for 2008. Assessment of the results in light of Council Directive it is obvious that only 42,7% of the samples for SCC and 10,7% for TVC fulfill the criteria of Council Directive having in mind different requirements vs. Book of rules.

  10. Biologic properties of viable deletion mutants of simian virus 40 (SV40) rescued from the cells of an SV40-induced hamster lymphocytic leukemia.

    Science.gov (United States)

    Diamandopoulos, G T; Carmichael, G

    1983-12-01

    A lymphocytic leukemia induced by the oncogenic DNA simian virus 40 (SV40) in an inbred LSH/SsLak Syrian golden hamster was evoked to produce infectious SV40 by fusion of the leukemia cells with grivet monkey kidney (GMK) cells and by exposure of the leukemia cells to the chemical inducers mitomycin C and cycloheximide. Plaque-purified viable substrains of the rescued SV40 when studied by restriction endonuclease digestion of viral DNA were found to contain small deletions within the Hind III restriction fragment C. These deletions lay near the viral origin of DNA replication. Ten plaque-purified substrains of the rescued virus identified by immunofluorescence as being SV40 were found, when compared to the wild-type SV40, to replicate slowly and to form small plaques. Although these substrains transformed NIH/3T3 cells as efficiently as the wild-type SV40 in tissue culture, they were generally less oncogenic in vivo--7 of the 10 failed to induce tumors. The 3 oncogenic SV40-rescued substrains were not found to exhibit "lymphocytotropism," i.e., the capacity to infect and neoplastically transform preferentially hamster lymphocytes. Thus the hamster lymphocytic leukemia originally induced by the wild-type SV40 was most likely a chance-stochastic event rather than the result of tropism-determinism mediated by the virus, as is usually the case with leukemogenic RNA viruses.

  11. Oxygen-Mass-Flow Calibration Cell

    Science.gov (United States)

    Martin, Robert E.

    1996-01-01

    Proposed calibration standard for mass flow rate of oxygen based on conduction of oxygen ions through solid electrolyte membrane made of zirconia and heated to temperature of 1,000 degrees C. Flow of oxygen ions proportional to applied electric current. Unaffected by variations in temperature and pressure, and requires no measurement of volume. Calibration cell based on concept used to calibrate variety of medical and scientific instruments required to operate with precise rates of flow of oxygen.

  12. Primordial germ cell-mediated chimera technology produces viable pure-line Houbara bustard offspring: potential for repopulating an endangered species.

    Directory of Open Access Journals (Sweden)

    Ulrich Wernery

    2010-12-01

    Full Text Available The Houbara bustard (Chlamydotis undulata is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring.Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs was injected into White Leghorn chicken (Gallus gallus domesticus embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16 gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster.This study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian

  13. Effects of water-filtered infrared-A and of heat on cell death, inflammation, antioxidative potential and of free radical formation in viable skin--first results.

    Science.gov (United States)

    Piazena, Helmut; Pittermann, Wolfgang; Müller, Werner; Jung, Katinka; Kelleher, Debra K; Herrling, Thomas; Meffert, Peter; Uebelhack, Ralf; Kietzmann, Manfred

    2014-09-05

    The effects of water-filtered infrared-A (wIRA) and of convective heat on viability, inflammation, inducible free radicals and antioxidative power were investigated in natural and viable skin using the ex vivo Bovine Udder System (BUS) model. Therefore, skin samples from differently treated parts of the udder of a healthy cow were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, by prostaglandin E2 (PGE2) measurement and by electron spin resonance (ESR) spectroscopy. Neither cell viability, the inflammation status, the radical status or the antioxidative defence systems of the skin were significantly affected by wIRA applied within 30 min by using an irradiance of 1900 W m(-2) which is of relevance for clinical use, but which exceeded the maximum solar IR-A irradiance at the Earth's surface more than 5 times and which resulted in a skin surface temperature of about 45 °C without cooling and of about 37 °C with convective cooling by air ventilation. No significant effects on viability and on inflammation were detected when convective heat was applied alone under equivalent conditions in terms of the resulting skin surface temperatures and exposure time. As compared with untreated skin, free radical formation was almost doubled, whereas the antioxidative power was reduced to about 50% after convective heating to about 45 °C. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Of energy and survival incognito: a relationship between viable but non-culturable cells formation and inorganic polyphosphate and formate metabolism in Campylobacter jejuni.

    Science.gov (United States)

    Kassem, Issmat I; Chandrashekhar, Kshipra; Rajashekara, Gireesh

    2013-01-01

    Campylobacter jejuni is a Gram-negative food-borne bacterium that can cause mild to serious diseases in humans. A variety of stress conditions including exposure to formic acid, a weak organic acid, can cause C. jejuni to form viable but non-culturable cells (VBNC), which was proposed as a potential survival mechanism. The inability to detect C. jejuni VBNC using standard culturing techniques may increase the risk of exposure to foods contaminated with this pathogen. However, little is known about the cellular mechanisms and triggers governing VBNC formation. Here, we discuss novel mechanisms that potentially affect VBNC formation in C. jejuni and emphasize the impact of formic acid on this process. Specifically, we highlight findings that show that impairing inorganic polyphosphate (poly-P) metabolism reduces the ability of C. jejuni to form VBNC in a medium containing formic acid. We also discuss the potential effect of poly-P and formate metabolism on energy homeostasis and cognate VBNC formation. The relationship between poly-P metabolism and VBNC formation under acid stress has only recently been identified and may represent a breakthrough in understanding this phenomenon and its impact on food safety.

  15. Inertial picobalance reveals fast mass fluctuations in mammalian cells

    Science.gov (United States)

    Martínez-Martín, David; Fläschner, Gotthold; Gaub, Benjamin; Martin, Sascha; Newton, Richard; Beerli, Corina; Mercer, Jason; Gerber, Christoph; Müller, Daniel J.

    2017-10-01

    The regulation of size, volume and mass in living cells is physiologically important, and dysregulation of these parameters gives rise to many diseases. Cell mass is largely determined by the amount of water, proteins, lipids, carbohydrates and nucleic acids present in a cell, and is tightly linked to metabolism, proliferation and gene expression. Technologies have emerged in recent years that make it possible to track the masses of single suspended cells and adherent cells. However, it has not been possible to track individual adherent cells in physiological conditions at the mass and time resolutions required to observe fast cellular dynamics. Here we introduce a cell balance (a ‘picobalance’), based on an optically excited microresonator, that measures the total mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity. Using our technique, we observe that the mass of living mammalian cells fluctuates intrinsically by around one to four per cent over timescales of seconds throughout the cell cycle. Perturbation experiments link these mass fluctuations to the basic cellular processes of ATP synthesis and water transport. Furthermore, we show that growth and cell cycle progression are arrested in cells infected with vaccinia virus, but mass fluctuations continue until cell death. Our measurements suggest that all living cells show fast and subtle mass fluctuations throughout the cell cycle. As our cell balance is easy to handle and compatible with fluorescence microscopy, we anticipate that our approach will contribute to the understanding of cell mass regulation in various cell states and across timescales, which is important in areas including physiology, cancer research, stem-cell differentiation and drug discovery.

  16. Clinical outcomes and histological findings of patients with advanced metastatic germ cell tumors undergoing post-chemotherapy resection of retroperitoneal lymph nodes and residual extraretroperitoneal masses.

    Science.gov (United States)

    Nakamura, Terukazu; Oishi, Masakatsu; Ueda, Takashi; Fujihara, Atsuko; Nakanishi, Hiroyuki; Kamoi, Kazumi; Naya, Yoshio; Hongo, Fumiya; Okihara, Koji; Miki, Tsuneharu

    2015-07-01

    To assess clinical outcomes of patients with advanced germ cell tumor undergoing post-chemotherapy retroperitoneal lymph node dissection with or without extraretroperitoneal mass resection. Between 1998 and 2013, 175 retroperitoneal lymph node dissections for advanced metastatic germ cell tumors were carried out at Kyoto Prefectural University of Medicine, Kyoto, Japan. Of patients receiving retroperitoneal lymph node dissections, 156 underwent post-chemotherapy retroperitoneal lymph node dissection with or without extraretroperitoneal mass resection as first surgery after completion of chemotherapy. Of these 156 patients, 47 underwent both post-chemotherapy retroperitoneal lymph node dissection and extraretroperitoneal mass resection. The histological findings were necrosis in 59.6%, teratoma in 31.4% and viable cancer in 9.0% at retroperitoneal lymph node. At extraretroperitoneal mass resection, necrosis was present in 59.6%, teratoma in 31.9% and viable cancer in 8.5%. Overall histological discordance between retroperitoneal lymph node and extraretroperitoneal mass was found in 31.9%. Five-year disease-free survival stratified by retroperitoneal lymph node histology in 156 patients was 91.3% for necrosis, 78.7% for teratoma and 63.5% for viable cancer (log-rank, P = 0.009). Antegrade ejaculation was preserved in 80.9%. In the worst histology of post-chemotherapy retroperitoneal lymph node dissection or extraretroperitoneal mass resection in 156 patients, 5-year disease-free survival was 93.2% for necrosis, 79.0% for teratoma and 63.4% for viable cancer (log-rank, P retroperitoneal lymph node histology and salvage chemotherapy. The presence of viable cancer at the retroperitoneal lymph node is an independent predictor of disease recurrence. In approximately one-third of cases, there is a histological discordance between retroperitoneal lymph node and extraretroperitoneal mass. Resection of residual retroperitoneal lymph node and extraretroperitoneal masses

  17. Where Do All the Phytoplankton Go? Challenges in Keeping Track of Viable Cells in Phytoplankton Communities Using Flow Cytometry and Cell Staining

    Science.gov (United States)

    Simmons, L. J.; Fobbe, D. J.; Berges, J. A.

    2016-02-01

    Understanding the dynamics of phytoplankton communities has traditionally focused on differences in growth and related processes among taxa. It is now appreciated that differences in mortality could be equally important in contributing to these dynamics. Studying mortality in communities is difficult, especially on relevant time scales, which could be as short as hours to days. Flow cytometry can potentially provide solutions, because it can allow discrimination of different taxa, and when combined with staining, distinguish live and dead cells. We applied flow cytometry and staining to phytoplankton communities in a model system: a small, well-studied, urban pond in southeastern Wisconsin. Using flow cytometry, it was possible to resolve up to six dominant taxa (most stain also affected other fluorescence channels, requiring compensation. Correlations of numbers of dead cells with environmental factors (e.g. temperature, nutrient concentrations, irradiance) were generally poor, suggesting the greater importance of biotic versus abiotic variables in community mortality dynamics. Ongoing work is focusing on the effects of viral pathogens, grazing and allelopathic interactions using experimental manipulations and individual-based modeling.

  18. Co-infusion of autologous adipose tissue derived neuronal differentiated mesenchymal stem cells and bone marrow derived hematopoietic stem cells, a viable therapy for post-traumatic brachial plexus injury: A case report

    Directory of Open Access Journals (Sweden)

    Umang G Thakkar

    2014-08-01

    Full Text Available Stem cell therapy is emerging as a viable approach in regenerative medicine. A 31-year-old male with brachial plexus injury had complete sensory-motor loss since 16 years with right pseudo-meningocele at C5-D1 levels and extra-spinal extension up to C7-D1, with avulsion on magnetic resonance imaging and irreversible damage. We generated adipose tissue derived neuronal differentiated mesenchymal stem cells (N-AD-MSC and bone marrow derived hematopoietic stem cells (HSC-BM. Neuronal stem cells expressed β-3 tubulin and glial fibrillary acid protein which was confirmed on immunofluorescence. On day 14, 2.8 ml stem cell inoculum was infused under local anesthesia in right brachial plexus sheath by brachial block technique under ultrasonography guidance with a 1.5-inch-long 23 gauge needle. Nucleated cell count was 2 × 10 4 /μl, CD34+ was 0.06%, and CD45-/90+ and CD45-/73+ were 41.63% and 20.36%, respectively. No untoward effects were noted. He has sustained recovery with re-innervation over a follow-up of 4 years documented on electromyography-nerve conduction velocity study.

  19. Potential of Escherichia coli 0157:H7 to persist and form viable but non-culturable cells on a food-contact surface subjected to cycles of soiling and chemical treatment

    DEFF Research Database (Denmark)

    Marouani-Gadri, Nesrine; Firmesse, Olivier; Chassaing, Danielle

    2010-01-01

    only, a further increase in this proportion occurred 24 h after the CT, suggesting that some of the surviving viable but non-culturable cells finally died. This study shows that conditions leading to E. coli O157:H7 persistence are not likely to arise when good refrigeration and hygiene practices...... no longer detectable after the first week. However, on 66-hour biofilms with 6.7 log CFU/cm², after initially decreasing, E. coli numbers reached 6.6 log CFU/cm² and 8.3 log viable cells/cm² on the 11th day. When E. coli was cultured with a Comamonas testosteroni previously shown to increase E. coli biofilm...

  20. The Chemically Synthesized Ageladine A-Derivative LysoGlow84 Stains Lysosomes in Viable Mammalian Brain Cells and Specific Structures in the Marine Flatworm Macrostomum lignano

    Directory of Open Access Journals (Sweden)

    Thorsten Mordhorst

    2015-02-01

    Full Text Available Based on the chemical structure and the known chemical synthesis of the marine sponge alkaloid ageladine A, we synthesized the ageladine A-derivative 4-(naphthalene-2-yl-1H-imidazo[4,5-c]pyridine trifluoroacetate (LysoGlow84. The two-step synthesis started with the Pictet-Spengler reaction of histamine and naphthalene-2-carbaldehyde to a tetrahydropyridine intermediate, which was dehydrogenated with activated manganese (IV oxide to LysoGlow84. Structure and purity of the synthesized LysoGlow84 were confirmed by NMR spectroscopy and mass spectrometry. The fluorescence intensity emitted by LysoGlow84 depended strongly on the pH of the solvent with highest fluorescence intensity recorded at pH 4. The fluorescence maximum (at 315 nm excitation was observed at 440 nm. Biocompatibility of LysoGlow84 was investigated using cultured rat brain astrocytes and the marine flatworm Macrostomum lignano. Exposure of the astrocytes for up to 6 h to micromolar concentrations of LysoGlow84 did not compromise cell viability, as demonstrated by several viability assays, but revealed a promising property of this compound for staining of cellular vesicles. Conventional fluorescence microscopy as well as confocal scanning microscopy of LysoGlow84-treated astrocytes revealed co-localization of LysoGlow84 fluorescence with that of LysoTracker® Red DND-99. LysoGlow84 stained unclear structures in Macrostomum lignano, which were identified as lysosomes by co-staining with LysoTracker. Strong fluorescence staining by LysoGlow84 was further observed around the worms’ anterior gut and the female genital pore which were not counterstained by LysoTracker Red. Thus, LysoGlow84 is a new promising dye that stains lysosomes and other acidic compartments in cultured cells and in worms.

  1. The chemically synthesized ageladine A-derivative LysoGlow84 stains lysosomes in viable mammalian brain cells and specific structures in the marine flatworm Macrostomum lignano.

    Science.gov (United States)

    Mordhorst, Thorsten; Awal, Sushil; Jordan, Sebastian; Petters, Charlotte; Sartoris, Linda; Dringen, Ralf; Bickmeyer, Ulf

    2015-02-11

    Based on the chemical structure and the known chemical synthesis of the marine sponge alkaloid ageladine A, we synthesized the ageladine A-derivative 4-(naphthalene-2-yl)-1H-imidazo[4,5-c]pyridine trifluoroacetate (LysoGlow84). The two-step synthesis started with the Pictet-Spengler reaction of histamine and naphthalene-2-carbaldehyde to a tetrahydropyridine intermediate, which was dehydrogenated with activated manganese (IV) oxide to LysoGlow84. Structure and purity of the synthesized LysoGlow84 were confirmed by NMR spectroscopy and mass spectrometry. The fluorescence intensity emitted by LysoGlow84 depended strongly on the pH of the solvent with highest fluorescence intensity recorded at pH 4. The fluorescence maximum (at 315 nm excitation) was observed at 440 nm. Biocompatibility of LysoGlow84 was investigated using cultured rat brain astrocytes and the marine flatworm Macrostomum lignano. Exposure of the astrocytes for up to 6 h to micromolar concentrations of LysoGlow84 did not compromise cell viability, as demonstrated by several viability assays, but revealed a promising property of this compound for staining of cellular vesicles. Conventional fluorescence microscopy as well as confocal scanning microscopy of LysoGlow84-treated astrocytes revealed co-localization of LysoGlow84 fluorescence with that of LysoTracker® Red DND-99. LysoGlow84 stained unclear structures in Macrostomum lignano, which were identified as lysosomes by co-staining with LysoTracker. Strong fluorescence staining by LysoGlow84 was further observed around the worms' anterior gut and the female genital pore which were not counterstained by LysoTracker Red. Thus, LysoGlow84 is a new promising dye that stains lysosomes and other acidic compartments in cultured cells and in worms.

  2. Mass Spectrometry of Polymer Electrolyte Membrane Fuel Cells

    Directory of Open Access Journals (Sweden)

    Viktor Johánek

    2016-01-01

    Full Text Available The chemical analysis of processes inside fuel cells under operating conditions in either direct or inverted (electrolysis mode and their correlation with potentiostatic measurements is a crucial part of understanding fuel cell electrochemistry. We present a relatively simple yet powerful experimental setup for online monitoring of the fuel cell exhaust (of either cathode or anode side downstream by mass spectrometry. The influence of a variety of parameters (composition of the catalyst, fuel type or its concentration, cell temperature, level of humidification, mass flow rate, power load, cell potential, etc. on the fuel cell operation can be easily investigated separately or in a combined fashion. We demonstrate the application of this technique on a few examples of low-temperature (70°C herein polymer electrolyte membrane fuel cells (both alcohol- and hydrogen-fed subjected to a wide range of conditions.

  3. Mass Spectrometry of Polymer Electrolyte Membrane Fuel Cells.

    Science.gov (United States)

    Johánek, Viktor; Ostroverkh, Anna; Fiala, Roman; Rednyk, Andrii; Matolín, Vladimír

    2016-01-01

    The chemical analysis of processes inside fuel cells under operating conditions in either direct or inverted (electrolysis) mode and their correlation with potentiostatic measurements is a crucial part of understanding fuel cell electrochemistry. We present a relatively simple yet powerful experimental setup for online monitoring of the fuel cell exhaust (of either cathode or anode side) downstream by mass spectrometry. The influence of a variety of parameters (composition of the catalyst, fuel type or its concentration, cell temperature, level of humidification, mass flow rate, power load, cell potential, etc.) on the fuel cell operation can be easily investigated separately or in a combined fashion. We demonstrate the application of this technique on a few examples of low-temperature (70°C herein) polymer electrolyte membrane fuel cells (both alcohol- and hydrogen-fed) subjected to a wide range of conditions.

  4. Osteocalcin Effect on Human β-Cells Mass and Function.

    Science.gov (United States)

    Sabek, Omaima M; Nishimoto, Satoru Ken; Fraga, Daniel; Tejpal, Neelam; Ricordi, Camillo; Gaber, A O

    2015-09-01

    The osteoblast-specific hormone osteocalcin (OC) was found to regulate glucose metabolism, fat mass, and β-cell proliferation in mice. Here, we investigate the effect of decarboxylated OC (D-OC) on human β-cell function and mass in culture and in vivo using a Nonobese diabetic-severe combined immunodeficiency mouse model. We found that D-OC at dose ranges from 1.0 to 15 ng/mL significantly augmented insulin content and enhanced human β-cell proliferation of cultured human islets. This was paralleled by increased expression of sulfonylurea receptor protein; a marker of β-cell differentiation and a component of the insulin-secretory apparatus. Moreover, in a Nonobese diabetic-severe combined immunodeficiency mouse model, systemic administration of D-OC at 4.5-ng/h significantly augmented production of human insulin and C-peptide from the grafted human islets. Finally, histological staining of the human islet grafts showed that the improvement in the β-cell function was attributable to an increase in β-cell mass as a result of β-cell proliferation indicated by MKI67 staining together with the increased β-cell number and decreased α-cell number data obtained using laser scanning cytometry. Our data for the first time show D-OC-enhanced β-cell function in human islets and support future exploitation of D-OC-mediated β-cell regulation for developing useful clinical treatments for patients with diabetes.

  5. Mass transport mechanism in porous fuel cell electrodes

    Science.gov (United States)

    Jonsson, I.; Lindholm, I.

    1969-01-01

    Results of experiments on hydrogen-oxygen fuel cells show that higher current densities are obtained with cell anodes having a 100 micron thin active layer of porous nickel containing silver electrocatalyst. Increase in current density is attributed to a convective mass transport mechanism.

  6. T cells recognizing a peptide contaminant undetectable by mass spectrometry

    DEFF Research Database (Denmark)

    Brezar, Vedran; Culina, Slobodan; Østerbye, Thomas

    2011-01-01

    Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility...... complex (MHC) Class I-restricted ß-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid...... chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(206-214) epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP(206-214) using a novel method confirmed the identity...

  7. Imaging the Beta-cell mass: why and how

    DEFF Research Database (Denmark)

    Saudek, Frantisek; Brogren, Carl-Henrik; Manohar, Srirang

    2008-01-01

    of the fate of beta-cells under disease and therapy conditions. This could pave the way for a new era of intervention by islet replacement and regeneration regimens. Monitoring the beta-cell mass requires a reliable method for noninvasive in vivo imaging. Such a method is not available at present due...... to the lack of a beta-cell-specific contrast agent. The only existing method to monitor islet cells in vivo consists of labeling islet transplants with iron nanoparticles prior to transplantation and visualization of the transplanted islets by magnetic resonance imaging (MRI). Therefore, accurate assessment...... of the native beta-cell mass is still limited to autopsy studies. Endeavors to find a biological structure specific for beta-cells led to the discovery of potential candidates that have been tested for noninvasive imaging. Among them are the ligand to the vesicular monoamine transporter type 2 (VMAT-2), which...

  8. Tumor-selective replication herpes simplex virus-based technology significantly improves clinical detection and prognostication of viable circulating tumor cells

    DEFF Research Database (Denmark)

    Zhang, Wen; Bao, Li; Yang, Shaoxing

    2016-01-01

    Detection of circulating tumor cells remains a significant challenge due to their vast physical and biological heterogeneity. We developed a cell-surface-marker-independent technology based on telomerase-specific, replication-selective oncolytic herpes-simplex-virus-1 that targets telomerase...

  9. Preparation of Single Cells for Imaging Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-10-24

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

  10. Enzymatic isolation of viable human odontoblasts.

    Science.gov (United States)

    Cuffaro, H M; Pääkkönen, V; Tjäderhane, L

    2016-05-01

    To improve an enzymatic method previously used for isolation of rat odontoblasts to isolate viable mature human odontoblasts. Collagenase I, collagenase I/hyaluronidase mixture and hyaluronidase were used to extract mature human odontoblasts from the pulp chamber. Detachment of odontoblasts from dentine was determined with field emission scanning electron microscopy (FESEM) and to analyse the significance of differences in tubular diameter, and the t-test was used. MTT-reaction was used to analyse cell viability, and nonparametric Kruskal-Wallis and Mann-Whitney post hoc tests were used to analyse the data. Immunofluorescent staining of dentine sialoprotein (DSP), aquaporin-4 (AQP4) and matrix metalloproteinase-20 (MMP-20) and quantitative PCR (qPCR) of dentine sialophosphoprotein (DSPP) were used to confirm the odontoblastic nature of the cells. MTT-reaction and FESEM demonstrated collagenase I/hyaluronidase resulted in more effective detachment and higher viability than collagenase I alone. Hyaluronidase alone was not able to detach odontoblasts. Immunofluorescence revealed the typical odontoblastic-morphology with one process, and DSP, AQP4 and MMP-20 were detected. Quantitative PCR of DSPP confirmed that the isolated cells expressed this odontoblast-specific gene. The isolation of viable human odontoblasts was successful. The cells demonstrated morphology typical for odontoblasts and expressed characteristic odontoblast-type genes and proteins. This method will enable new approaches, such as apoptosis analysis, for studies using fully differentiated odontoblasts. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  11. The binding of rhBMP-2 to the receptors of viable MC3T3-E1 cells and the question of cooperativity

    Energy Technology Data Exchange (ETDEWEB)

    Wiemann, M.; Rumpf, H.M.; Bingmann, D.; Jennissen, H.P. [Universitaetsklinikum Essen (Germany). Inst. fuer Physiologie; Universitaetsklinikum Essen (Germany). Inst. fuer Physiologiesche Chemie

    2001-12-01

    The binding of rhBMP-2 to its receptors, the signal transduction cascade and the final responses of bone cells, osteoprogenitor cells and derived cell lines is of high fundamental and clinical interest. In this report concentration-response curves of the osteoblast cell line MC3T3-E1 under influence of rhBMP-2 was investigated. The biological response of the cells (corresponding to a down-stream effect of the receptor state-function) was monitored in pilot experiments by the MC3T3-cell alkaline phosphatase-induction test (MC3T3-cell ALP-induction test). It is shown that the MC3T3-cell ALP-induction test is a good tool for measuring biologically active recombinant human BMP-2 (rhBMP-2) in crude extracts of E. coli as well as in highly purified form. In addition this test is very sensitive to chemically induced structural changes of rhBMP-2 such as those resulting from a radiolabeling of rhBMP-2 by the Bolton-Hunter procedure. The latter procedure reduces the biological activity of rhBMP-2 by a factor of 3-4. The measured concentration-response curves could all be non-linearly fitted to a rectangular hyperbola. The half-maximal saturation, K{sub 0.5}, is found between 30-100 nM rhBMP-2 (= 0.8-2.5 {mu}g/ml). The effect of rhBMP-2 shows a plateau i.e. maximal response at ca. 300-1000 nM rhBMP-2 (= 8-25 {mu}g/ml). The data thus indicate a non-cooperative binding-response behavior. This was unexpected since BMP-2 binds simultaneously to two cooperating receptors of type 1 and type 2. However in the very low concentration range of employed rhBMP-2 a variable response of the cells was measured so that a full exclusion of cooperativity cannot be concluded at the present time. This will be clarified by future experiments. (orig.)

  12. Enumeration of viable and non-viable larvated Ascaris eggs with quantitative PCR.

    Science.gov (United States)

    Raynal, Maria; Villegas, Eric N; Nelson, Kara L

    2012-12-01

    The goal of this study was to further develop an incubation-quantitative polymerase chain reaction (qPCR) method for quantifying viable Ascaris eggs by characterizing the detection limit and number of template copies per egg, determining the specificity of the method, and testing the method with viable and inactivated larvated eggs. The number of template copies per cell was determined by amplifying DNA from known numbers of eggs at different development stages; the value was estimated to be 32 copies. The specificity of the method was tested against a panel of bacteria, fungi, protozoa and helminths, and no amplification was found with non-target DNA. Finally, fully larvated eggs were inactivated by four different treatments: 254 nm ultraviolet light, 2,000 ppm NH(3)-N at pH 9, moderate heat (48 °C) and high heat (70 °C). Concentrations of treated eggs were measured by direct microscopy and incubation-qPCR. The qPCR signal decreased following all four treatments, and was in general agreement with the decrease in viable eggs determined by microscopy. The incubation-qPCR method for enumerating viable Ascaris eggs is a promising approach that can produce results faster than direct microscopy, and may have benefits for applications such as assessing biosolids.

  13. Cell density modulates intracellular mass transport in neural networks.

    Science.gov (United States)

    Cintora, Patricia; Arikkath, Jyothi; Kandel, Mikhail; Popescu, Gabriel; Best-Popescu, Catherine

    2017-05-01

    In order to fully understand brain connectivity and elucidate the mechanisms involved in central nervous system disease, the field of neuroscience depends on quantitative studies of neuronal structure and function. Cell morphology and neurite (axonal and dendritic) arborization are typically studied by immunohistochemical and fluorescence techniques. However, dry mass content and intracellular mass transport rates have largely been under-investigated given the inherent difficulties in their measurement. Here, spatial light interference microscopy (SLIM) and dispersion-relation phase spectroscopy (DPS) were used to measure pathlength fluctuations that report on the dry mass and transport within cultured primary neurons across low, medium, and high cell density conditions. It was found that cell density (confluence) affects significantly both the growth rate and mass transport. The analysis method is label-free and does not require neuronal tracing, particle tracking, or neuron reconstruction. Since SLIM can upgrade any existing phase contrast microscope and the imaging and analysis are high-throughput, we anticipate that this approach will be embraced by neuroscientists for broad scale studies. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  14. Mass spectrometric production of heterogeneous metal clusters using Knudsen cell

    Directory of Open Access Journals (Sweden)

    Veljković Filip M.

    2016-01-01

    Full Text Available Knudsen effusion mass spectrometry or high-temperature method of mass spectrometry for decades gives new information about saturated vapor of hardly volatile compounds and it is an important method in the discovery of many new molecules, radicals, ions and clusters present in the gas phase. Since pioneering works until now, this method has been successfully applied to a large number of systems (ores, oxides, ceramics, glass materials, borides, carbides, sulfides, nitrates, metals, fullerenes, etc which led to the establishment of various research branches such as chemistry of clusters. This paper describes the basic principles of Knudsen cell use for both identification of chemical species created in the process of evaporation and determination of their ionization energies. Depending on detected ions intensities and the partial pressure of each gaseous component, as well as on changes in partial pressure with temperature, Knudsen cell mass spectrometry enables the determination of thermodynamic parameters of the tested system. A special attention is paid to its application in the field of small heterogeneous and homogeneous clusters of alkali metals. Furthermore, experimental results for thermodynamic parameters of some clusters, as well as capabilities of non-standard ways of using Knudsen cells in the process of synthesis of new clusters are presented herein. [Projekat Ministarstva nauke Republike Srbije, br. 172019

  15. Regulation of fat cell mass by insulin in Drosophila melanogaster.

    Science.gov (United States)

    DiAngelo, Justin R; Birnbaum, Morris J

    2009-12-01

    A phylogenetically conserved response to nutritional abundance is an increase in insulin signaling, which initiates a set of biological responses dependent on the species. Consequences of augmented insulin signaling include developmental progression, cell and organ growth, and the storage of carbohydrates and lipids. Here, we address the evolutionary origins of insulin's positive effects on anabolic lipid metabolism by selectively modulating insulin signaling in the fat body of the fruit fly, Drosophila melanogaster. Analogous to the actions of insulin in higher vertebrates, those in Drosophila include expansion of the insect fat cell mass both by increasing the adipocyte number and by promoting lipid accumulation. The ability of insulin to accomplish the former depends on its capacity to bring about phosphorylation and inhibition of the transcription factor Drosophila FOXO (dFOXO) and the serine/threonine protein kinase shaggy, the fly ortholog of glycogen synthase kinase 3 (GSK3). Increasing the amount of triglyceride per cell also depends on the phosphorylation of shaggy but is independent of dFOXO. Thus, the findings of this study provide evidence that the control of fat mass by insulin is a conserved process and place dFOXO and shaggy/GSK3 downstream of the insulin receptor in controlling adipocyte cell number and triglyceride storage, respectively.

  16. Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture

    Science.gov (United States)

    Ducos, Jean-Paul; Terrier, Bénédicte; Courtois, Didier

    Different types of bioreactors are used at Nestlé R&D Centre - Tours for mass propagation of selected plant varieties by somatic embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has recently been set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-L glass bioreactors. An improved process has been developed using a 10-L disposable bioreactor consisting of a bag containing a rigid plastic box ('Box-in-Bag' bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design. For large scale cell culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 L working volumes, validated with several plant species ('Wave and Undertow' and 'Slug Bubble' bioreactors). The advantages and the limits of these new types of bioreactor are discussed, based mainly on our own experience on coffee somatic embryogenesis and mass cell culture of soya and tobacco.

  17. Viable Syntax: Rethinking Minimalist Architecture

    Directory of Open Access Journals (Sweden)

    Ken Safir

    2010-03-01

    Full Text Available Hauser et al. (2002 suggest that the human language faculty emerged as a genetic innovation in the form of what is called here a ‘keystone factor’—a single, simple, formal mental capability that, interacting with the pre-existing faculties of hominid ancestors, caused a cascade of effects resulting in the language faculty in modern humans. They take Merge to be the keystone factor, but instead it is posited here that Merge is the pre-existing mechanism of thought made viable by a principle that permits relations interpretable at the interfaces to be mapped onto c-command. The simplified minimalist architecture proposed here respects the keystone factor as closely as possible, but is justified on the basis of linguistic analyses it makes available, including a relativized intervention theory applicable across Case, scope, agreement, selection and linearization, a derivation of the A/A’-distinction from Case theory, and predictions such as why in situ wh-interpretation is island-insensitive, but susceptible to intervention effects.

  18. Experimental design for the optimization of propidium monoazide treatment to quantify viable and non-viable bacteria in piggery effluents.

    Science.gov (United States)

    Desneux, Jérémy; Chemaly, Marianne; Pourcher, Anne-Marie

    2015-08-16

    Distinguishing between viable and dead bacteria in animal and urban effluents is a major challenge. Among existing methods, propidium monoazide (PMA)-qPCR is a promising way to quantify viable cells. However, its efficiency depends on the composition of the effluent, particularly on total suspended solids (TSS)) and on methodological parameters. The aim of this study was evaluate the influence of three methodological factors (concentration of PMA, incubation time and photoactivation time) on the efficiency of PMA-qPCR to quantify viable and dead cells of Listeria monocytogenes used as a microorganism model, in two piggery effluents (manure and lagoon effluent containing 20 and 0.4 TSS g.kg(-1), respectively). An experimental design strategy (Doehlert design and desirability function) was used to identify the experimental conditions to achieve optimal PMA-qPCR results. The quantification of viable cells of L. monocytogenes was mainly influenced by the concentration of PMA in the manure and by the duration of photoactivation in the lagoon effluent. Optimal values differed with the matrix: 55 μM PMA, 5 min incubation and 56 min photoactivation for manure and 20 μM PMA, 20 min incubation and 30 min photoactivation for lagoon effluent. Applied to five manure and four lagoon samples, these conditions resulted in satisfactory quantification of viable and dead cells. PMA-qPCR can be used on undiluted turbid effluent with high levels of TSS, provided preliminary tests are performed to identify the optimal conditions.

  19. On-line cell mass monitoring of Saccharomyces cerevisiae cultivations by multi-wavelength fluorescence

    DEFF Research Database (Denmark)

    Haack, Martin Brian; Eliasson, Anna; Olsson, Lisbeth

    2004-01-01

    in a decomposition of the multivariate fluorescent landscape, whereby underlying spectra of the individual intrinsic fluorophors present in the cell mass were estimated. Furthermore, gravimetrically determined cell mass concentration was used together with the fluorescence spectra for calibration and validation...

  20. Live-cell mass profiling: an emerging approach in quantitative biophysics.

    Science.gov (United States)

    Zangle, Thomas A; Teitell, Michael A

    2014-12-01

    Cell mass, volume and growth rate are tightly controlled biophysical parameters in cellular development and homeostasis, and pathological cell growth defines cancer in metazoans. The first measurements of cell mass were made in the 1950s, but only recently have advances in computer science and microfabrication spurred the rapid development of precision mass-quantifying approaches. Here we discuss available techniques for quantifying the mass of single live cells with an emphasis on relative features, capabilities and drawbacks for different applications.

  1. Fuel cell collector plates with improved mass transfer channels

    Science.gov (United States)

    Gurau, Vladimir; Barbir, Frano; Neutzler, Jay K.

    2003-04-22

    A fuel cell collector plate can be provided with one or more various channel constructions for the transport of reactants to the gas diffusion layer and the removal of water therefrom. The outlet channel can be arranged to have a reduced volume compared to the inlet channel, in both interdigitated and discontinuous spiral applications. The land width between an inlet channel and outlet channel can be reduced to improved mass flow rate in regions of deleted reactant concentrations. Additionally or alternatively, the depth of the inlet channel can be reduced in the direction of flow to reduce the diffusion path as the concentration of reactant is reduced.

  2. Intracellular water exchange for measuring the dry mass, water mass and changes in chemical composition of living cells.

    Directory of Open Access Journals (Sweden)

    Francisco Feijó Delgado

    Full Text Available We present a method for direct non-optical quantification of dry mass, dry density and water mass of single living cells in suspension. Dry mass and dry density are obtained simultaneously by measuring a cell's buoyant mass sequentially in an H2O-based fluid and a D2O-based fluid. Rapid exchange of intracellular H2O for D2O renders the cell's water content neutrally buoyant in both measurements, and thus the paired measurements yield the mass and density of the cell's dry material alone. Utilizing this same property of rapid water exchange, we also demonstrate the quantification of intracellular water mass. In a population of E. coli, we paired these measurements to estimate the percent dry weight by mass and volume. We then focused on cellular dry density - the average density of all cellular biomolecules, weighted by their relative abundances. Given that densities vary across biomolecule types (RNA, DNA, protein, we investigated whether we could detect changes in biomolecular composition in bacteria, fungi, and mammalian cells. In E. coli, and S. cerevisiae, dry density increases from stationary to exponential phase, consistent with previously known increases in the RNA/protein ratio from up-regulated ribosome production. For mammalian cells, changes in growth conditions cause substantial shifts in dry density, suggesting concurrent changes in the protein, nucleic acid and lipid content of the cell.

  3. Paper-Based Electrochemical Cell Coupled to Mass Spectrometry.

    Science.gov (United States)

    Liu, Yao-Min; Perry, Richard H

    2015-10-01

    On-line coupling of electrochemistry (EC) to mass spectrometry (MS) is a powerful approach for identifying intermediates and products of EC reactions in situ. In addition, EC transformations have been used to increase ionization efficiency and derivatize analytes prior to MS, improving sensitivity and chemical specificity. Recently, there has been significant interest in developing paper-based electroanalytical devices as they offer convenience, low cost, versatility, and simplicity. This report describes the development of tubular and planar paper-based electrochemical cells (P-EC) coupled to sonic spray ionization (SSI) mass spectrometry (P-EC/SSI-MS). The EC cells are composed of paper sandwiched between two mesh stainless steel electrodes. Analytes and reagents can be added directly to the paper substrate along with electrolyte, or delivered via the SSI microdroplet spray. The EC cells are decoupled from the SSI source, allowing independent control of electrical and chemical parameters. We utilized P-EC/SSI-MS to characterize various EC reactions such as oxidations of cysteine, dopamine, polycyclic aromatic hydrocarbons, and diphenyl sulfide. Our results show that P-EC/SSI-MS has the ability to increase ionization efficiency, to perform online EC transformations, and to capture intermediates of EC reactions with a response time on the order of hundreds of milliseconds. The short response time allowed detection of a deprotonated diphenyl sulfide intermediate, which experimentally confirms a previously proposed mechanism for EC oxidation of diphenyl sulfide to pseudodimer sulfonium ion. This report introduces paper-based EC/MS via development of two device configurations (tubular and planar electrodes), as well as discusses the capabilities, performance, and limitations of the technique.

  4. Detection of viable toxigenic Vibrio cholerae and virulent Shigella ...

    African Journals Online (AJOL)

    A rapid and sensitive assay was developed for the detection of low numbers of viable Vibrio cholerae and Shigella spp. cells in environmental and drinking water samples. Water samples were filtered, and the filters were enriched in a non-selective medium. The enrichment cultures were prepared for polymerase chain ...

  5. Detection of viable toxigenic Vibrio cholerae and virulent Shigella ...

    African Journals Online (AJOL)

    DRINIE

    2003-04-02

    Apr 2, 2003 ... A rapid and sensitive assay was developed for the detection of low numbers of viable Vibrio cholerae and Shigella spp. cells in environmental and drinking water samples. Water samples were filtered, and the filters were enriched in a non-selective medium. The enrichment cultures were prepared for ...

  6. Bimetric gravity is cosmologically viable

    Directory of Open Access Journals (Sweden)

    Yashar Akrami

    2015-09-01

    Full Text Available Bimetric theory describes gravitational interactions in the presence of an extra spin-2 field. Previous work has suggested that its cosmological solutions are generically plagued by instabilities. We show that by taking the Planck mass for the second metric, Mf, to be small, these instabilities can be pushed back to unobservably early times. In this limit, the theory approaches general relativity with an effective cosmological constant which is, remarkably, determined by the spin-2 interaction scale. This provides a late-time expansion history which is extremely close to ΛCDM, but with a technically-natural value for the cosmological constant. We find Mf should be no larger than the electroweak scale in order for cosmological perturbations to be stable by big-bang nucleosynthesis. We further show that in this limit the helicity-0 mode is no longer strongly-coupled at low energy scales.

  7. Single-Cell Mass Cytometry Analysis of Human Tonsil T Cell Remodeling by Varicella Zoster Virus

    Directory of Open Access Journals (Sweden)

    Nandini Sen

    2014-07-01

    Full Text Available Although pathogens must infect differentiated host cells that exhibit substantial diversity, documenting the consequences of infection against this heterogeneity is challenging. Single-cell mass cytometry permits deep profiling based on combinatorial expression of surface and intracellular proteins. We used this method to investigate varicella-zoster virus (VZV infection of tonsil T cells, which mediate viral transport to skin. Our results indicate that VZV induces a continuum of changes regardless of basal phenotypic and functional T cell characteristics. Contrary to the premise that VZV selectively infects T cells with skin trafficking profiles, VZV infection altered T cell surface proteins to enhance or induce these properties. Zap70 and Akt signaling pathways that trigger such surface changes were activated in VZV-infected naive and memory cells by a T cell receptor (TCR-independent process. Single-cell mass cytometry is likely to be broadly relevant for demonstrating how intracellular pathogens modulate differentiated cells to support pathogenesis in the natural host.

  8. Radiomanganese PET Detects Changes in Functional β-cell Mass in Mouse Models of Diabetes

    DEFF Research Database (Denmark)

    Hernandez, Reinier; Graves, Stephen A.; Gregg, Trillian

    2017-01-01

    from pancreatic sections. 52Mn2+-PET also reported the expected increase in functional β-cell mass in the ob/ob model of pre-type 2 diabetes, a result corroborated by histological β-cell mass measurements and live-cell imaging of β-cell Ca2+ oscillations. These results indicate that 52Mn2+-PET...

  9. Mice carrying a complete deletion of the talin2 coding sequence are viable and fertile

    Energy Technology Data Exchange (ETDEWEB)

    Debrand, Emmanuel; Conti, Francesco J.; Bate, Neil; Spence, Lorraine; Mazzeo, Daniela; Pritchard, Catrin A.; Monkley, Susan J. [Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN (United Kingdom); Critchley, David R., E-mail: drc@le.ac.uk [Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN (United Kingdom)

    2012-09-21

    Highlights: Black-Right-Pointing-Pointer Mice lacking talin2 are viable and fertile with only a mildly dystrophic phenotype. Black-Right-Pointing-Pointer Talin2 null fibroblasts show no major defects in proliferation, adhesion or migration. Black-Right-Pointing-Pointer Maintaining a colony of talin2 null mice is difficult indicating an underlying defect. -- Abstract: Mice homozygous for several Tln2 gene targeted alleles are viable and fertile. Here we show that although the expression of talin2 protein is drastically reduced in muscle from these mice, other tissues continue to express talin2 albeit at reduced levels. We therefore generated a Tln2 allele lacking the entire coding sequence (Tln2{sup cd}). Tln2{sup cd/cd} mice were viable and fertile, and the genotypes of Tln2{sup cd/+} intercrosses were at the expected Mendelian ratio. Tln2{sup cd/cd} mice showed no major difference in body mass or the weight of the major organs compared to wild-type, although they displayed a mildly dystrophic phenotype. Moreover, Tln2{sup cd/cd} mouse embryo fibroblasts showed no obvious defects in cell adhesion, migration or proliferation. However, the number of Tln2{sup cd/cd} pups surviving to adulthood was variable suggesting that such mice have an underlying defect.

  10. Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses.

    Science.gov (United States)

    Cho, Myung Soo; Kim, Sang Jin; Ku, Seung-Yup; Park, Jung Hyun; Lee, Haksup; Yoo, Dae Hoon; Park, Un Chul; Song, Seul Ae; Choi, Young Min; Yu, Hyeong Gon

    2012-09-01

    Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Pure Quantum Interpretations Are not Viable

    Science.gov (United States)

    Schmelzer, I.

    2011-02-01

    Pure interpretations of quantum theory, which throw away the classical part of the Copenhagen interpretation without adding new structure to its quantum part, are not viable. This is a consequence of a non-uniqueness result for the canonical operators.

  12. Therapeutic Approaches for Preserving or Restoring Pancreatic β-Cell Function and Mass

    Directory of Open Access Journals (Sweden)

    Kyong Yeun Jung

    2014-12-01

    Full Text Available The goal for the treatment of patients with diabetes has today shifted from merely reducing glucose concentrations to preventing the natural decline in β-cell function and delay the progression of disease. Pancreatic β-cell dysfunction and decreased β-cell mass are crucial in the development of diabetes. The β-cell defects are the main pathogenesis in patients with type 1 diabetes and are associated with type 2 diabetes as the disease progresses. Recent studies suggest that human pancreatic β-cells have a capacity for increased proliferation according to increased demands for insulin. In humans, β-cell mass has been shown to increase in patients showing insulin-resistance states such as obesity or in pregnancy. This capacity might be useful for identifying new therapeutic strategies to reestablish a functional β-cell mass. In this context, therapeutic approaches designed to increase β-cell mass might prove a significant way to manage diabetes and prevent its progression. This review describes the various β-cell defects that appear in patients with diabetes and outline the mechanisms of β-cell failure. We also review common methods for assessing β-cell function and mass and methodological limitations in vivo. Finally, we discuss the current therapeutic approaches to improve β-cell function and increase β-cell mass.

  13. Identification of Viable Helicobacter pylori in Drinking Water Supplies by Cultural and Molecular Techniques.

    Science.gov (United States)

    Santiago, Paula; Moreno, Yolanda; Ferrús, M Antonía

    2015-08-01

    Helicobacter pylori is one of the most common causes of chronic bacterial infection in humans, directly related to peptic ulcer and gastric cancer. It has been suggested that H. pylori can be acquired through different transmission routes, including water. In this study, culture and qPCR were used to detect and identify the presence of H. pylori in drinking water. Furthermore, the combined techniques PMA-qPCR and DVC-FISH were applied for detection of viable cells of H. pylori. Among 24 drinking water samples, 16 samples were positive for the presence of H. pylori, but viable cells were only detected in six samples. Characteristic colonies, covered by a mass of bacterial unspecific growth, were observed on selective agar plates from an only sample, after enrichment. The mixed culture was submitted to DVC-FISH and qPCR analysis, followed by sequencing of the amplicons. Molecular techniques confirmed the growth of H. pylori on the agar plate. Our results demonstrate for the first time that H. pylori can survive and be potentially infective in drinking water, showing that water distribution systems could be a potential route for H. pylori transmission. © 2015 John Wiley & Sons Ltd.

  14. Grazing of particle-associated bacteria-an elimination of the non-viable fraction.

    Science.gov (United States)

    Gonsalves, Maria-Judith; Fernandes, Sheryl Oliveira; Priya, Madasamy Lakshmi; LokaBharathi, Ponnapakkam Adikesavan

    Quantification of bacteria being grazed by microzooplankton is gaining importance since they serve as energy subsidies for higher trophic levels which consequently influence fish production. Hence, grazing pressure on viable and non-viable fraction of free and particle-associated bacteria in a tropical estuary controlled mainly by protist grazers was estimated using the seawater dilution technique. In vitro incubations over a period of 42h showed that at the end of 24h, growth coefficient (k) of particle-associated bacteria was 9 times higher at 0.546 than that of free forms. Further, 'k' value of viable cells on particles was double that of free forms at 0.016 and 0.007, respectively. While bacteria associated with particles were grazed (coefficient of removal (g)=0.564), the free forms were relatively less grazed indicating that particle-associated bacteria were exposed to grazers in these waters. Among the viable and non-viable forms, 'g' of non-viable fraction (particle-associated bacteria=0.615, Free=0.0086) was much greater than the viable fraction (particle-associated bacteria=0.056, Free=0.068). Thus, grazing on viable cells was relatively low in both the free and attached states. These observations suggest that non-viable forms of particle-associated bacteria were more prone to grazing and were weeded out leaving the viable cells to replenish the bacterial standing stock. Particle colonization could thus be a temporary refuge for the "persistent variants" where the viable fraction multiply and release their progeny. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  15. Using Generic Examples to Make Viable Arguments

    Science.gov (United States)

    Adams, Anne E.; Ely, Rob; Yopp, David

    2017-01-01

    The twenty-first century has seen an increased call to train students to craft mathematical arguments. The third of the Common Core's (CCSS) Standards for Mathematical Practice (SMP 3) (CCSSI 2010) calls for all mathematically proficient students to "construct viable arguments" to support the truth of their ideas and to "critique…

  16. Removal of viable bacteria and endotoxins by Electro Deionization (EDI).

    Science.gov (United States)

    Harada, Norimitsu; Otomo, Teruo; Watabe, Tomoichi; Ase, Tomonobu; Takemura, Takuto; Sato, Toshio

    2011-09-01

    Viable bacteria and endotoxins in water sometimes cause problems for human health. Endotoxins are major components of the outer cell wall of gram-negative bacteria (lipopolysaccharides). In medical procedures, especially haemodialysis (HD) and related therapies (haemodiafiltration (HDF), haemofiltration (HF)), endotoxins in the water for haemodialysis can permeate through the haemodialysis membrane and cause microinflammation or various haemodialysis-related illnesses. To decrease such a biological risk, RO and UF membranes are generally used. Also, hot water disinfection or the chemical disinfection is regularly executed to kill bacteria which produce endotoxins. However, simple treatment methods and equipment may be able to decrease the biological risk more efficiently. In our experiments, we confirmed that viable bacteria and endotoxins were removed by Electro Deionization (EDI) technology and also clarified the desorption mechanisms.

  17. Cdk4 regulates recruitment of quiescent beta-cells and ductal epithelial progenitors to reconstitute beta-cell mass.

    Directory of Open Access Journals (Sweden)

    Ji-Hyeon Lee

    2010-01-01

    Full Text Available Insulin-producing pancreatic islet beta cells (beta-cells are destroyed, severely depleted or functionally impaired in diabetes. Therefore, replacing functional beta-cell mass would advance clinical diabetes management. We have previously demonstrated the importance of Cdk4 in regulating beta-cell mass. Cdk4-deficient mice display beta-cell hypoplasia and develop diabetes, whereas beta-cell hyperplasia is observed in mice expressing an active Cdk4R24C kinase. While beta-cell replication appears to be the primary mechanism responsible for beta-cell mass increase, considerable evidence also supports a contribution from the pancreatic ductal epithelium in generation of new beta-cells. Further, while it is believed that majority of beta-cells are in a state of 'dormancy', it is unclear if and to what extent the quiescent cells can be coaxed to participate in the beta-cell regenerative response. Here, we address these queries using a model of partial pancreatectomy (PX in Cdk4 mutant mice. To investigate the kinetics of the regeneration process precisely, we performed DNA analog-based lineage-tracing studies followed by mathematical modeling. Within a week after PX, we observed considerable proliferation of islet beta-cells and ductal epithelial cells. Interestingly, the mathematical model showed that recruitment of quiescent cells into the active cell cycle promotes beta-cell mass reconstitution in the Cdk4R24C pancreas. Moreover, within 24-48 hours post-PX, ductal epithelial cells expressing the transcription factor Pdx-1 dramatically increased. We also detected insulin-positive cells in the ductal epithelium along with a significant increase of islet-like cell clusters in the Cdk4R24C pancreas. We conclude that Cdk4 not only promotes beta-cell replication, but also facilitates the activation of beta-cell progenitors in the ductal epithelium. In addition, we show that Cdk4 controls beta-cell mass by recruiting quiescent cells to enter the cell

  18. Monotone viable trajectories for functional differential inclusions

    Science.gov (United States)

    Haddad, Georges

    This paper is a study on functional differential inclusions with memory which represent the multivalued version of retarded functional differential equations. The main result gives a necessary and sufficient equations. The main result gives a necessary and sufficient condition ensuring the existence of viable trajectories; that means trajectories remaining in a given nonempty closed convex set defined by given constraints the system must satisfy to be viable. Some motivations for this paper can be found in control theory where F( t, φ) = { f( t, φ, u)} uɛU is the set of possible velocities of the system at time t, depending on the past history represented by the function φ and on a control u ranging over a set U of controls. Other motivations can be found in planning procedures in microeconomics and in biological evolutions where problems with memory do effectively appear in a multivalued version. All these models require viability constraints represented by a closed convex set.

  19. Mass Cytometry for Detection of Silver at the Bacterial Single Cell Level

    Directory of Open Access Journals (Sweden)

    Yuting Guo

    2017-07-01

    Full Text Available Background: Mass cytometry (Cytometry by Time of Flight, CyTOF allows single-cell characterization on the basis of specific metal-based cell markers. In addition, other metals in the mass range such as silver can be detected per cell. Bacteria are known to be sensible to silver and a protocol was developed to measure both the number of affected cells per population and the quantities of silver per cell.Methods: For mass cytometry ruthenium red was used as a marker for all cells of a population while parallel application of cisplatin discriminated live from dead cells. Silver quantities per cell and frequencies of silver containing cells in a population were measured by mass cytometry. In addition, live/dead subpopulations were analyzed by flow cytometry and distinguished by cell sorting based on ruthenium red and propidium iodide double staining. Verification of the cells’ silver load was performed on the bulk level by using ICP-MS in combination with cell sorting. The protocol was developed by conveying both, fast and non-growing Pseudomonas putida cells as test organisms.Results: A workflow for labeling bacteria in order to be analyzed by mass cytometry was developed. Three different parameters were tested: ruthenium red provided counts for all bacterial cells in a population while consecutively applied cisplatin marked the frequency of dead cells. Apparent population heterogeneity was detected by different frequencies of silver containing cells. Silver quantities per cell were also well measurable. Generally, AgNP-10 treatment caused higher frequencies of dead cells, higher frequencies of silver containing cells and higher per-cell silver quantities. Due to an assumed chemical equilibrium of free and bound silver ions live and dead cells were associated with silver in equal quantities and this preferably during exponential growth. With ICP-MS up to 1.5 fg silver per bacterial cell were detected.Conclusion: An effective mass cytometry

  20. Ethnic Similarities and Differences in the Relationship between Beta Cell Mass and Diabetes

    Directory of Open Access Journals (Sweden)

    Jun Inaishi

    2017-11-01

    Full Text Available Recent evidence has revealed that a change of functional beta cell mass is an essential factor of the pathophysiology of type 2 diabetes (T2DM. Since beta cell dysfunction is not only present in T2DM but also progressively worsens with disease duration, to preserve or recover functional beta cell mass is important in both prevention of the development of T2DM and therapeutic strategies for T2DM. Furthermore, ethnic difference in functional beta cell mass may also need to be taken into account. Recent evidences suggest that Asians have less beta cell functional capacity compared with Caucasians. Preservation or recovery of functional beta cell mass seems to be further emphasized for Asians because of the limited capacity of beta cell. This review summarizes the current knowledge on beta cell dysfunction in T2DM and discusses the similarities and differences in functional beta cell mass between ethnicities in the face of obesity and T2DM.

  1. Transgenic overexpression of active calcineurin in beta-cells results in decreased beta-cell mass and hyperglycemia.

    Directory of Open Access Journals (Sweden)

    Ernesto Bernal-Mizrachi

    2010-08-01

    Full Text Available Glucose modulates beta-cell mass and function through an initial depolarization and Ca(2+ influx, which then triggers a number of growth regulating signaling pathways. One of the most important downstream effectors in Ca(2+ signaling is the calcium/Calmodulin activated serine threonine phosphatase, calcineurin. Recent evidence suggests that calcineurin/NFAT is essential for beta-cell proliferation, and that in its absence loss of beta-cells results in diabetes. We hypothesized that in contrast, activation of calcineurin might result in expansion of beta-cell mass and resistance to diabetes.To determine the role of activation of calcineurin signaling in the regulation of pancreatic beta-cell mass and proliferation, we created mice that expressed a constitutively active form of calcineurin under the insulin gene promoter (caCn(RIP. To our surprise, these mice exhibited glucose intolerance. In vitro studies demonstrated that while the second phase of Insulin secretion is enhanced, the overall insulin secretory response was conserved. Islet morphometric studies demonstrated decreased beta-cell mass suggesting that this was a major component responsible for altered Insulin secretion and glucose intolerance in caCn(RIP mice. The reduced beta-cell mass was accompanied by decreased proliferation and enhanced apoptosis.Our studies identify calcineurin as an important factor in controlling glucose homeostasis and indicate that chronic depolarization leading to increased calcineurin activity may contribute, along with other genetic and environmental factors, to beta-cell dysfunction and diabetes.

  2. Hypoglycemia Reduces Vascular Endothelial Growth Factor A Production by Pancreatic Beta Cells as a Regulator of Beta Cell Mass*

    Science.gov (United States)

    Xiao, Xiangwei; Guo, Ping; Chen, Zean; El-Gohary, Yousef; Wiersch, John; Gaffar, Iljana; Prasadan, Krishna; Shiota, Chiyo; Gittes, George K.

    2013-01-01

    VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need. PMID:23378532

  3. Hypoglycemia reduces vascular endothelial growth factor A production by pancreatic beta cells as a regulator of beta cell mass.

    Science.gov (United States)

    Xiao, Xiangwei; Guo, Ping; Chen, Zean; El-Gohary, Yousef; Wiersch, John; Gaffar, Iljana; Prasadan, Krishna; Shiota, Chiyo; Gittes, George K

    2013-03-22

    VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need.

  4. Laparoscopic resection of a residual retroperitoneal tumor mass of nonseminomatous testicular germ cell tumors

    NARCIS (Netherlands)

    Ozturk, Cigdem; van Ginkel, Robert J.; Krol, Ruby M.; Gietema, Jourik A.; Hofker, Hendrik S.; Hoekstra, Harald J.

    Resection of a residual retroperitoneal tumor mass (RRRTM) is standard procedure after combination chemotherapy for metastatic nonseminomatous testicular germ cell tumors (NSTGCT). At the University Medical Center Groningen, 79 consecutive patients with disseminated NSTGCT were treated with

  5. Identity and Diversity of Human Peripheral Th and T Regulatory Cells Defined by Single-Cell Mass Cytometry.

    Science.gov (United States)

    Kunicki, Matthew A; Amaya Hernandez, Laura C; Davis, Kara L; Bacchetta, Rosa; Roncarolo, Maria-Grazia

    2018-01-01

    Human CD3+CD4+ Th cells, FOXP3+ T regulatory (Treg) cells, and T regulatory type 1 (Tr1) cells are essential for ensuring peripheral immune response and tolerance, but the diversity of Th, Treg, and Tr1 cell subsets has not been fully characterized. Independent functional characterization of human Th1, Th2, Th17, T follicular helper (Tfh), Treg, and Tr1 cells has helped to define unique surface molecules, transcription factors, and signaling profiles for each subset. However, the adequacy of these markers to recapitulate the whole CD3+CD4+ T cell compartment remains questionable. In this study, we examined CD3+CD4+ T cell populations by single-cell mass cytometry. We characterize the CD3+CD4+ Th, Treg, and Tr1 cell populations simultaneously across 23 memory T cell-associated surface and intracellular molecules. High-dimensional analysis identified several new subsets, in addition to the already defined CD3+CD4+ Th, Treg, and Tr1 cell populations, for a total of 11 Th cell, 4 Treg, and 1 Tr1 cell subsets. Some of these subsets share markers previously thought to be selective for Treg, Th1, Th2, Th17, and Tfh cells, including CD194 (CCR4)+FOXP3+ Treg and CD183 (CXCR3)+T-bet+ Th17 cell subsets. Unsupervised clustering displayed a phenotypic organization of CD3+CD4+ T cells that confirmed their diversity but showed interrelation between the different subsets, including similarity between Th1-Th2-Tfh cell populations and Th17 cells, as well as similarity of Th2 cells with Treg cells. In conclusion, the use of single-cell mass cytometry provides a systems-level characterization of CD3+CD4+ T cells in healthy human blood, which represents an important baseline reference to investigate abnormalities of different subsets in immune-mediated pathologies. Copyright © 2017 by The American Association of Immunologists, Inc.

  6. The Means: Cytometry and Mass Spectrometry Converge in a Single Cell Deep Profiling Platform

    Science.gov (United States)

    Weis-Garcia, Frances; Bandura, Dmitry; Baranov, Vladimir; Ornatsky, Olga; Tanner, Scott

    2013-01-01

    Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a distinct flavor of mass spectrometry that has had little association with cell biology: it remains the state of the art for the determination of the atomic composition of materials. Unrelatedly, flow cytometry is the superior method for distinguishing the heterogeneity of cells through the determination of antigen signatures using tagged antibodies. Simply replacing fluorophore tags with stable isotopes of the heavy metals, and measuring these cell-by-cell with ICP-MS, dramatically increases the number of probes that can be simultaneously measured in cytometry and enables a transformative increase in the resolution of rare cell populations in complex biological samples. While this can be thought of as a novel incarnation of single-cell targeted proteomics, the metal-labeling reagents, ICP-MS of single cells, and accompanying informatics comprise a new field of technology termed Mass Cytometry. While the conception of mass cytometry is simple the embodiment to address the issues of multi-parameter flow cytometry has been far more challenging. There are many elements, and many more stable isotopes of those elements, that might be used as distinct reporter tags. Still, there are many approaches to conjugating metals to antibodies (or other affinity reagents) and work in this area along with developing new applications is ongoing. The mass resolution and linear (quantitative) dynamic range of ICP-MS allows those many stable isotopes to be measured simultaneously and without the spectral overlap issues that limit fluorescence assay. However, the adaptation of ICP-MS to allow high-speed simultaneous measurement with single cell distinction at high throughput required innovation of the cell introduction system, ion optics (sampling, transmission and beam-shaping), mass analysis, and signal handling and processing. An overview of “the nuts and bolts” of Mass Cytometry is presented.

  7. Air-spore in Cartagena, Spain: viable and non-viable sampling methods.

    Science.gov (United States)

    Elvira-Rendueles, Belen; Moreno, Jose; Garcia-Sanchez, Antonio; Vergara, Nuria; Martinez-Garcia, Maria Jose; Moreno-Grau, Stella

    2013-01-01

    In the presented study the airborne fungal spores of the semiarid city of Cartagena, Spain, are identified and quantified by means of viable or non-viable sampling methods. Airborne fungal samples were collected simultaneously using a filtration method and a pollen and particle sampler based on the Hirst methodology. This information is very useful for elucidating geographical patterns of hay fever and asthma. The qualitative results showed that when the non-viable methodology was employed, Cladosporium, Ustilago, and Alternaria were the most abundant spores identified in the atmosphere of Cartagena, while the viable methodology showed that the most abundant taxa were: Cladosporium, Penicillium, Aspergillus and Alternaria. The quantitative results of airborne fungal spores identified by the Hirst-type air sampler (non-viable method), showed that Deuteromycetes represented 74% of total annual spore counts, Cladosporium being the major component of the fungal spectrum (62.2%), followed by Alternaria (5.3%), and Stemphylium (1.3%). The Basidiomycetes group represented 18.9% of total annual spore counts, Ustilago (7.1%) being the most representative taxon of this group and the second most abundant spore type. Ascomycetes accounted for 6.9%, Nectria (2.3%) being the principal taxon. Oomycetes (0.2%) and Zygomycestes and Myxomycestes (0.06%) were scarce. The prevailing species define our bioaerosol as typical of dry air. The viable methodology was better at identifying small hyaline spores and allowed for the discrimination of the genus of some spore types. However, non-viable methods revealed the richness of fungal types present in the bioaerosol. Thus, the use of both methodologies provides a more comprehensive characterization of the spore profile.

  8. Parejas viables que perduran en el tiempo

    OpenAIRE

    Juan José Cuervo Rodríguez

    2013-01-01

    El presente artículo científico presenta resultados del proceso llevado a cabo en el proyecto de investigación docente "Mecanismos de autorregulación en parejas viables que perduran en el tiempo". Se soporta en una mirada compleja de la psicología basada en una epistemología de la construcción. En el ámbito metodológico, se inscribe en los estudios de terapia familiar desde una perspectiva de la comunicación humana como un todo integrado. Participaron nueve parejas. Los criterios de inclusión...

  9. Relative quantitation in single-cell metabolomics by laser ablation electrospray mass spectrometry.

    Science.gov (United States)

    Shrestha, Bindesh; Vertes, Akos

    2014-01-01

    Single-cell analysis of metabolites by mass spectrometry (MS) is challenging due to the very limited volume and inherent molecular complexity of the sample. Quantitative metabolomic analysis of individual cells provides information on the metabolic heterogeneity of cells unattainable by aggregate analysis of multiple cells. Depending on the ionization method, MS can offer quantitative analysis for a broad class of metabolites exhibiting both high sensitivity and selectivity. Laser ablation electrospray ionization (LAESI) has been successfully exploited to analyze metabolites from broad range of biological samples, including single cells and small cell populations. In this work, we describe a protocol for the relative quantitation of metabolites in single cells by LAESI-mass spectrometry.

  10. A rapid biosensor for viable B. anthracis spores.

    Science.gov (United States)

    Baeumner, Antje J; Leonard, Barbara; McElwee, John; Montagna, Richard A

    2004-09-01

    A simple membrane-strip-based biosensor assay has been combined with a nucleic acid sequence-based amplification (NASBA) reaction for rapid (4 h) detection of a small number (ten) of viable B. anthracis spores. The biosensor is based on identification of a unique mRNA sequence from one of the anthrax toxin genes, the protective antigen ( pag), encoded on the toxin plasmid, pXO1, and thus provides high specificity toward B. anthracis. Previously, the anthrax toxins activator ( atxA) mRNA had been used in our laboratory for the development of a biosensor for the detection of a single B. anthracis spore within 12 h. Changing the target sequence to the pag mRNA provided the ability to shorten the overall assay time significantly. The vaccine strain of B. anthracis (Sterne strain) was used in all experiments. A 500-microL sample containing as few as ten spores was mixed with 500 microL growth medium and incubated for 30 min for spore germination and mRNA production. Thus, only spores that are viable were detected. Subsequently, RNA was extracted from lysed cells, selectively amplified using NASBA, and rapidly identified by the biosensor. While the biosensor assay requires only 15 min assay time, the overall process takes 4 h for detection of ten viable B. anthracis spores, and is shortened significantly if more spores are present. The biosensor is based on an oligonucleotide sandwich-hybridization assay format. It uses a membrane flow-through system with an immobilized DNA probe that hybridizes with the target sequence. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye sulforhodamine B. The amount of liposomes captured in the detection zone can be read visually or quantified with a hand-held reflectometer. The biosensor can detect as little as 1 fmol target mRNA (1 nmol L(-1)). Specificity analysis revealed no cross-reactivity with 11 organisms tested, among them closely

  11. Regulating the beta cell mass as a strategy for type-2 diabetes treatment.

    Science.gov (United States)

    Song, Imane; Muller, Christo; Louw, Johan; Bouwens, Luc

    2015-01-01

    The incidence of type 2 diabetes (T2D) increases dramatically worldwide and has created an enormous health care burden. Obesity, dyslipidemia and insulin resistance are major risk factors for the development of T2D, but the major factor leading to the disease is failure of the insulin-producing beta cell mass to compensate for increasing insulin demands of the body. Progression of the disease further diminishes the beta cell mass as a result of lipotoxicity and glucotoxicity for which beta cells are particularly sensitive. Hence, treatment aiming to prevent beta cell loss or increase the number of beta cells could inhibit diabetes progression or lead to restoration of normal metabolism. Whereas current and new antidiabetic drugs are mainly targeting insulin secretion and action or glucose uptake, newer interventions must be found that prevent beta cell loss or increase beta cell number. The targets for this are beta cell proliferation, neogenesis and survival. This review examines major evidence from animal experiments suggesting that it is feasible to regulate the beta cell mass by bioactive compounds like growth factors, cytokines, hormones, phytochemicals and small molecules. Often the mode of action remains unclear due to inadequate methods to assess the effects of the compounds on the beta cell dynamics. Furthermore, a major challenge is to identify compounds with sufficient specificity in order to avoid unwanted effects on other cell types. Provided such safety issues can be solved, this may provide a curative approach for diabetes treatment.

  12. Timing of Ca2+ response in pancreatic beta-cells is related to mitochondrial mass

    DEFF Research Database (Denmark)

    Gustavsson, N; Abedi, G; Larsson-Nyrén, G

    2006-01-01

    and Fluo-3 to study how the amount of active mitochondria is related to the lag-time and the magnitude of calcium response to 20mM glucose in isolated beta-cells and in cells within intact lean and ob/ob mouse islets. Results show that the mitochondrial mass is inversely correlated with the lag...

  13. Prospective clinical and radiographic evaluation of an allogeneic bone matrix containing stem cells (Trinity Evolution® Viable Cellular Bone Matrix) in patients undergoing two-level anterior cervical discectomy and fusion.

    Science.gov (United States)

    Peppers, Timothy A; Bullard, Dennis E; Vanichkachorn, Jed S; Stanley, Scott K; Arnold, Paul M; Waldorff, Erik I; Hahn, Rebekah; Atkinson, Brent L; Ryaby, James T; Linovitz, Raymond J

    2017-04-26

    Trinity Evolution® (TE), a viable cellular bone allograft, previously demonstrated high fusion rates and no safety-related concerns after single-level anterior cervical discectomy and fusion (ACDF) procedures. This prospective multicenter clinical study was performed to assess the radiographic and clinical outcomes of TE in subjects undergoing two-level ACDF procedures. In a prospective, multicenter study, 40 subjects that presented with symptomatic cervical degeneration at two adjacent vertebral levels underwent instrumented ACDF using TE autograft substitute in a polyetherethereketone (PEEK) cage. At 12 months, radiographic fusion status was evaluated by dynamic motion plain radiographs and thin cut CT with multiplanar reconstruction by a panel that was blinded to clinical outcome. Fusion success was defined by angular motion (≤4°) and the presence of bridging bone across the adjacent vertebral endplates. Clinical pain and function assessments included the Neck Disability Index (NDI), neck and arm pain as evaluated by visual analog scales (VAS), and SF-36 at both 6 and 12 months. At both 6 and 12 months, all clinical outcome scores (SF-36, NDI, and VAS pain) improved significantly (p Trinity Evolution in Anterior Cervical Disectomy and Fusion (ACDF) NCT00951938.

  14. Beyond Body Mass Index. Is the Body Cell Mass Index (BCMI) a useful prognostic factor to describe nutritional, inflammation and muscle mass status in hospitalized elderly?: Body Cell Mass Index links in elderly.

    Science.gov (United States)

    Rondanelli, Mariangela; Talluri, Jacopo; Peroni, Gabriella; Donelli, Chiara; Guerriero, Fabio; Ferrini, Krizia; Riggi, Emilia; Sauta, Elisabetta; Perna, Simone; Guido, Davide

    2017-03-24

    The aim of this study was to establish the effectiveness of Body Cell Mass Index (BCMI) as a prognostic index of (mal)nutrition, inflammation and muscle mass status in the elderly. A cross-sectional observational study has been conducted on 114 elderly patients (80 women and 34 men), with mean age equal to 81.07 ± 6.18 years. We performed a multivariate regression model by Structural Equation Modelling (SEM) framework. We detected the effects over a Mini Nutritional Assessment (MNA) stratification, by performing a multi-group multivariate regression model (via SEM) in two MNA nutritional strata, less and bigger (or equal) than 17. BCMI had a significant effect on albumin (β = +0.062, P = 0.001), adjusting for the other predictors of the model as Body Mass Index (BMI), age, sex, fat mass and cognitive condition. An analogous result is maintained in MNAmass (FFM) (β = +0.480, P Index (SMI) (β = +0.265, P < 0.001), assessed by DXA. BCMI also returned suggestive evidences (0.05 < P < 0.10) for both the effect on FFM and on SMI in overall sample. The main result of this study is that the BCMI, compared to BMI, proved to be significantly related to an important marker as albumin in geriatric population. Then, assessing the BCMI could be a valuable, inexpensive, easy to perform tool to investigate the inflammation status of elderly patients. Copyright © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

  15. Ultrastructural and molecular distinctions between the porcine inner cell mass and epiblast reveal unique pluripotent cell states

    DEFF Research Database (Denmark)

    Hall, V. J.; Jacobsen, Janus Valentin; Rasmussen, M. A.

    2010-01-01

    pluripotent cell population as it develops from the ICM to the late epiblast. The ultrastructural observations revealed that the outer cells of the ICM have a high nuclear:cytoplasmic ratio but are transcriptionally inactive and contain mitochondria with few cristae. In contrast, the epiblast cells have......Characterization of the pluripotent cell populations within the porcine embryo is essential for understanding pluripotency and self-renewal regulation in the inner cell mass (ICM) and epiblast. In this study, we perform detailed ultrastructural and molecular characterization of the developing...

  16. In situ metabolic profiling of single cells by laser ablation electrospray ionization mass spectrometry.

    Science.gov (United States)

    Shrestha, Bindesh; Vertes, Akos

    2009-10-15

    Depending on age, phase in the cell cycle, nutrition, and environmental factors, individual cells exhibit large metabolic diversity. To explore metabolic variations in cell populations, laser ablation electrospray ionization (LAESI) mass spectrometry (MS) was used for the in situ analysis of individual cells at atmospheric pressure. Single cell ablation was achieved by delivering mid-IR laser pulses through the etched tip of a GeO(2)-based glass fiber. Metabolic analysis was performed from single cells and small cell populations of Allium cepa and Narcissus pseudonarcissus bulb epidermis, as well as single eggs of Lytechinus pictus. Of the 332 peaks detected for A. cepa, 35 were assigned to metabolites with the help of accurate ion masses and tandem MS. The metabolic profiles from single cells of the two plant species included a large variety of oligosaccharides including possibly fructans in A. cepa, and alkaloids, e.g., lycorine in N. pseudonarcissus. Analysis of adjacent individual cells with a difference in pigmentation showed that, in addition to essential metabolites found in both variants, the pigmented cells contained anthocyanidins, other flavonoids, and their glucosides. Analysis of single epidermal cells from different scale leaves in an A. cepa bulb showed metabolic differences corresponding to their age. Our results indicate the feasibility of using LAESI-MS for the in situ analysis of metabolites in single cells with potential applications in studying cell differentiation, changes due to disease states, and response to xenobiotics.

  17. Fingerprinting Breast Cancer vs. Normal Mammary Cells by Mass Spectrometric Analysis of Volatiles

    Science.gov (United States)

    He, Jingjing; Sinues, Pablo Martinez-Lozano; Hollmén, Maija; Li, Xue; Detmar, Michael; Zenobi, Renato

    2014-06-01

    There is increasing interest in the development of noninvasive diagnostic methods for early cancer detection, to improve the survival rate and quality of life of cancer patients. Identification of volatile metabolic compounds may provide an approach for noninvasive early diagnosis of malignant diseases. Here we analyzed the volatile metabolic signature of human breast cancer cell lines versus normal human mammary cells. Volatile compounds in the headspace of conditioned culture medium were directly fingerprinted by secondary electrospray ionization-mass spectrometry. The mass spectra were subsequently treated statistically to identify discriminating features between normal vs. cancerous cell types. We were able to classify different samples by using feature selection followed by principal component analysis (PCA). Additionally, high-resolution mass spectrometry allowed us to propose their chemical structures for some of the most discriminating molecules. We conclude that cancerous cells can release a characteristic odor whose constituents may be used as disease markers.

  18. PMA-Linked Fluorescence for Rapid Detection of Viable Bacterial Endospores

    Science.gov (United States)

    LaDuc, Myron T.; Venkateswaran, Kasthuri; Mohapatra, Bidyut

    2012-01-01

    The most common approach for assessing the abundance of viable bacterial endospores is the culture-based plating method. However, culture-based approaches are heavily biased and oftentimes incompatible with upstream sample processing strategies, which make viable cells/spores uncultivable. This shortcoming highlights the need for rapid molecular diagnostic tools to assess more accurately the abundance of viable spacecraft-associated microbiota, perhaps most importantly bacterial endospores. Propidium monoazide (PMA) has received a great deal of attention due to its ability to differentiate live, viable bacterial cells from dead ones. PMA gains access to the DNA of dead cells through compromised membranes. Once inside the cell, it intercalates and eventually covalently bonds with the double-helix structures upon photoactivation with visible light. The covalently bound DNA is significantly altered, and unavailable to downstream molecular-based manipulations and analyses. Microbiological samples can be treated with appropriate concentrations of PMA and exposed to visible light prior to undergoing total genomic DNA extraction, resulting in an extract comprised solely of DNA arising from viable cells. This ability to extract DNA selectively from living cells is extremely powerful, and bears great relevance to many microbiological arenas.

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

    Science.gov (United States)

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

    2011-12-21

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

  20. Roots of success: cultivating viable community forestry

    Energy Technology Data Exchange (ETDEWEB)

    MacQueen, Duncan

    2009-05-15

    Is community forestry emerging from the shadows? The evidence shows that locally controlled enterprises can be economically viable, and often build on stronger social and environmental foundations than the big private-sector players. Certainly this is an industry in need of a shakeup. Many forests have become flashpoints where agro-industry, large-scale logging concerns and conservation interests clash, while forest-dependent communities are left out in the cold. Meanwhile, governments – driven by concerns over the climate impacts of deforestation – are having to gear up for legal, sustainable forestry production. Community forestry could be crucial to solving many of these challenges. By building on local core capabilities and developing strategic partnerships, they are forging key new business models that could transform the sector.

  1. Uptake, efflux, and mass transfer coefficient of fluorescent PAMAM dendrimers into pancreatic cancer cells.

    Science.gov (United States)

    Opitz, Armin W; Czymmek, Kirk J; Wickstrom, Eric; Wagner, Norman J

    2013-02-01

    Targeted delivery of imaging agents to cells can be optimized with the understanding of uptake and efflux rates. Cellular uptake of macromolecules is studied frequently with fluorescent probes. We hypothesized that the internalization and efflux of fluorescently labeled macromolecules into and out of mammalian cells could be quantified by confocal microscopy to determine the rate of uptake and efflux, from which the mass transfer coefficient is calculated. The cellular influx and efflux of a third generation poly(amido amine) (PAMAM) dendrimer labeled with an Alexa Fluor 555 dye was measured in Capan-1 pancreatic cancer cells using confocal fluorescence microscopy. The Capan-1 cells were also labeled with 5-chloromethylfluorescein diacetate (CMFDA) green cell tracker dye to delineate cellular boundaries. A dilution curve of the fluorescently labeled PAMAM dendrimer enabled quantification of the concentration of dendrimer in the cell. A simple mass transfer model described the uptake and efflux behavior of the PAMAM dendrimer. The effective mass transfer coefficient was found to be 0.054±0.043μm/min, which corresponds to a rate constant of 0.035±0.023min(-1) for uptake of the PAMAM dendrimer into the Capan-1 cells. The effective mass transfer coefficient was shown to predict the efflux behavior of the PAMAM dendrimer from the cell if the fraction of labeled dendrimer undergoing non-specific binding is accounted for. This work introduces a novel method to quantify the mass transfer behavior of fluorescently labeled macromolecules into mammalian cells. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Deletion of the mitochondrial flavoprotein apoptosis inducing factor (AIF induces beta-cell apoptosis and impairs beta-cell mass.

    Directory of Open Access Journals (Sweden)

    Fabienne T Schulthess

    Full Text Available BACKGROUND: Apoptosis is a hallmark of beta-cell death in both type 1 and type 2 diabetes mellitus. Understanding how apoptosis contributes to beta-cell turnover may lead to strategies to prevent progression of diabetes. A key mediator of apoptosis, mitochondrial function, and cell survival is apoptosis inducing factor (AIF. In the present study, we investigated the role of AIF on beta-cell mass and survival using the Harlequin (Hq mutant mice, which are hypomorphic for AIF. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical evaluation of pancreata from Hq mutant mice displayed much smaller islets compared to wild-type mice (WT. Analysis of beta-cell mass in these mice revealed a greater than 4-fold reduction in beta-cell mass together with an 8-fold increase in beta-cell apoptosis. Analysis of cell cycle dynamics, using BrdU pulse as a marker for cells in S-phase, did not detect significant differences in the frequency of beta-cells in S-phase. In contrast, double staining for phosphorylated Histone H3 and insulin showed a 3-fold increase in beta-cells in the G2 phase in Hq mutant mice, but no differences in M-phase compared to WT mice. This suggests that the beta-cells from Hq mutant mice are arrested in the G2 phase and are unlikely to complete the cell cycle. beta-cells from Hq mutant mice display increased sensitivity to hydrogen peroxide-induced apoptosis, which was confirmed in human islets in which AIF was depleted by siRNA. AIF deficiency had no effect on glucose stimulated insulin secretion, but the impaired effect of hydrogen peroxide on beta-cell function was potentiated. CONCLUSIONS/SIGNIFICANCE: Our results indicate that AIF is essential for maintaining beta-cell mass and for oxidative stress response. A decrease in the oxidative phosphorylation capacity may counteract the development of diabetes, despite its deleterious effects on beta-cell survival.

  3. Polymerase chain reaction-based discrimination of viable from non-viable Mycoplasma gallisepticum

    Directory of Open Access Journals (Sweden)

    Ching Giap Tan

    2014-02-01

    Full Text Available The present study was based on the reverse transcription polymerase chain reaction (RT-PCR of the 16S ribosomal nucleic acid (rRNA of Mycoplasma for detection of viable Mycoplasma gallisepticum. To determine the stability of M. gallisepticum 16S rRNA in vitro, three inactivation methods were used and the suspensions were stored at different temperatures. The 16S rRNA of M. gallisepticum was detected up to approximately 20–25 h at 37 °C, 22–25 h at 16 °C, and 23–27 h at 4 °C. The test, therefore, could detect viable or recently dead M. gallisepticum (< 20 h. The RT-PCR method was applied during an in vivo study of drug efficacy under experimental conditions, where commercial broiler-breeder eggs were inoculated with M. gallisepticum into the yolk. Hatched chicks that had been inoculated in ovo were treated with Macrolide 1. The method was then applied in a flock of day 0 chicks with naturally acquired vertical transmission of M. gallisepticum, treated with Macrolide 2. Swabs of the respiratory tract were obtained for PCR and RT-PCR evaluations to determine the viability of M. gallisepticum. This study proved that the combination of both PCR and RT-PCR enables detection and differentiation of viable from non-viable M. gallisepticum.

  4. Intravascular large B-cell lymphoma with multi-organ failure presenting as a pancreatic mass: a case with atypical presentation and definite diagnosis postmortem.

    Science.gov (United States)

    Ronny, Faisal Mahmudul Huq; Black, Margaret Ann; Arbini, Arnaldo A

    2017-01-01

    Intravascular large B-cell lymphoma (IVLBCL) is a very rare extra nodal lymphoma that tends to proliferate within small blood vessels, particularly capillaries and postcapillary venules while sparing the organ parenchyma. The cause of its affinity for the vascular bed remains unknown. Because of its rarity and unremarkable clinical presentation, a timely diagnosis of IVLBCL is very challenging. Here, we describe a case of IVLBCL presenting as pancreatic mass that was ultimately diagnosed at autopsy. A 71-year-old Caucasian female presented with a 3-month history of fatigue, abdominal pain, and weight loss. She was referred to the emergency room with a new diagnosis of portal vein thrombosis and lactic acidosis. During her hospital course she was found to have a 1.9 × 1.8 cm lesion in the pancreatic tail on imaging; The cytologic specimen on the mass showed a high-grade lymphoma. A bone marrow biopsy showed no involvement. The patient's condition rapidly deteriorated and she, later, died due to multi-organ failure. An autopsy revealed diffuse intravascular invasion in multiple organs by the lymphoma cells. Based on our literature review-and to the best of our knowledge-there are virtually no reports describing the presentation of this lymphoma with a discernible tissue mass and associated multi-organ failure. The immunophenotypic studies performed revealed de novo CD5+ intravascular large B-cell lymphoma, which is known to be aggressive with very poor prognosis. Although it is a very rare lymphoma, it should be considered as a potential cause of multi-organ failure when no other cause has been identified. A prompt tissue diagnosis, appropriate high-dose chemotherapy and stem cell transplantation remain the only viable alternative to achieve some kind of remission.

  5. Giardia-filled Pancreatic Mass in a Patient With Recently Treated T-cell-rich B-cell Lymphoma.

    Science.gov (United States)

    Shah, Raj; Asif, Talal; Johnson, Richard

    2017-02-09

    Giardia lamblia (G. lamblia)-filled pancreatic masses are a rarely reported entity. Furthermore, there are only a few case reports in literature on the association of these masses with cancer. We present a case of a G. lamblia-filled pancreatic cystic mass in a patient with a history of T-cell-rich B-cell lymphoma. The authors performed a PubMed search using (Medical Subject Headings) MeSH terms of pancreas, mass, Giardia, and lymphoma.  A 53-year-old male with past medical and surgical history of T-cell-rich B-cell lymphoma, status post R-CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab) therapy with positron emission tomography (PET) scan showing no residual disease, essential hypertension, and alcohol use disorder presented to the emergency department (ED) with epigastric pain and nausea for one week. Computed tomography (CT) scan of the abdomen showed a 2.3 cm hypodense pancreatic cystic mass. This was a new finding when compared to his prior abdominal imaging. Fine needle aspiration (FNA) biopsy of the mass showed lymphocytes, reactive atypical epithelial cells, and numerous organisms consistent with Giardia lamblia. He was treated with metronidazole 250 mg by mouth three times a day (TID) for five days. Follow-up magnetic resonance imaging (MRI) and magnetic resonance cholangiopancreatography (MRCP) showed complete resolution of the pancreatic mass. There are only a few case reports on G. lamblia in the pancreas. The pathologist indicated sheets of numerous Giardia in the sample, making small bowel contamination less likely and G. lamblia aspirate from the pancreas more probable as the source. The authors hypothesize that this patient may have had chronic G. lamblia infection as a potential cause for the T-cell-rich B-cell lymphoma manifestation. The patient reported travel to an area with possible exposure to G. lamblia one year prior to presentation with the lymphoma. During that time he had increasing abdominal

  6. Effects of solution mass transport on the ECC ozonesonde background current. [Electrochemical Concentration Cell

    Science.gov (United States)

    Thornton, D. C.; Niazy, N.

    1983-01-01

    A technique is developed to measure the effective mass transport parameter for the electrochemical concentration cell (ECC) ozonesonde in order to determine the mass transport rate constant for the ECC as a function of pressure. It is shown that a pressure dependent factor in the background current originates in a convective mass transport parameter. It is determined that for atmospheric pressures greater than 100 mb the mass transport parameter is a constant, while at pressures less than 100 mb it decreases logarithmically with pressure. It is suggested that the background current correction is directly correlated to the mass transport parameter pressure dependence. The presently used background current correction, which is based on the partial pressure of oxygen, is found to lead to an overestimation of the integrated ozone value in the troposphere for the ECC ozonesonde data.

  7. Phospho-BAD BH3 Mimicry Protects β Cells and Restores Functional β Cell Mass in Diabetes

    Directory of Open Access Journals (Sweden)

    Sanda Ljubicic

    2015-02-01

    Full Text Available Strategies that simultaneously enhance the survival and glucose responsiveness of insulin-producing β cells will greatly augment β cell replacement therapies in type 1 diabetes (T1D. We show that genetic and pharmacologic mimetics of the phosphorylated BCL-2 homology 3 (BH3 domain of BAD impart β-cell-autonomous protective effects in the face of stress stimuli relevant to β cell demise in T1D. Importantly, these benefits translate into improved engraftment of donor islets in transplanted diabetic mice, increased β cell viability in islet grafts, restoration of insulin release, and diabetes reversal. Survival of β cells in this setting is not merely due to the inability of phospho-BAD to suppress prosurvival BCL-2 proteins but requires its activation of the glucose-metabolizing enzyme glucokinase. Thus, BAD phospho-BH3 mimetics may prove useful in the restoration of functional β cell mass in diabetes.

  8. Two-Dimensional Simulation of Mass Transfer in Unitized Regenerative Fuel Cells under Operation Mode Switching

    Directory of Open Access Journals (Sweden)

    Lulu Wang

    2016-01-01

    Full Text Available A two-dimensional, single-phase, isothermal, multicomponent, transient model is built to investigate the transport phenomena in unitized regenerative fuel cells (URFCs under the condition of switching from the fuel cell (FC mode to the water electrolysis (WE mode. The model is coupled with an electrochemical reaction. The proton exchange membrane (PEM is selected as the solid electrolyte of the URFC. The work is motivated by the need to elucidate the complex mass transfer and electrochemical process under operation mode switching in order to improve the performance of PEM URFC. A set of governing equations, including conservation of mass, momentum, species, and charge, are considered. These equations are solved by the finite element method. The simulation results indicate the distributions of hydrogen, oxygen, water mass fraction, and electrolyte potential response to the transient phenomena via saltation under operation mode switching. The hydrogen mass fraction gradients are smaller than the oxygen mass fraction gradients. The average mass fractions of the reactants (oxygen and hydrogen and product (water exhibit evident differences between each layer in the steady state of the FC mode. By contrast, the average mass fractions of the reactant (water and products (oxygen and hydrogen exhibit only slight differences between each layer in the steady state of the WE mode. Under either the FC mode or the WE mode, the duration of the transient state is only approximately 0.2 s.

  9. Beta cell count instead of beta cell mass to assess and localize growth in beta cell population following pancreatic duct ligation in mice.

    Directory of Open Access Journals (Sweden)

    Marie Chintinne

    Full Text Available BACKGROUND: Pancreatic-tail duct ligation (PDL in adult rodents has been reported to induce beta cell generation and increase beta cell mass but increases in beta cell number have not been demonstrated. This study examines whether PDL increases beta cell number and whether this is caused by neogenesis of small clusters and/or their growth to larger aggregates. METHODOLOGY: Total beta cell number and its distribution over small (100 µm clusters was determined in pancreatic tails of 10-week-old mice, 2 weeks after PDL or sham. PRINCIPAL FINDINGS: PDL increased total beta cell mass but not total beta cell number. It induced neogenesis of small beta cell clusters (2.2-fold higher number which contained a higher percent proliferating beta cells (1.9% Ki67+cells than sham tails (<0.2%; their higher beta cell number represented <5% of total beta cell number and was associated with a similar increase in alpha cell number. It is unknown whether the regenerative process is causally related to the inflammatory infiltration in PDL-tails. Human pancreases with inflammatory infiltration also exhibited activation of proliferation in small beta cell clusters. CONCLUSIONS/SIGNIFICANCE: The PDL model illustrates the advantage of direct beta cell counts over beta cell mass measurements when assessing and localizing beta cell regeneration in the pancreas. It demonstrates the ability of the adult mouse pancreas for neogenesis of small beta cell clusters with activated beta cell proliferation. Further studies should investigate conditions under which neoformed small beta cell clusters grow to larger aggregates and hence to higher total beta cell numbers.

  10. Research Techniques Made Simple: Experimental Methodology for Single-Cell Mass Cytometry.

    Science.gov (United States)

    Matos, Tiago R; Liu, Hongye; Ritz, Jerome

    2017-04-01

    Growing recognition of the complexity of interactions within cellular systems has fueled the development of mass cytometry. The precision of time-of-flight mass spectrometry combined with the labeling of specific ligands with mass tags enables detection and quantification of more than 40 markers at a single-cell resolution. The 135 available detection channels allow simultaneous study of additional characteristics of complex biological systems across millions of cells. Cutting-edge mass cytometry by time-of-flight (CyTOF) can profoundly affect our knowledge of cell population heterogeneity and hierarchy, cellular state, multiplexed signaling pathways, proteolysis products, and mRNA transcripts. Although CyTOF is currently scarcely used within the field of investigative dermatology, we aim to highlight CyTOF's utility and demystify the technique. CyTOF may, for example, uncover the immunological heterogeneity and differentiation of Langerhans cells, delineate the signaling pathways responsible for each phase of the hair cycle, or elucidate which proteolysis products from keratinocytes promote skin inflammation. However, the success of mass cytometry experiments depends on fully understanding the methods and how to control for variations when making comparisons between samples. Here, we review key experimental methods for CyTOF that enable accurate data acquisition by optimizing signal detection and minimizing background noise and sample-to-sample variation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Reverse transcriptase real-time PCR for detection and quantification of viable Campylobacter jejuni directly from poultry faecal samples

    DEFF Research Database (Denmark)

    Bui, Thanh Xuan; Wolff, Anders; Madsen, Mogens

    2012-01-01

    and quantification of viable Campylobacter jejuni directly from chicken faecal samples. The results of this method anda DNA-based quantitative real-time PCR (qPCR) method were compared with those of a bacterial culture method. Using bacterial culture andRT-qPCR methods, viable C. jejuni cells could be detected...

  12. Allogeneic cardiospheres delivered via percutaneous transendocardial injection increase viable myocardium, decrease scar size, and attenuate cardiac dilatation in porcine ischemic cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Kristine Yee

    Full Text Available Epicardial injection of heart-derived cell products is safe and effective post-myocardial infarction (MI, but clinically-translatable transendocardial injection has never been evaluated. We sought to assess the feasibility, safety and efficacy of percutaneous transendocardial injection of heart-derived cells in porcine chronic ischemic cardiomyopathy.We studied a total of 89 minipigs; 63 completed the specified protocols. After NOGA-guided transendocardial injection, we quantified engraftment of escalating doses of allogeneic cardiospheres or cardiosphere-derived cells in minipigs (n = 22 post-MI. Next, a dose-ranging, blinded, randomized, placebo-controlled ("dose optimization" study of transendocardial injection of the better-engrafting product was performed in infarcted minipigs (n = 16. Finally, the superior product and dose (150 million cardiospheres were tested in a blinded, randomized, placebo-controlled ("pivotal" study (n = 22. Contrast-enhanced cardiac MRI revealed that all cardiosphere doses preserved systolic function and attenuated remodeling. The maximum feasible dose (150 million cells was most effective in reducing scar size, increasing viable myocardium and improving ejection fraction. In the pivotal study, eight weeks post-injection, histopathology demonstrated no excess inflammation, and no myocyte hypertrophy, in treated minipigs versus controls. No alloreactive donor-specific antibodies developed over time. MRI showed reduced scar size, increased viable mass, and attenuation of cardiac dilatation with no effect on ejection fraction in the treated group compared to placebo.Dose-optimized injection of allogeneic cardiospheres is safe, decreases scar size, increases viable myocardium, and attenuates cardiac dilatation in porcine chronic ischemic cardiomyopathy. The decreases in scar size, mirrored by increases in viable myocardium, are consistent with therapeutic regeneration.

  13. The Conceptual Mechanism for Viable Organizational Learning Based on Complex System Theory and the Viable System Model

    Science.gov (United States)

    Sung, Dia; You, Yeongmahn; Song, Ji Hoon

    2008-01-01

    The purpose of this research is to explore the possibility of viable learning organizations based on identifying viable organizational learning mechanisms. Two theoretical foundations, complex system theory and viable system theory, have been integrated to provide the rationale for building the sustainable organizational learning mechanism. The…

  14. Deletion of ultraconserved elements yields viable mice

    Energy Technology Data Exchange (ETDEWEB)

    Ahituv, Nadav; Zhu, Yiwen; Visel, Axel; Holt, Amy; Afzal, Veena; Pennacchio, Len A.; Rubin, Edward M.

    2007-07-15

    Ultraconserved elements have been suggested to retainextended perfect sequence identity between the human, mouse, and ratgenomes due to essential functional properties. To investigate thenecessities of these elements in vivo, we removed four non-codingultraconserved elements (ranging in length from 222 to 731 base pairs)from the mouse genome. To maximize the likelihood of observing aphenotype, we chose to delete elements that function as enhancers in amouse transgenic assay and that are near genes that exhibit markedphenotypes both when completely inactivated in the mouse as well as whentheir expression is altered due to other genomic modifications.Remarkably, all four resulting lines of mice lacking these ultraconservedelements were viable and fertile, and failed to reveal any criticalabnormalities when assayed for a variety of phenotypes including growth,longevity, pathology and metabolism. In addition more targeted screens,informed by the abnormalities observed in mice where genes in proximityto the investigated elements had been altered, also failed to revealnotable abnormalities. These results, while not inclusive of all thepossible phenotypic impact of the deleted sequences, indicate thatextreme sequence constraint does not necessarily reflect crucialfunctions required for viability.

  15. Is Greenberg's "Macro-Carib" viable?

    Directory of Open Access Journals (Sweden)

    Spike Gildea

    Full Text Available In his landmark work Language in the Americas, Greenberg (1987 proposed that Macro-Carib was one of the major low-level stocks of South America, which together with Macro-Panoan and Macro-Ge-Bororo were claimed to comprise the putative Ge-Pano-Carib Phylum. His Macro-Carib includes the isolates Andoke and Kukura, and the Witotoan, Peba-Yaguan, and Cariban families. Greenberg's primary evidence came from person-marking paradigms in individual languages, plus scattered words from individual languages collected into 79 Macro-Carib 'etymologies' and another 64 Amerind 'etymologies'. The goal of this paper is to re-evaluate Greenberg's Macro-Carib claim in the light of the much more extensive and reliable language data that has become available largely since 1987. Based on full person-marking paradigms for Proto-Cariban, Yagua, Bora and Andoke, we conclude that Greenberg's morphological claims are unfounded. For our lexical comparison, we created lexical lists for Proto-Cariban, Proto-Witotoan, Yagua and Andoke, for both Greenberg's 143 putative etymologies and for the Swadesh 100 list. From both lists, a total of 23 potential cognates were found, but no consonantal correspondences were repeated even once. We conclude that our greatly expanded and improved database does not provide sufficient evidence to convince the skeptic that the Macro-Carib hypothesis is viable

  16. Economically viable large-scale hydrogen liquefaction

    Science.gov (United States)

    Cardella, U.; Decker, L.; Klein, H.

    2017-02-01

    The liquid hydrogen demand, particularly driven by clean energy applications, will rise in the near future. As industrial large scale liquefiers will play a major role within the hydrogen supply chain, production capacity will have to increase by a multiple of today’s typical sizes. The main goal is to reduce the total cost of ownership for these plants by increasing energy efficiency with innovative and simple process designs, optimized in capital expenditure. New concepts must ensure a manageable plant complexity and flexible operability. In the phase of process development and selection, a dimensioning of key equipment for large scale liquefiers, such as turbines and compressors as well as heat exchangers, must be performed iteratively to ensure technological feasibility and maturity. Further critical aspects related to hydrogen liquefaction, e.g. fluid properties, ortho-para hydrogen conversion, and coldbox configuration, must be analysed in detail. This paper provides an overview on the approach, challenges and preliminary results in the development of efficient as well as economically viable concepts for large-scale hydrogen liquefaction.

  17. Beta Cell Mass Restoration in Alloxan-Diabetic Mice Treated with EGF and Gastrin.

    Directory of Open Access Journals (Sweden)

    Imane Song

    Full Text Available One week of treatment with EGF and gastrin (EGF/G was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, however it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treatment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogenesis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experiments, respectively. Acinar cell lineage tracing failed to show an important contribution of acinar cells to the newly formed beta cells. No appearance of transitional cells co-expressing insulin and glucagon, a hallmark for alpha-to-beta cell conversion, was found, suggesting that alpha cells did not significantly contribute to the regeneration. An important fraction of the beta cells significantly lost insulin positivity after alloxan administration, which was restored to normal after one week of EGF/G treatment. Alloxan-only mice showed more pronounced beta cell neogenesis and proliferation, even though beta cell mass remained significantly depleted, suggesting ongoing beta cell death in that group. After one week, macrophage infiltration was significantly reduced in EGF/G-treated group compared to the alloxan-only group. Our results suggest that EGF/G-induced beta cell regeneration in alloxan-diabetic mice is driven by beta cell neogenesis, proliferation and recovery of

  18. Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study.

    Science.gov (United States)

    Ibáñez, Clara; Simó, Carolina; Valdés, Alberto; Campone, Luca; Piccinelli, Anna Lisa; García-Cañas, Virginia; Cifuentes, Alejandro

    2015-06-10

    In this work, the optimization of an effective protocol for cell metabolomics is described with special emphasis in the sample preparation and subsequent analysis of intracellular metabolites from adherent mammalian cells by capillary electrophoresis-mass spectrometry. As case study, colon cancer HT-29 cells, a human cell model to investigate colon cancer, are employed. The feasibility of the whole method for cell metabolomics is demonstrated via a fast and sensitive profiling of the intracellular metabolites HT-29 cells by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF MS). The suitability of this methodology is further corroborated through the examination of the metabolic changes in the polyamines pathway produced in colon cancer HT-29 cells by difluoromethylornithine (DFMO), a known potent ornithine decarboxylase inhibitor. The selection of the optimum extraction conditions allowed a higher sample volume injection that led to an increase in CE-TOF MS sensitivity. Following a non-targeted metabolomics approach, 10 metabolites (namely, putrescine, ornithine, gamma-aminobutyric acid (GABA), oxidized and reduced glutathione, 5'-deoxy-5'-(methylthio)adenosine, N-acetylputrescine, cysteinyl-glycine, spermidine and an unknown compound) were found to be significantly altered by DFMO (p<0.05) in HT-29 cells. In addition to the effect of DFMO on polyamine metabolism, minor modifications of other metabolic pathways (e.g., related to intracellular thiol redox state) were observed. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Is telomerase a viable target in cancer?

    Science.gov (United States)

    Buseman, C.M.; Wright, W.E.; Shay, J.W.

    2012-01-01

    The ideal cancer treatment would specifically target cancer cells yet have minimal or no adverse effects on normal somatic cells. Telomerase, the ribonucleoprotein reverse transcriptase that maintains the ends of human chromosome, is an attractive cancer therapeutic target for exactly this reason [1]. Telomerase is expressed in more than 85% of cancer cells, making it a nearly universal cancer marker, while the majority of normal somatic cells are telomerase negative. Telomerase activity confers limitless replicative potential to cancer cells, a hallmark of cancer which must be attained for the continued growth that characterizes almost all advanced neoplasms [2]. In this review we will summarize the role of telomeres and telomerase in cancer cells, and how properties of telomerase are being exploited to create targeted cancer therapies including telomerase inhibitors, telomerase-targeted immunotherapies and telomerase-driven virotherapies. A frank and balanced assessment of the current state of telomerase inhibitors with caveats and potential limitations will be included. PMID:21802433

  20. Cell-patterned glass spray for direct drug assay using mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jing [School of Science, China University of Geosciences (Beijing), Beijing 100083 (China); Wang, Shiqi; Chen, Qiushui [Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084 (China); Jiang, Hao; Liang, Shuping [School of Science, China University of Geosciences (Beijing), Beijing 100083 (China); Lin, Jin-Ming, E-mail: jmlin@mail.tsinghua.edu.cn [Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084 (China)

    2015-09-10

    In this work, the establishment of a glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was described. Cell co-culture, drug-induced cell apoptosis, proliferation analysis and intracellular drug absorption measurement were performed simultaneously on this specifically designed platform. Two groups of co-cultured cells (NIH-3T3/HepG2 and HepG2/MCF-7) were cultivated and they showed high viability within 3 days. The biocompatibility of the platform facilitated the subsequent bioassays, in which, cyclophosphamide (CPA) and genistein were used as the model drugs. The distinctions of cell apoptosis and proliferation between the mono-cultured and co-cultured cells were clearly observed and well explained by in situ GS-MS measurements. A satisfactory linearity of the calibration curve between the relative MS intensity and CPA concentrations was obtained using stable isotope labeling method (y = 0.16545 + 0.0985x, R{sup 2} = 0.9937). The variations in the quantity of absorbed drug were detected and the results were consistent with the concentration-dependence of cell apoptosis. All the results demonstrated that direct cell-based drug assay could be performed on the stable isotope labeling assisted GS-MS platform in a facile and quantitative manner. - Highlights: • A versatile glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was developed in this paper. • It has characteristics of the atmospheric pressure ionization method. • It is multifunctional for cell co-culture, bioassays, qualitative and quantitative intracellular drug absorption measurement. • GS-MS has the potential to increase the use of mass spectrometry in biological analysis.

  1. Research Resource: A Dual Proteomic Approach Identifies Regulated Islet Proteins During β-Cell Mass Expansion In Vivo

    DEFF Research Database (Denmark)

    Horn, Signe; Kirkegaard, Jeannette S.; Hoelper, Soraya

    2016-01-01

    Diabetes is characterized by insulin insufficiency due to a relative paucity of functional β-cell mass. Thus, strategies for increasing β-cell mass in situ are sought-after for therapeutic purposes. Pregnancy is a physiological state capable of inducing robust β-cell mass expansion, however, the ...... as upstream regulators of the observed expressional changes. As the first characterization of the islet-proteome during pregnancy, this study provides novel insight into the mechanisms involved in promoting pregnancy-induced β-cell mass expansion and function.[on SciFinder (R)]...

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

    Energy Technology Data Exchange (ETDEWEB)

    Fung, N.

    1998-03-27

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

  3. Metastatic Renal Cell Carcinoma Presenting as a Paranasal Sinus Mass: The Importance of Differential Diagnosis

    Directory of Open Access Journals (Sweden)

    Massimo Ralli

    2017-01-01

    Full Text Available Metastases in the paranasal sinuses are rare; renal cell carcinoma is the most common cancer that metastasizes to this region. We present the case of a patient with a 4-month history of a rapidly growing mass of the nasal pyramid following a nasal trauma, associated with spontaneous epistaxis and multiple episodes of hematuria. Cranial CT scan and MRI showed an ethmoid mass extending to the choanal region, the right orbit, and the right frontal sinus with an initial intracranial extension. Patient underwent surgery with a trans-sinusal frontal approach using a bicoronal incision combined with an anterior midfacial degloving; histological exam was compatible with a metastasis of clear cell renal cell carcinoma. Following histological findings, a total body CT scan showed a solitary 6 cm mass in the upper posterior pole of the left kidney identified as the primary tumor. Although rare, metastatic renal cell carcinoma should always be suspected in patients with nasal or paranasal masses, especially if associated with symptoms suggestive of a systemic involvement such as hematuria. A correct early-stage diagnosis of metastatic RCC can considerably improve survival rate in these patients; preoperative differential diagnosis with contrast-enhanced imaging is fundamental for the correct treatment and follow-up strategy.

  4. Biomolecular Imaging Mass Spectrometry : mapping molecular distributions in cells and tissue sections

    NARCIS (Netherlands)

    Altelaar, A.F.M.

    2007-01-01

    Imaging mass spectrometry (IMS) allows the investigation of both identity and localization of the molecular content directly from tissue sections, single cells and many other surfaces. To further develop the application of IMS, different approaches to IMS will be described in this thesis and the

  5. Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay.

    Science.gov (United States)

    Silva, Filomena S G; Starostina, Irina G; Ivanova, Vilena V; Rizvanov, Albert A; Oliveira, Paulo J; Pereira, Susana P

    2016-05-04

    The identification of rapid, reliable, and highly reproducible biological assays that can be standardized and routinely used in preclinical tests constitutes a promising approach to reducing drug discovery costs and time. This unit details a tandem, rapid, and reliable cell viability method for preliminary screening of chemical compounds. This assay measures metabolic activity and cell mass in the same cell sample using a dual resazurin/sulforhodamine B assay, eliminating the variation associated with cell seeding and excessive manipulations in assays that test different cell samples across plates. The procedure also reduces the amount of cells, test compound, and reagents required, as well as the time expended in conventional tests, thus resulting in a more confident prediction of toxic thresholds for the tested compounds. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  6. Precise Temporal Profiling of Signaling Complexes in Primary Cells Using SWATH Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Etienne Caron

    2017-03-01

    Full Text Available Spatiotemporal organization of protein interactions in cell signaling is a fundamental process that drives cellular functions. Given differential protein expression across tissues and developmental stages, the architecture and dynamics of signaling interaction proteomes is, likely, highly context dependent. However, current interaction information has been almost exclusively obtained from transformed cells. In this study, we applied an advanced and robust workflow combining mouse genetics and affinity purification (AP-SWATH mass spectrometry to profile the dynamics of 53 high-confidence protein interactions in primary T cells, using the scaffold protein GRB2 as a model. The workflow also provided a sufficient level of robustness to pinpoint differential interaction dynamics between two similar, but functionally distinct, primary T cell populations. Altogether, we demonstrated that precise and reproducible quantitative measurements of protein interaction dynamics can be achieved in primary cells isolated from mammalian tissues, allowing resolution of the tissue-specific context of cell-signaling events.

  7. Dynamic analysis of CO₂ labeling and cell respiration using membrane-inlet mass spectrometry.

    Science.gov (United States)

    Yang, Tae Hoon

    2014-01-01

    Here, we introduce a mass spectrometry-based analytical method and relevant technical details for dynamic cell respiration and CO2 labeling analysis. Such measurements can be utilized as additional information and constraints for model-based (13)C metabolic flux analysis. Dissolved dynamics of oxygen consumption and CO2 mass isotopomer evolution from (13)C-labeled tracer substrates through different cellular processes can be precisely measured on-line using a miniaturized reactor system equipped with a membrane-inlet mass spectrometer. The corresponding specific rates of physiologically relevant gases and CO2 mass isotopomers can be quantified within a short-term range based on the liquid-phase dynamics of dissolved fermentation gases.

  8. Parejas viables que perduran en el tiempo

    Directory of Open Access Journals (Sweden)

    Juan José Cuervo Rodríguez

    2013-01-01

    Full Text Available El presente artículo científico presenta resultados del proceso llevado a cabo en el proyecto de investigación docente "Mecanismos de autorregulación en parejas viables que perduran en el tiempo". Se soporta en una mirada compleja de la psicología basada en una epistemología de la construcción. En el ámbito metodológico, se inscribe en los estudios de terapia familiar desde una perspectiva de la comunicación humana como un todo integrado. Participaron nueve parejas. Los criterios de inclusión fueron: cinco o más años de convivencia, participación voluntaria, no presentar (ni haber presentado problemáticas especiales que ameriten intervención psicoterapéutica y la obtención de un porcentaje significativo en el uso de estrategias de comunicación asertiva en la resolución de conflictos. El método general utilizado fue el análisis de la comunicación en tarea de conversación. Los principales hallazgos señalan una estrecha relación entre el contexto de desarrollo de las parejas, la emergencia de códigos comunicacionales propios y la posibilidad de perdurar en el tiempo; también, se resalta el tipo de comunicación asertiva o constructiva, la construcción de valores como el respeto y la aceptación de las diferencias, y el deseo por vivir y construir bienestar común, como elementos constitutivos de su identidad como pareja.

  9. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    Directory of Open Access Journals (Sweden)

    Christian Niehage

    Full Text Available Multipotent mesenchymal stromal cells (MSCs are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2% or high (10% serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention.

  10. Vitrification of bovine blastocysts produced in vitro inflicts selective damage to the inner cell mass.

    Science.gov (United States)

    Gómez, E; Muñoz, M; Rodríguez, A; Caamaño, J N; Facal, N; Díez, C

    2009-04-01

    In contrast to the embryos derived from live animals, the embryos produced in vitro undergo increased damage and reduced survival after cryopreservation, particularly when produced with serum. In medium containing serum, retinoic acid increases cell numbers in the inner cell mass and the trophectoderm without altering their relative proportions in the bovine blastocyst. In this work, in medium without serum, we analyzed the contribution of retinoic acid to the development of blastocyst and survival to vitrification, and found a strong cell reduction in the inner mass when compared to the trophectoderm. Day-6 in vitro-produced morulae were treated for 24 h with retinoic acid (0.7 and 1.4 microm) and subsequently cultured without additives for a further 24 h period. Day-8 blastocyst production and cell counts in hatched blastocysts were unaffected by retinoic acid. However, Day-7 expanded, vitrified embryos produced with retinoic acid 1.4 microm survived at lower rates than controls when cultured after warming. Vitrification greatly reduced cell numbers in the inner mass (p vitro survival to cryopreservation is sometimes scarcely informative on the viability of the embryo after transfer to recipients.

  11. Development, growth and maintenance of β-cell mass: models are also part of the story.

    Science.gov (United States)

    Khadra, Anmar; Schnell, Santiago

    2015-04-01

    Pancreatic β-cells in the islets of Langerhans play a crucial role in regulating glucose homeostasis in the circulation. Loss of β-cell mass or function due to environmental, genetic and immunological factors leads to the manifestation of diabetes mellitus. The mechanisms regulating the dynamics of pancreatic β-cell mass during normal development and diabetes progression are complex. To fully unravel such complexity, experimental and clinical approaches need to be combined with mathematical and computational models. In the natural sciences, mathematical and computational models have aided the identification of key mechanisms underlying the behavior of systems comprising multiple interacting components. A number of mathematical and computational models have been proposed to explain the development, growth and death of pancreatic β-cells. In this review, we discuss some of these models and how their predictions provide novel insight into the mechanisms controlling β-cell mass during normal development and diabetes progression. Lastly, we discuss a handful of the major open questions in the field. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Importance of oestrogen receptors to preserve functional β-cell mass in diabetes.

    Science.gov (United States)

    Tiano, Joseph P; Mauvais-Jarvis, Franck

    2012-02-14

    Protecting the functional mass of insulin-producing β cells of the pancreas is a major therapeutic challenge in patients with type 1 (T1DM) or type 2 diabetes mellitus (T2DM). The gonadal hormone 17β-oestradiol (E2) is involved in reproductive, bone, cardiovascular and neuronal physiology. In rodent models of T1DM and T2DM, treatment with E2 protects pancreatic β cells against oxidative stress, amyloid polypeptide toxicity, lipotoxicity and apoptosis. Three oestrogen receptors (ERs)--ERα, ERβ and the G protein-coupled ER (GPER)--have been identified in rodent and human β cells. Whereas activation of ERα enhances glucose-stimulated insulin biosynthesis, reduces islet toxic lipid accumulation and promotes β-cell survival from proapoptotic stimuli, activation of ERβ increases glucose-stimulated insulin secretion. However, activation of GPER protects β cells from apoptosis, raises glucose-stimulated insulin secretion and lipid homeostasis without affecting insulin biosynthesis. Oestrogens are also improving islet engraftment in rodent models of pancreatic islet transplantation. This Review describes developments in the role of ERs in islet insulin biosynthesis and secretion, lipid homeostasis and survival. Moreover, we discuss why and how enhancing ER action in β cells without the undesirable effect of general oestrogen therapy is a therapeutic avenue to preserve functional β-cell mass in patients with diabetes mellitus.

  13. Cell mass and cell cycle dynamics of an asynchronous budding yeast population

    DEFF Research Database (Denmark)

    Lencastre Fernandes, Rita; Carlquist, Magnus; Lundin, Luisa

    2013-01-01

    consumption observed during batch cultivation. The good agreement between the proposed multi-scale model (a population balance model [PBM] coupled to an unstructured model) and experimental data (both the overall physiology and cell size and cell cycle distributions) indicates that a mechanistic model...... in experimental single-cell studies has taken place in the last decades. It has however not been fully accompanied by similar contributions within data analysis and mathematical modeling. Indeed, literature reporting, for example, quantitative analyses of experimental single-cell observations and validation...... of model predictions for cell property distributions against experimental data is scarce. This study focuses on the experimental and mathematical description of the dynamics of cell size and cell cycle position distributions, of a population of Saccharomyces cerevisiae, in response to the substrate...

  14. Oncologic Efficacy of Radio Frequency Ablation for Small Renal Masses: Clear Cell vs Papillary Subtype.

    Science.gov (United States)

    Lay, Aaron H; Faddegon, Stephen; Olweny, Ephrem O; Morgan, Monica; Lorber, Gideon; Trimmer, Clayton; Leveillee, Raymond; Cadeddu, Jeffrey A; Gahan, Jeffrey C

    2015-09-01

    Current radio frequency ablation series do not distinguish renal cell carcinoma subtypes when reporting oncologic efficacy. Papillary neoplasms may be more amenable to radio frequency ablation than clear cell carcinoma because they are less vascular, which may limit heat energy loss. We report the long-term outcomes of patients treated with radio frequency ablation for small renal masses by renal cell carcinoma subtype. The records of patients undergoing radio frequency ablation for small renal masses (cT1a) at 2 institutions from March 2007 to July 2012 were retrospectively reviewed. Patients were included in analysis if they had biopsy confirmed clear cell or papillary renal cell carcinoma histology. Patients had at least 1 contrast enhanced cross-sectional image following radio frequency ablation. Demographic data between tumor subtypes were compared using the paired t-test. Oncologic outcomes were determined by Kaplan-Meier survival analysis and survivor curves were compared with the log rank test. A total of 229 patients met study inclusion criteria. There were 181 clear cell tumors and 48 papillary tumors. Median followup was 33.2 months. There was no difference between tumor groups based on patient age, tumor size or grade, or months of followup. Five-year disease-free survival was 89.7% for clear cell tumors and 100% for papillary tumors (p = 0.041). There was no significant difference in overall survival (88.4% vs 89.6%, p = 0.764). Radio frequency ablation outcomes seem to be determined in part by renal cell carcinoma subtype with clear cell renal tumors having less favorable outcomes. We hypothesize that this is due to differences in tumor vascularity. Our experience suggests that future tumor ablation studies should consider reporting outcomes based on tumor cell types. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  15. MALDI Orbitrap Mass Spectrometry Profiling of Dysregulated Sulfoglycosphingolipids in Renal Cell Carcinoma Tissues

    Science.gov (United States)

    Jirásko, Robert; Holčapek, Michal; Khalikova, Maria; Vrána, David; Študent, Vladimír; Prouzová, Zuzana; Melichar, Bohuslav

    2017-08-01

    Matrix-assisted laser desorption/ionization coupled with Orbitrap mass spectrometry (MALDI-Orbitrap-MS) is used for the clinical study of patients with renal cell carcinoma (RCC), as the most common type of kidney cancer. Significant changes in sulfoglycosphingolipid abundances between tumor and autologous normal kidney tissues are observed. First, sulfoglycosphingolipid species in studied RCC samples are identified using high mass accuracy full scan and tandem mass spectra. Subsequently, optimization, method validation, and statistical evaluation of MALDI-MS data for 158 tissues of 80 patients are discussed. More than 120 sulfoglycosphingolipids containing one to five hexosyl units are identified in human RCC samples based on the systematic study of their fragmentation behavior. Many of them are recorded here for the first time. Multivariate data analysis (MDA) methods, i.e., unsupervised principal component analysis (PCA) and supervised orthogonal partial least square discriminant analysis (OPLS-DA), are used for the visualization of differences between normal and tumor samples to reveal the most up- and downregulated lipids in tumor tissues. Obtained results are closely correlated with MALDI mass spectrometry imaging (MSI) and histologic staining. Important steps of the present MALDI-Orbitrap-MS approach are also discussed, such as the selection of best matrix, correct normalization, validation for semiquantitative study, and problems with possible isobaric interferences on closed masses in full scan mass spectra.

  16. Reactivity and analytical performance of oxygen as cell gas in inductively coupled plasma tandem mass spectrometry

    Science.gov (United States)

    Virgilio, Alex; Amais, Renata S.; Amaral, Clarice D. B.; Fialho, Lucimar L.; Schiavo, Daniela; Nóbrega, Joaquim A.

    2016-12-01

    The reactivity and analytical performance of O2 as cell gas in inductively coupled plasma tandem mass spectrometry was investigated. Selected analytes in a wide mass range were divided in three groups according to their reactivity: G1 represents elements with high oxygen affinity (Ce, La, P, Sc, Ti, and Y), G2 contains elements that may partially react with oxygen (As, Ba, Mo, Si, Sr, and V), and G3 comprises elements expected to be less reactive towards oxygen (Al, Bi, Cu, Mg, Pb, and Pd). On-mass and mass-shift modes were evaluated by monitoring atomic and metal oxide ions, respectively. Analytical signal profiles, oxide percentages, sensitivities and limits of detection for oxygen flow rates varying from 0.1 to 1.0 mL min- 1 were also studied. Group 1 elements plus As and V presented better sensitivities and LODs when measuring oxides, which were the major species for all flow rates evaluated. Molybdenum and Si oxides presented intermediate behavior and MoO fraction was up to 47% and limit of detection was the same as that obtained in on-mass mode. For others G2 and G3 elements, on-mass mode presented higher sensitivity and better LODs, with estimated oxide contents lower than 10%. In most cases, increasing oxygen flow rates led to lower sensitivities and worse LODs.

  17. Effect of ultra-low Pt loading on mass activity of polymer electrolyte membrane fuel cells

    Science.gov (United States)

    Kriston, Ákos; Xie, Tianyuan; Gamliel, David; Ganesan, Prabhu; Popov, Branko N.

    2013-12-01

    The mass activity is intensively used as characterization parameters for evaluation of the effectiveness of the cathode catalyst in polymer electrolyte membrane (PEM) fuel cells. In this work, the dependence of mass activity on platinum loading was studied at the cathode. The results indicated that the mass activity and the utilized electrochemical surface area of the catalyst are not independent of the catalyst loading. The electrochemical specific surface area (ECSA) and the mass activity increase as the loading is decreased. The increase of the ECSA is attributed to the increase of the utilization of the catalyst. The commonly applied Tafel-approximation cannot be used to fit the result because the mass activity is controlled not only by RΩ and the ORR kinetics, but also the utilization of the catalyst, which in turn depends on catalyst loading, the structure of the catalyst layer, the degree of agglomeration, and screening of the catalyst particles. A detailed and more precise definition of mass activity (MA) is given to elucidate the variation of MA with catalyst loading.

  18. Shaper-Based Filters for the compensation of the load cell response in dynamic mass measurement

    Science.gov (United States)

    Richiedei, Dario; Trevisani, Alberto

    2018-01-01

    This paper proposes a novel model-based signal filtering technique for dynamic mass measurement through load cells. Load cells are sensors with an underdamped oscillatory response which usually imposes a long settling time. Real-time filtering is therefore necessary to compensate for such a dynamics and to quickly retrieve the mass of the measurand (which is the steady state value of the load cell response) before the measured signal actually settles. This problem has a big impact on the throughput of industrial weighing machines. In this paper a novel solution to this problem is developed: a model-based filtering technique is proposed to ensure accurate, robust and rapid estimation of the mass of the measurand. The digital filters proposed are referred to as Shaper-Based Filters (SBFs) and are based on the convolution of the load cell output signal with a sequence of few impulses (typically, between 2 and 5). The amplitudes and the instants of application of such impulses are computed through the analytical development of the load cell step response, by imposing the admissible residual oscillation in the steady-state filtered signal and by requiring the desired sensitivity of the filter. The inclusion of robustness specifications tackles effectively the unavoidable uncertainty and variability in the load cell frequency and damping. The effectiveness of the proposed filters is proved experimentally through an industrial set up: the load-cell-instrumented weigh bucket of a multihead weighing machine for packaging. A performance comparison with other benchmark filters is provided and discussed too.

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

    Science.gov (United States)

    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.

  20. Breast Cancer Mimic: Cutaneous B-Cell Lymphoma Presenting as an Isolated Breast Mass

    Directory of Open Access Journals (Sweden)

    Margaret Taghavi

    2014-10-01

    Full Text Available Background: Primary cutaneous B-cell lymphoma typically localizes to the skin, and dissemination to internal organs is rare. Lymphomatous involvement of the breasts is also rare. We describe the clinical and radiological findings of an unusual case of primary cutaneous B-cell lymphoma presenting as an isolated breast mass without associated skin changes. Case Presentation: The patient was a 55-year-old Caucasian female who initially presented with cutaneous B-cell lymphoma around her eyes and forehead with recurrence involving the skin between her breasts. Three years after terminating treatment due to a lack of symptoms, she presented for an annual screening mammogram that found a new mass in her upper inner right breast without imaging signs of cutaneous extension. On physical examination, there were no corresponding skin findings. Due to the suspicious imaging features of the mass that caused concern for primary breast malignancy, she underwent a core biopsy which revealed cutaneous B-cell lymphoma. Conclusion: When evaluating patients with a systemic disease who present with findings atypical for that process, it is important to still consider the systemic disease as a potential etiology, particularly with lymphoma given its reputation as a great mimicker.

  1. Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

    Directory of Open Access Journals (Sweden)

    Katrin Bomans

    Full Text Available Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS. However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day manner.

  2. Palladium-based Mass-Tag Cell Barcoding with a Doublet-Filtering Scheme and Single Cell Deconvolution Algorithm

    Science.gov (United States)

    Zunder, Eli R.; Finck, Rachel; Behbehani, Gregory K.; Amir, El-ad D.; Krishnaswamy, Smita; Gonzalez, Veronica D.; Lorang, Cynthia G.; Bjornson, Zach; Spitzer, Matthew H.; Bodenmiller, Bernd; Fantl, Wendy J.; Pe’er, Dana; Nolan, Garry P.

    2015-01-01

    SUMMARY Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption, and shortens instrument measurement time. Here, we present an optimized MCB protocol with several improvements over previously described methods. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell-based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3–4 h, not including sample acquisition time of ~1 h per million cells. PMID:25612231

  3. Quantitative Imaging of Cell Membrane-associated Effective Mass Density Using Photonic Crystal Enhanced Microscopy (PCEM).

    Science.gov (United States)

    Zhuo, Yue; Choi, Ji Sun; Marin, Thibault; Yu, Hojeong; Harley, Brendan A; Cunningham, Brian T

    2016-11-01

    Adhesion is a critical cellular process that contributes to migration, apoptosis, differentiation, and division. It is followed by the redistribution of cellular materials at the cell membrane or at the cell-surface interface for cells interacting with surfaces, such as basement membranes. Dynamic and quantitative tracking of changes in cell adhesion mass redistribution is challenging because cells are rapidly moving, inhomogeneous, and nonequilibrium objects, whose physical and mechanical properties are difficult to measure or predict. Here, we report a novel biosensor based microscopy approach termed Photonic Crystal Enhanced Microscopy (PCEM) that enables the movement of cellular materials at the plasma membrane of individual live cells to be dynamically monitored and quantitatively imaged. PCEM utilizes a photonic crystal biosensor surface, which can be coated with arbitrary extracellular matrix materials to facilitate cellular interactions, within a modified brightfield microscope with a low intensity non-coherent light source. Benefiting from the high sensitivity, narrow resonance peak, and tight spatial confinement of the evanescent field atop the photonic crystal biosensor, PCEM enables label-free live cell imaging with high sensitivity and high lateral and axial spatial-resolution, thereby allowing dynamic adhesion phenotyping of single cells without the use of fluorescent tags or stains. We apply PCEM to investigate adhesion and the early stage migration of different types of stem cells and cancer cells. By applying image processing algorithms to analyze the complex spatiotemporal information generated by PCEM, we offer insight into how the plasma membrane of anchorage dependent cells is dynamically organized during cell adhesion. The imaging and analysis results presented here provide a new tool for biologists to gain a deeper understanding of the fundamental mechanisms involved with cell adhesion and concurrent or subsequent migration events.

  4. Quantitative Imaging of Cell Membrane-associated Effective Mass Density Using Photonic Crystal Enhanced Microscopy (PCEM)

    Science.gov (United States)

    Zhuo, Yue; Choi, Ji Sun; Marin, Thibault; Yu, Hojeong; Harley, Brendan A.; Cunningham, Brian T.

    2017-01-01

    Adhesion is a critical cellular process that contributes to migration, apoptosis, differentiation, and division. It is followed by the redistribution of cellular materials at the cell membrane or at the cell-surface interface for cells interacting with surfaces, such as basement membranes. Dynamic and quantitative tracking of changes in cell adhesion mass redistribution is challenging because cells are rapidly moving, inhomogeneous, and nonequilibrium objects, whose physical and mechanical properties are difficult to measure or predict. Here, we report a novel biosensor based microscopy approach termed Photonic Crystal Enhanced Microscopy (PCEM) that enables the movement of cellular materials at the plasma membrane of individual live cells to be dynamically monitored and quantitatively imaged. PCEM utilizes a photonic crystal biosensor surface, which can be coated with arbitrary extracellular matrix materials to facilitate cellular interactions, within a modified brightfield microscope with a low intensity non-coherent light source. Benefiting from the high sensitivity, narrow resonance peak, and tight spatial confinement of the evanescent field atop the photonic crystal biosensor, PCEM enables label-free live cell imaging with high sensitivity and high lateral and axial spatial-resolution, thereby allowing dynamic adhesion phenotyping of single cells without the use of fluorescent tags or stains. We apply PCEM to investigate adhesion and the early stage migration of different types of stem cells and cancer cells. By applying image processing algorithms to analyze the complex spatiotemporal information generated by PCEM, we offer insight into how the plasma membrane of anchorage dependent cells is dynamically organized during cell adhesion. The imaging and analysis results presented here provide a new tool for biologists to gain a deeper understanding of the fundamental mechanisms involved with cell adhesion and concurrent or subsequent migration events. PMID

  5. Quantitative assay of element mass inventories in single cell biological systems with micro-PIXE

    Energy Technology Data Exchange (ETDEWEB)

    Ogrinc, Nina [Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia); LOTRIČ Metrology, Selca 163, SI-4227 Selca (Slovenia); Pelicon, Primož, E-mail: primoz.pelicon@ijs.si [Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia); Vavpetič, Primož; Kelemen, Mitja; Grlj, Nataša; Jeromel, Luka [Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia); Tomić, Sergej [Medical Faculty of the Military Medical Academy, University of Defense, Crnotravska 17, Belgrade (Serbia); Čolić, Miodrag [Medical Faculty of the Military Medical Academy, University of Defense, Crnotravska 17, Belgrade (Serbia); Medical Faculty, University of Niš, Boulevard of Dr. Zoran Djindjić 81, 18000 Niš (Serbia); Beran, Alfred [Dipartimento di Oceanografia Biologica, Istituto Nazionale di Oceanografia e Geofisica Sperimentale, Via Auguste Piccard 54, 34151 Trieste (Italy)

    2013-07-01

    Elemental concentrations in micro-PIXE (Particle Induced X-ray Emission) maps of elements in biological tissue slices have been determined using auxiliary information on the sample matrix composition from EBS (Elastic Backscattering Spectroscopy) and STIM (Scanning Transmission Ion Microscopy). The thin sample approximation may be used for evaluating micro-PIXE data in cases, where X-ray absorption in the sample can be neglected and the mass of elements in a selected area can be estimated. The resulting sensitivity amounts to an impressive 10{sup −12} g of the selected elements. Two cases are presented as examples. In the first, we determined the total mass of gold nanoparticles internalized by human monocyte-derived dendritic cells (MDDC). In the second, an inventory of the mass of elements in the micro-particulate material adsorbed at the wall of the lorica of the microzooplankton species Tintinnopsis radix has been created.

  6. Determination of viable Salmonellae from potable and source water through PMA assisted qPCR.

    Science.gov (United States)

    Singh, Gulshan; Vajpayee, Poornima; Bhatti, Saurabh; Ronnie, Nirmala; Shah, Nimish; McClure, Peter; Shanker, Rishi

    2013-07-01

    Resource constrained countries identified as endemic zones for pathogenicity of Salmonella bear an economic burden due to recurring expenditure on medical treatment. qPCR used for Salmonella detection could not discriminate between viable and nonviable cells. Propidium monoazide (PMA) that selectively penetrates nonviable cells to cross-link their DNA, was coupled with ttr gene specific qPCR for quantifying viable salmonellae in source/potable waters collected from a north Indian city. Source water (raw water for urban potable water supply) and urban potable water exhibited viable salmonellae in the range of 2.1×10(4)-2.6×10(6) and 2-7160CFU/100mL, respectively. Potable water at water works exhibited DNA from dead cells but no viable cells were detected. PMA assisted qPCR could specifically detect low numbers of live salmonellae in Source and potable waters. This strategy can be used in surveillance of urban potable water distribution networks to map contamination points for better microbial risk management. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Lipidomics: analysis of the lipid composition of cells and subcellular organelles by electrospray ionization mass spectrometry.

    Science.gov (United States)

    Brügger, Britta

    2014-01-01

    Lipidomics aims to quantitatively define lipid classes, including their molecular species, in biological systems. Lipidomics has experienced rapid progress, mainly because of continuous technical advances in instrumentation that are now enabling quantitative lipid analyses with an unprecedented level of sensitivity and precision. The still-growing category of lipids includes a broad diversity of chemical structures with a wide range of physicochemical properties. Reflecting this diversity, different methods and strategies are being applied to the quantification of lipids. Here, I review state-of-the-art electrospray ionization tandem mass spectrometric approaches and direct infusion to quantitatively assess lipid compositions of cells and subcellular fractions. Finally, I discuss a few examples of the power of mass spectrometry-based lipidomics in addressing cell biological questions.

  8. Modeling and experimental validation of water mass balance in a PEM fuel cell stack

    DEFF Research Database (Denmark)

    Liso, Vincenzo; Araya, Samuel Simon; Olesen, Anders Christian

    2016-01-01

    management in PEM fuel cell is crucial in order to avoid an imbalance between water production and water removal from the fuel cell. In the present study, a novel mathematical zero-dimensional model has been formulated for the water mass balance and hydration of a polymer electrolyte membrane. This model...... is validated against experimental data. In the results it is shown that the fuel cell water balance calculated by this model shows better fit with experimental data-points compared with model where only steady state operation were considered. We conclude that this discrepancy is due a different rate of water......Polymer electrolyte membrane (PEM) fuel cells require good hydration in order to deliver high performance and ensure long life operation. Water is essential for proton conductivity in the membrane which increases by nearly six orders of magnitude from dry to fully hydrated. Adequate water...

  9. Mass transport aspects of polymer electrolyte fuel cells under two-phase flow conditions

    Energy Technology Data Exchange (ETDEWEB)

    Kramer, D.

    2007-03-15

    This well-illustrated, comprehensive dissertation by Dr. Ing. Denis Kramer takes an in-depth look at polymer electrolyte fuel cells (PEFC) and the possibilities for their application. First of all, the operating principles of polymer electrolyte fuel cells are described and discussed, whereby thermodynamics aspects and loss mechanisms are examined. The mass transport diagnostics made with respect to the function of the cells are discussed. Field flow geometry, gas diffusion layers and, amongst other things, liquid distribution, the influence of flow direction and the low-frequency behaviour of air-fed PEFCs are discussed. Direct methanol fuel cells are examined, as are the materials chosen. The documentation includes comprehensive mathematical and graphical representations of the mechanisms involved.

  10. Using Spores for Fusarium spp. Classification by MALDI-Based Intact Cell/Spore Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Wolfgang Winkler

    2012-01-01

    Full Text Available Fusarium is a widespread genus of filamentous fungi and a member of the soil microbial community. Certain subspecies are health threatening because of their mycotoxin production that affects the human and animal food chain. Thus, for early and effective pest control, species identification is of particular interest; however, differentiation on the subspecies level is challenging and time-consuming for this fungus. In the present study, we show the possibilities of intact cell mass spectrometry for spore analysis of 22 different Fusarium strains belonging to six Fusarium subspecies. We found that species differentiation is possible if mass spectrometric analyses are performed under well-defined conditions with fixed parameters. A critical point for analysis is a proper sample preparation of spores, which increases the quality of mass spectra with respect to signal intensity and m/z value variations. It was concluded that data acquistion has to be performed automatically; otherwise, user-specific variations are introduced generating data which cannot fit the existing datasets. Data that show clearly that matrix-assisted laser desorption ionization-based intact cell/intact spore mass spectrometry (IC/ISMS can be applied to differentiate closely related Fusarium spp. are presented. Results show a potential to build a database on Fusarium species for accurate species identification, for fast response in the case of infections in the cornfield. We furthermore demonstrate the high precision of our approach in classification of intact Fusarium species according to the location of their collection.

  11. Cost Analysis of Direct Methanol Fuel Cell Stacks for Mass Production

    Directory of Open Access Journals (Sweden)

    Mauro Francesco Sgroi

    2016-11-01

    Full Text Available Fuel cells are very promising technologies for efficient electrical energy generation. The development of enhanced system components and new engineering solutions is fundamental for the large-scale deployment of these devices. Besides automotive and stationary applications, fuel cells can be widely used as auxiliary power units (APUs. The concept of a direct methanol fuel cell (DMFC is based on the direct feed of a methanol solution to the fuel cell anode, thus simplifying safety, delivery, and fuel distribution issues typical of conventional hydrogen-fed polymer electrolyte fuel cells (PEMFCs. In order to evaluate the feasibility of concrete application of DMFC devices, a cost analysis study was carried out in the present work. A 200 W-prototype developed in the framework of a European Project (DURAMET was selected as the model system. The DMFC stack had a modular structure allowing for a detailed evaluation of cost characteristics related to the specific components. A scale-down approach, focusing on the model device and projected to a mass production, was used. The data used in this analysis were obtained both from research laboratories and industry suppliers specialising in the manufacturing/production of specific stack components. This study demonstrates that mass production can give a concrete perspective for the large-scale diffusion of DMFCs as APUs. The results show that the cost derived for the DMFC stack is relatively close to that of competing technologies and that the introduction of innovative approaches can result in further cost savings.

  12. Laryngeal Langerhans Cell Histiocytosis Presenting with Neck Mass in an Adult Woman

    Directory of Open Access Journals (Sweden)

    Hesam Jahandideh

    2016-01-01

    Full Text Available Langerhans cell histiocytosis (LCH is a very rare condition that commonly affects the head and neck region. There are very few cases of isolated laryngeal involvement by LCH, mostly reported in pediatric patients. Here, we report a case of laryngeal LCH in a 62-year-old woman presenting with a neck mass several weeks ago. The clinical and histopathological findings are reported with a brief discussion about the disease.

  13. Optical volume and mass measurements show that mammalian cells swell during mitosis.

    Science.gov (United States)

    Zlotek-Zlotkiewicz, Ewa; Monnier, Sylvain; Cappello, Giovanni; Le Berre, Mael; Piel, Matthieu

    2015-11-23

    The extent, mechanism, and function of cell volume changes during specific cellular events, such as cell migration and cell division, have been poorly studied, mostly because of a lack of adequate techniques. Here we unambiguously report that a large range of mammalian cell types display a significant increase in volume during mitosis (up to 30%). We further show that this increase in volume is tightly linked to the mitotic state of the cell and not to its spread or rounded shape and is independent of the presence of an intact actomyosin cortex. Importantly, this volume increase is not accompanied by an increase in dry mass and thus corresponds to a decrease in cell density. This mitotic swelling might have important consequences for mitotic progression: it might contribute to produce strong pushing forces, allowing mitotic cells to round up; it might also, by lowering cytoplasmic density, contribute to the large change of physicochemical properties observed in mitotic cells. © 2015 Zlotek-Zlotkiewicz et al.

  14. Dynamic mass redistribution assay decodes differentiation of a neural progenitor stem cell.

    Science.gov (United States)

    Pai, Sadashiva; Verrier, Florence; Sun, Haiyan; Hu, Haibei; Ferrie, Ann M; Eshraghi, Azita; Fang, Ye

    2012-10-01

    Stem cells hold great potential in drug discovery and development. However, challenges remain to quantitatively measure the functions of stem cells and their differentiated products. Here, we applied fluorescent imaging, quantitative real-time PCR, and label-free dynamic mass redistribution (DMR) assays to characterize the differentiation process of the ReNcell VM human neural progenitor stem cell. Immunofluorescence imaging showed that after growth factor withdrawal, the neuroprogenitor stem cell was differentiated into dopaminergic neurons, astrocytes, and oligodendrocytes, thus creating a neuronal cell system. High-performance liquid chromatography analysis showed that the differentiated cell system released dopamine upon depolarization with KCl. In conjunction with quantitative real-time PCR, DMR assays using a G-protein-coupled receptor agonist library revealed that a subset of receptors, including dopamine D(1) and D(4) receptors, underwent marked alterations in both receptor expression and signaling pathway during the differentiation process. These findings suggest that DMR assays can decode the differentiation process of stem cells at the cell system level.

  15. Sustained beta-cell dysfunction but normalized islet mass in aged thrombospondin-1 deficient mice.

    Directory of Open Access Journals (Sweden)

    Carl Johan Drott

    Full Text Available Pancreatic islet endothelial cells have in recent years been shown to support beta-cell mass and function by paracrine interactions. Recently, we identified an islets endothelial-specific glycoprotein, thrombospondin-1 (TSP-1, that showed to be of importance for islet angiogenesis and beta-cell function in young mice. The present study aimed to investigate long-term consequences for islet morphology and beta-cell function of TSP-1 deficiency. Islet and beta-cell mass were observed increased at 10-12 weeks of age in TSP-1 deficient mice, but were normalized before 16 weeks of age when compared to wild-type controls. Islet vascularity was normal in 10-12 and 16-week-old TSP-1 deficient animals, whereas islets of one-year-old animals lacking TSP-1 were hypervascular. Beta-cell dysfunction in TSP-1 deficient animals was present at similar magnitudes between 10-12 and 52 weeks of age, as evaluated by glucose tolerance tests. The insulin secretion capacity in vivo of islets in one-year-old TSP-1 deficient animals was only ∼15% of that in wild-type animals. Using a transplantation model, we reconstituted TSP-1 in adult TSP-deficient islets. In contrast to neonatal TSP-1 deficient islets that we previously reported to regain function after TSP-1 reconstitution, adult islets failed to recover. We conclude that TSP-1 deficiency in islets causes changing vascular and endocrine morphological alterations postnatally, but is coupled to a chronic beta-cell dysfunction. The beta-cell dysfunction induced by TSP-1 deficiency is irreversible if not substituted early in life.

  16. Asouzu's Complementary Ontology as a Foundation for a Viable ...

    African Journals Online (AJOL)

    This paper on “Asouzu's Complementary Ontology as a foundation for a viable Ethic of the Environment”, posits that an ethic of the environment can be seen as viable if it considers the whole of reality as ontologically relevant. This point of view would free environmental ethics of anthropocentric bias and its attendant ...

  17. Conjunctival mass as an initial presentation of mantle cell lymphoma: a case report

    Directory of Open Access Journals (Sweden)

    Khanlari Mahsa

    2012-12-01

    Full Text Available Abstract Background To describe a rare manifestation of mantle cell lymphoma (MCL in conjunctiva, with clinical, hisologic, immunohistologic and genetic findings together with review of the Literature. Case presentation Most ocular adnexal lymphomas are extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (MALT. A few cases of ocular adnexal mantle cell lymphomas have been reported in the literature. We present a case of mantle cell lymphoma presenting as right conjunctival mass of at least three months duration in a 64-year-old man. Histopathologic examination showed a proliferation of monomorphous small-to-medium-sized lymphoid cells with cleaved nuclei in the subconjunctiva. By immunohistochemistry, the infiltrate was positive for CD20, CD5, BCL-2, cyclin D1, and the transcription factor SOX11. Fluorescent in situ hybridization demonstrated the presence of IGH-CCND1 fusion indicating t(11;14. Conclusion A rigorous approach to initial diagnosis and staging of small cell lymphomas of the ocular adnexa is needed. The recognition of ocular MCL requires appropriate immunohistochemical staining and/or genetic confirmation to differentiate this rare form of presentation of MCL from other more frequent small cell lymphomas.

  18. High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.

    Science.gov (United States)

    Hemmen, Katherina; Reinl, Tobias; Buttler, Kerstin; Behler, Friederike; Dieken, Hauke; Jänsch, Lothar; Wilting, Jörg; Weich, Herbert A

    2011-05-01

    Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.

  19. An increase in immature β-cells lacking Glut2 precedes the expansion of β-cell mass in the pregnant mouse.

    Directory of Open Access Journals (Sweden)

    Christine A Beamish

    Full Text Available A compensatory increase in β-cell mass occurs during pregnancy to counter the associated insulin resistance, and a failure in adaptation is thought to contribute to gestational diabetes. Insulin-expressing but glucose-transporter-2-low (Ins+Glut2LO progenitor cells are present in mouse and human pancreas, being predominantly located in extra-islet β-cell clusters, and contribute to the regeneration of the endocrine pancreas following induced ablation. We therefore sought to investigate the contribution of Ins+Glut2LO cells to β-cell mass expansion during pregnancy. Female C57Bl/6 mice were time mated and pancreata were collected at gestational days (GD 6, 9, 12, 15, and 18, and postpartum D7 (n = 4/time-point and compared to control (non-pregnant animals. Beta cell mass, location, proliferation (Ki67+, and proportion of Ins+Glut2LO cells were measured using immunohistochemistry and bright field or confocal microscopy. Beta cell mass tripled by GD18 and β-cell proliferation peaked at GD12 in islets (≥6 β-cells and small β-cell clusters (1-5 β-cells. The proportion and fraction of Ins+Glut2LO cells undergoing proliferation increased significantly at GD9 in both islets and clusters, preceding the increase in β-cell mass and proliferation, and their proliferation within clusters persisted until GD15. The overall number of clusters increased significantly at GD9. Quantitative PCR showed a significant increase in Pdx1 presence at GD9 vs. GD18 or control pancreas, and Pdx1 was visualized by immunohistochemistry within both Ins+Glut2LO and Ins+Glut2HI cells within clusters. These results indicate that Ins+Glut2LO cells are likely to contribute to β-cell mass expansion during pregnancy.

  20. Separation of viable and non-viable tomato (Solanum lycopersicum L.) seeds using single seed near-infrared spectroscopy

    DEFF Research Database (Denmark)

    Shrestha, Santosh; Deleuran, Lise Christina; Gislum, René

    2017-01-01

    -viable tomato seeds of two cultivars using chemometrics. The data exploration were performed by principal component analysis (PCA). Subsequently, viable and non-viable seeds were classified by partial least squares-discriminant analysis (PLS-DA) and interval PLS-DA (iPLS-DA). The indication of clustering...... of viable and non-viable seeds were observed in the PCA of each cultivar and the pooled samples. However, the PCA did not exhibit a pattern of separation among the early, normal and late germinated tomato seeds. The NIR spectral regions of 1160–1170, 1383–1397, 1647–1666, 1860–1884 and 1915–1940 nm were...... identified as important for classification of viable and non-viable tomato seeds by iPLS-DA. The sensitivity i.e. ability to correctly identify the positive samples and specificity i.e. ability to reject the negative samples of the (iPLS-DA) model on identified spectral regions for prediction of viable...

  1. Cosmic constraint on massive neutrinos in viable f( R) gravity with producing Λ CDM background expansion

    Science.gov (United States)

    Lu, Jianbo; Liu, Molin; Wu, Yabo; Wang, Yan; Yang, Weiqiang

    2016-12-01

    Tensions between several cosmic observations were found recently, such as the inconsistent values of H0 (or σ 8) were indicated by the different cosmic observations. Introducing the massive neutrinos in Λ CDM could potentially solve the tensions. Viable f( R) gravity producing Λ CDM background expansion with massive neutrinos is investigated in this paper. We fit the current observational data: Planck-2015 CMB, RSD, BAO, and SNIa to constrain the mass of neutrinos in viable f( R) theory. The constraint results at 95% confidence level are: Σ m_ν case, m_{ν , sterile}^effcase. For the effects due to the mass of the neutrinos, the constraint results on model parameter at 95% confidence level become f_{R0}× 10^{-6}> -1.89 and f_{R0}× 10^{-6}> -2.02 for two cases, respectively. It is also shown that the fitting values of several parameters much depend on the neutrino properties, such as the cold dark matter density, the cosmological quantities at matter-radiation equality, the neutrino density and the fraction of baryonic mass in helium. Finally, the constraint result shows that the tension between direct and CMB measurements of H_0 gets slightly weaker in the viable f( R) model than that in the base Λ CDM model.

  2. Heat and mass transfer effects in a direct methanol fuel cell: A 1D model

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, V.B.; Falcao, D.S.; Pinto, A.M.F.R. [Centro de Estudos de Fenomenos de Transporte, Departamento de Eng. Quimica, Faculdade de Engenharia da Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto (Portugal); Rangel, C.M. [INETI - Unidade de Electroquimica e Materiais, Paco do Lumiar, 22,1649-038 (Portugal)

    2008-07-15

    Models are a fundamental tool for the design process of fuel cells and fuel cell systems. In this work, a steady-state, one-dimensional model accounting for coupled heat and mass transfer, along with the electrochemical reactions occurring in the DMFC, is presented. The model output is the temperature profile through the cell and the water balance and methanol crossover between the anode and the cathode. The model predicts the correct trends for the influence of current density and methanol feed concentration on both methanol and water crossover. The model estimates the net water transfer coefficient through the membrane, {alpha}, a very important parameter to describe water management in the DMFC. Suitable operating ranges can be set up for different MEA structures maintaining the crossover of methanol and water within acceptable levels. The model is rapidly implemented and is therefore suitable for inclusion in real-time system level DMFC calculations. (author)

  3. Biochemical and Mass Spectrometry-Based Approaches to Profile SUMOylation in Human Cells

    DEFF Research Database (Denmark)

    Kessler, Benedikt M; Bursomanno, Sara; McGouran, Joanna F

    2017-01-01

    Posttranslational modification of proteins with the small ubiquitin-like modifier (SUMO) regulates protein function in the context of cell cycle and DNA repair. The occurrence of SUMOylation is less frequent as compared to protein modification with ubiquitin, and appears to be controlled by a sma...... of the latest experimental approaches to the study of SUMOylation, and also describe hands-on protocols using a combination of biochemistry and mass spectrometry-based technologies to profile proteins that are SUMOylated in human cells.......Posttranslational modification of proteins with the small ubiquitin-like modifier (SUMO) regulates protein function in the context of cell cycle and DNA repair. The occurrence of SUMOylation is less frequent as compared to protein modification with ubiquitin, and appears to be controlled...

  4. The masses of nobelium and lawrencium isotopes, the mass difference between {sup 180}W and {sup 180}Hf, and a characterization of the future cryogenic stopping cell of the online mass spectrometer SHIPTRAP

    Energy Technology Data Exchange (ETDEWEB)

    Droese, Christian

    2015-01-29

    This work describes the recent scientific and technical achievements obtained at the high-precision Penning trap mass spectrometer SHIPTRAP. The scientific focus of the SHIPTRAP experiment are mass measurements of short-lived nuclides with proton number larger than 100. The masses of these isotopes are usually determined via extrapolations, systematic trends, predictions based on theoretical models or alpha-decay spectroscopy. In several experiments the masses of the isotopes {sup 252-255}No and {sup 255,256}Lr have been measured directly. With the obtained results the region of enhanced nuclear stability at the deformed shell closure at the neutron number 152 was investigated. Furthermore, the masses have been used to benchmark theoretical mass models. The measured masses were compared selected mass models which revealed differences between few keV/c² up to several MeV/c² depending on the investigated nuclide and model. In order to perform mass measurements on superheavy nuclei with lower production rates, the efficiency of the SHIPTRAP setup needs to be increased. Currently, the efficiency is 2% and mainly limited by the stopping- and extraction efficiency of the buffer gas cell. The stopping and extraction efficiency of the current buffer gas cell is 12%. To this end, a modified version of the buffer gas cell was developed and characterized with {sup 223}Ra ion source. Besides a larger stopping volume and a coaxial injection the new buffer gas cell is operated at a temperature of 40 K. The operation at cryogenic temperatures increases the cleanliness of the buffer gas. From extraction measurements and simulations an overall efficiency of 62(3)% was determined which results in an increase by a factor of 5 in comparison to the current buffer gas cell. Aside from high-precision mass measurements of heavy radionuclides the mass differences of metastable isobars was measured to identify candidates for the neutrinoless double-electron capture. Neutrinoless double

  5. Metastatic Sarcomatoid Squamous Cell Carcinoma of the Cervix Presenting with Chest Mass

    Directory of Open Access Journals (Sweden)

    Lilit Karapetyan

    2017-01-01

    Full Text Available Background. Sarcomatoid squamous cell carcinoma is a rare and aggressive form of cervical cancer. We report a case of metastatic sarcomatoid squamous cell carcinoma (SSCC of cervix that presented with an anterior chest wall mass. Case. A 43-year-old Hispanic female presented with a two-month history of a central chest wall mass. The patient’s only past medical history was SSCC of the cervix, stage IIB, diagnosed two years priorly. She underwent neoadjuvant chemoradiation therapy (CRT with cisplatin followed by radical hysterectomy. Surgical margins were positive which led to adjuvant CRT with carboplatin and paclitaxel. PET scan 4 months after the postoperative treatment was negative for recurrence and metastatic disease. On current presentation, the CT chest revealed anterior mediastinal destructive soft tissue mass involving sternum, and the biopsy showed SSCC. The patient received palliative radiation therapy to her chest with improvement in pain and ability to swallow. After discussing the prognosis she refused further chemotherapy and decided on hospice care. Conclusion. Despite good response to first-line therapy, SSCC tends to recur early and does not respond to second-line therapy. Radiation therapy seems to be the most effective modality for treatment, but randomized controlled trials of therapy are impractical.

  6. Exercise and amino acid anabolic cell signaling and the regulation of skeletal muscle mass.

    Science.gov (United States)

    Pasiakos, Stefan M

    2012-07-01

    A series of complex intracellular networks influence the regulation of skeletal muscle protein turnover. In recent years, studies have examined how cellular regulators of muscle protein turnover modulate metabolic mechanisms contributing to the loss, gain, or conservation of skeletal muscle mass. Exercise and amino acids both stimulate anabolic signaling potentially through several intracellular pathways including the mammalian target of rapamycin complex 1 and the mitogen activated protein kinase cell signaling cascades. As novel molecular regulators of muscle integrity continue to be explored, a contemporary analysis of the literature is required to understand the metabolic mechanisms by which contractile forces and amino acids affect cellular process that contribute to long-term adaptations and preservation of muscle mass. This article reviews the literature related to how exercise and amino acid availability affect cellular regulators of skeletal muscle mass, especially highlighting recent investigations that have identified mechanisms by which contractile forces and amino acids modulate muscle health. Furthermore, this review will explore integrated exercise and nutrition strategies that promote the maintenance of muscle health by optimizing exercise, and amino acid-induced cell signaling in aging adults susceptible to muscle loss.

  7. Exercise and Amino Acid Anabolic Cell Signaling and the Regulation of Skeletal Muscle Mass

    Directory of Open Access Journals (Sweden)

    Stefan M. Pasiakos

    2012-07-01

    Full Text Available A series of complex intracellular networks influence the regulation of skeletal muscle protein turnover. In recent years, studies have examined how cellular regulators of muscle protein turnover modulate metabolic mechanisms contributing to the loss, gain, or conservation of skeletal muscle mass. Exercise and amino acids both stimulate anabolic signaling potentially through several intracellular pathways including the mammalian target of rapamycin complex 1 and the mitogen activated protein kinase cell signaling cascades. As novel molecular regulators of muscle integrity continue to be explored, a contemporary analysis of the literature is required to understand the metabolic mechanisms by which contractile forces and amino acids affect cellular process that contribute to long-term adaptations and preservation of muscle mass. This article reviews the literature related to how exercise and amino acid availability affect cellular regulators of skeletal muscle mass, especially highlighting recent investigations that have identified mechanisms by which contractile forces and amino acids modulate muscle health. Furthermore, this review will explore integrated exercise and nutrition strategies that promote the maintenance of muscle health by optimizing exercise, and amino acid-induced cell signaling in aging adults susceptible to muscle loss.

  8. Inductively coupled plasma mass spectrometer with axial field in a quadrupole reaction cell.

    Science.gov (United States)

    Bandura, Dmitry R; Baranov, Vladimir I; Tanner, Scott D

    2002-10-01

    A novel reaction cell for ICP-MS with an electric field provided inside the quadrupole along its axis is described. The field is implemented via a DC bias applied to additional auxiliary electrodes inserted between the rods of the quadrupole. The field reduces the settling time of the pressurized quadrupole when its mass bandpass is dynamically tuned. It also improves the transmission of analyte ions. It is shown that for the pressurized cell with the field activated, the recovery time for a change in quadrupole operating parameters is reduced to 610 times, respectively. The use of a retarding axial field for in-situ energy discrimination against cluster and polyatomic ions is shown. When the cell is pressurized with O2 for suppression of 129Xe+, the formation of 127IH2+ by reactions with gas impurities limits the detection of 129I to isotopic abundance of approximately 10(-6). In-cell energy discrimination against 127IH2+ utilizing a retarding axial field is shown to reduce the abundance of the background at m/z = 129 to ca. 3 x 10(-8) of the 127I+ signal. In-cell energy discrimination against 127IH2+ is shown to cause less I+ loss than a post-cell potential energy barrier for the same degree of 127IH2+ suppression.

  9. Extracellular mass transport considerations for space flight research concerning suspended and adherent in vitro cell cultures

    Science.gov (United States)

    Klaus, David M.; Benoit, Michael R.; Nelson, Emily S.; Hammond, Timmothy G.

    2004-01-01

    Conducting biological research in space requires consideration be given to isolating appropriate control parameters. For in vitro cell cultures, numerous environmental factors can adversely affect data interpretation. A biological response attributed to microgravity can, in theory, be explicitly correlated to a specific lack of weight or gravity-driven motion occurring to, within or around a cell. Weight can be broken down to include the formation of hydrostatic gradients, structural load (stress) or physical deformation (strain). Gravitationally induced motion within or near individual cells in a fluid includes sedimentation (or buoyancy) of the cell and associated shear forces, displacement of cytoskeleton or organelles, and factors associated with intra- or extracellular mass transport. Finally, and of particular importance for cell culture experiments, the collective effects of gravity must be considered for the overall system consisting of the cells, their environment and the device in which they are contained. This does not, however, rule out other confounding variables such as launch acceleration, on orbit vibration, transient acceleration impulses or radiation, which can be isolated using onboard centrifuges or vibration isolation techniques. A framework is offered for characterizing specific cause-and-effect relationships for gravity-dependent responses as a function of the above parameters.

  10. Characterization of the Viable but Nonculturable (VBNC State in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Mohammad Salma

    Full Text Available The Viable But Non Culturable (VBNC state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable population and the viable population demonstrated the presence of viable cells that were non culturable. In addition, removal of the stress by increasing the pH of the medium at different time intervals into the VBNC state allowed the VBNC S. cerevisiae cells to "resuscitate". The similarity between the cell cycle profiles of VBNC cells and cells exiting the VBNC state together with the generation rate of cells exiting VBNC state demonstrated the absence of cellular multiplication during the exit from the VBNC state. This provides evidence of a true VBNC state. To get further insight into the molecular mechanism pertaining to the VBNC state, we studied the involvement of the SSU1 gene, encoding a sulfite pump in S. cerevisiae. The physiological behavior of wild-type S. cerevisiae was compared to those of a recombinant strain overexpressing SSU1 and null Δssu1 mutant. Our results demonstrated that the SSU1 gene is only implicated in the first stages of sulfite resistance but not per se in the VBNC phenotype. Our study clearly demonstrated the existence of an SO2-induced VBNC state in S. cerevisiae and that the stress removal allows the "resuscitation" of VBNC cells during the VBNC state.

  11. Changes in cancer cell metabolism revealed by direct sample analysis with MALDI mass spectrometry.

    Directory of Open Access Journals (Sweden)

    David A Pirman

    Full Text Available Biomarker discovery using mass spectrometry (MS has recently seen a significant increase in applications, mainly driven by the rapidly advancing field of metabolomics. Instrumental and data handling advancements have allowed for untargeted metabolite analyses which simultaneously interrogate multiple biochemical pathways to elucidate disease phenotypes and therapeutic mechanisms. Although most MS-based metabolomic approaches are coupled with liquid chromatography, a few recently published studies used matrix-assisted laser desorption (MALDI, allowing for rapid and direct sample analysis with minimal sample preparation. We and others have reported that prostaglandin E3 (PGE3, derived from COX-2 metabolism of the omega-3 fatty acid eicosapentaenoic acid (EPA, inhibited the proliferation of human lung, colon and pancreatic cancer cells. However, how PGE3 metabolism is regulated in cancer cells, particularly human non-small cell lung cancer (NSCLC cells, is not fully understood. Here, we successfully used MALDI to identify differences in lipid metabolism between two human non-small-cell lung cancer (NSCLC cell lines, A549 and H596, which could contribute to their differential response to EPA treatment. Analysis by MALDI-MS showed that the level of EPA incorporated into phospholipids in H596 cells was 4-fold higher than A549 cells. Intriguingly, H596 cells produced much less PGE3 than A549 cells even though the expression of COX-2 was similar in these two cell lines. This appears to be due to the relatively lower expression of cytosolic phospholipase A2 (cPLA2 in H596 cells than that of A549 cells. Additionally, the MALDI-MS approach was successfully used on tumor tissue extracts from a K-ras transgenic mouse model of lung cancer to enhance our understanding of the mechanism of action of EPA in the in vivo model. These results highlight the utility of combining a metabolomics workflow with MALDI-MS to identify the biomarkers that may regulate the

  12. Human beta cell mass and function in diabetes: Recent advances in knowledge and technologies to understand disease pathogenesis

    Directory of Open Access Journals (Sweden)

    Chunguang Chen

    2017-09-01

    Major Conclusions: In contrast to early disease models, recent studies suggest that in type 1 and type 2 diabetes impairment of beta cell function is an early feature of disease pathogenesis while a substantial decrease in beta cell mass occurs more closely to clinical manifestation. This suggests that, in addition to beta cell mass replacement for late stage therapies, the development of novel strategies for protection and recovery of beta cell function could be most promising for successful diabetes treatment and prevention. The use of today's developing and wide range of technologies and platforms for the study of human beta cells will allow for a more detailed investigation of the underlying mechanisms and will facilitate development of treatment approaches to specifically target human beta cell mass and function.

  13. Non-viable Borrelia burgdorferi induce inflammatory mediators and apoptosis in human oligodendrocytes.

    Science.gov (United States)

    Parthasarathy, Geetha; Fevrier, Helene B; Philipp, Mario T

    2013-11-27

    In previous studies, exposure to live Borrelia burgdorferi was shown to induce inflammation and apoptosis of human oligodendrocytes. In this study we assessed the ability of non-viable bacteria (heat killed or sonicated) to induce inflammatory mediators and cell death. Both heat-killed and sonicated bacteria induced release of CCL2, IL-6, and CXCL8 from oligodendrocytes in a dose dependent manner. In addition, non-viable B. burgdorferi also induced cell death as evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and another cell viability assay. These results suggest that spirochetal residues left after bacterial demise, due to treatment or otherwise, may continue to be pathogenic to the central nervous system. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. Comparison of clustering methods for high-dimensional single-cell flow and mass cytometry data.

    Science.gov (United States)

    Weber, Lukas M; Robinson, Mark D

    2016-12-01

    Recent technological developments in high-dimensional flow cytometry and mass cytometry (CyTOF) have made it possible to detect expression levels of dozens of protein markers in thousands of cells per second, allowing cell populations to be characterized in unprecedented detail. Traditional data analysis by "manual gating" can be inefficient and unreliable in these high-dimensional settings, which has led to the development of a large number of automated analysis methods. Methods designed for unsupervised analysis use specialized clustering algorithms to detect and define cell populations for further downstream analysis. Here, we have performed an up-to-date, extensible performance comparison of clustering methods for high-dimensional flow and mass cytometry data. We evaluated methods using several publicly available data sets from experiments in immunology, containing both major and rare cell populations, with cell population identities from expert manual gating as the reference standard. Several methods performed well, including FlowSOM, X-shift, PhenoGraph, Rclusterpp, and flowMeans. Among these, FlowSOM had extremely fast runtimes, making this method well-suited for interactive, exploratory analysis of large, high-dimensional data sets on a standard laptop or desktop computer. These results extend previously published comparisons by focusing on high-dimensional data and including new methods developed for CyTOF data. R scripts to reproduce all analyses are available from GitHub (https://github.com/lmweber/cytometry-clustering-comparison), and pre-processed data files are available from FlowRepository (FR-FCM-ZZPH), allowing our comparisons to be extended to include new clustering methods and reference data sets. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC. © 2016 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

  15. Neurotransmitters and Neuropeptides: New Players in the Control of Islet of Langerhans' Cell Mass and Function.

    Science.gov (United States)

    Di Cairano, Eliana S; Moretti, Stefania; Marciani, Paola; Sacchi, Vellea Franca; Castagna, Michela; Davalli, Alberto; Folli, Franco; Perego, Carla

    2016-04-01

    Islets of Langerhans control whole body glucose homeostasis, as they respond, releasing hormones, to changes in nutrient concentrations in the blood stream. The regulation of hormone secretion has been the focus of attention for a long time because it is related to many metabolic disorders, including diabetes mellitus. Endocrine cells of the islet use a sophisticate system of endocrine, paracrine and autocrine signals to synchronize their activities. These signals provide a fast and accurate control not only for hormone release but also for cell differentiation and survival, key aspects in islet physiology and pathology. Among the different categories of paracrine/autocrine signals, this review highlights the role of neurotransmitters and neuropeptides. In a manner similar to neurons, endocrine cells synthesize, accumulate, release neurotransmitters in the islet milieu, and possess receptors able to decode these signals. In this review, we provide a comprehensive description of neurotransmitter/neuropetide signaling pathways present within the islet. Then, we focus on evidence supporting the concept that neurotransmitters/neuropeptides and their receptors are interesting new targets to preserve β-cell function and mass. A greater understanding of how this network of signals works in physiological and pathological conditions would advance our knowledge of islet biology and physiology and uncover potentially new areas of pharmacological intervention. J. Cell. Physiol. 231: 756-767, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  16. Regeneration of pancreatic beta-cell mass for the treatment of diabetes.

    Science.gov (United States)

    Domínguez-Bendala, Juan; Inverardi, Luca; Ricordi, Camillo

    2012-06-01

    The study of the endocrine compartment of the pancreas (the islets of Langerhans) is of great translational interest, as strategies aimed at restoring its mass could become therapies for glycemic dysregulation in type 1 and 2 diabetes mellitus, drug-related diabetes following diabetogenic therapies or hyperglycemic disturbances following the treatment of cancer and nesidioblastosis. Such strategies generally fall under one of the 'three Rs,' namely, replacement (islet transplantation and stem cell differentiation), reprogramming (chiefly from the exocrine compartment of the pancreas) and regeneration (replication and induction of endogenous stem cells). This expert opinion focuses on the latter, as islets are known to regenerate under specific circumstances of physiological (e.g., pregnancy), pathological (e.g., obesity, hyperglycemia, mutations in the glucose-sensing pathway) or experimental (e.g., partial pancreatectomy, cellophane wrapping, partial duct ligation) nature. This review presents the different models of pancreatic regeneration, which encompass the replication of existing beta-cells, reversible epithelial-to-mesenchymal transition and the reactivation of resident stem cells. Rather than a set mechanism, the pancreas appears to possess a wide range of facultative regeneration pathways. These are discussed in the context of the development of potential strategies aimed at restoring beta-cell function in insulin-dependent diabetes.

  17. Identification of biomolecule mass transport and binding rate parameters in living cells by inverse modeling

    Directory of Open Access Journals (Sweden)

    Shirmohammadi Adel

    2006-10-01

    Full Text Available Abstract Background Quantification of in-vivo biomolecule mass transport and reaction rate parameters from experimental data obtained by Fluorescence Recovery after Photobleaching (FRAP is becoming more important. Methods and results The Osborne-Moré extended version of the Levenberg-Marquardt optimization algorithm was coupled with the experimental data obtained by the Fluorescence Recovery after Photobleaching (FRAP protocol, and the numerical solution of a set of two partial differential equations governing macromolecule mass transport and reaction in living cells, to inversely estimate optimized values of the molecular diffusion coefficient and binding rate parameters of GFP-tagged glucocorticoid receptor. The results indicate that the FRAP protocol provides enough information to estimate one parameter uniquely using a nonlinear optimization technique. Coupling FRAP experimental data with the inverse modeling strategy, one can also uniquely estimate the individual values of the binding rate coefficients if the molecular diffusion coefficient is known. One can also simultaneously estimate the dissociation rate parameter and molecular diffusion coefficient given the pseudo-association rate parameter is known. However, the protocol provides insufficient information for unique simultaneous estimation of three parameters (diffusion coefficient and binding rate parameters owing to the high intercorrelation between the molecular diffusion coefficient and pseudo-association rate parameter. Attempts to estimate macromolecule mass transport and binding rate parameters simultaneously from FRAP data result in misleading conclusions regarding concentrations of free macromolecule and bound complex inside the cell, average binding time per vacant site, average time for diffusion of macromolecules from one site to the next, and slow or rapid mobility of biomolecules in cells. Conclusion To obtain unique values for molecular diffusion coefficient and

  18. Prognostic value of body mass index before treatment for laryngeal squamous cell carcinoma.

    Science.gov (United States)

    Li, Zhao-Qu; Zou, Lan; Liu, Tian-Run; Yang, An-Kui

    2015-12-01

    Patients with head and neck cancer often suffer from malnutrition. This study aims to investigate the influence of body mass index (BMI) on the prognosis of laryngeal squamous cell carcinoma (LSCC). A total of 473 patients with LSCC initially treated at Sun Yat-sen University Cancer Center between January 2005 and July 2009 were retrospectively reviewed. Survival analysis was performed by the Kaplan-Meier method and Cox regression model. Low BMI before treatment was significantly associated with poor overall survival in patients with LSCC (Pnutritional status is favorable to improve survival in patients with LSCC.

  19. Mass and charge transfer on various relevant scales in polymer electrolyte fuel cells[Dissertation 16991

    Energy Technology Data Exchange (ETDEWEB)

    Freunberger, S. A.

    2007-07-01

    This dissertation is concerned with the development, experimental diagnostics and mathematical modelling and simulation of polymer electrolyte fuel cells (PEFC). The central themes throughout this thesis are the closely interlinked phenomena of mass and charge transfer. In the face of developing a PEFC system for vehicle propulsion these phenomena are scrutinized on a broad range of relevant scales. Starting from the material related level of the membrane and the gas diffusion layer (GDL) we turn to length scales, where structural features of the cell additionally come into play. These are the scale of flow channels and ribs, the single cell and the cell stack followed by the cell, stack, and system development for an automotive power train. In Chapter 3 selected fundamental material models and properties, respectively, are explored that are crucial for the mathematical modelling and simulation of PEFC, as needed in some succeeding parts of this work. First, established mathematical models for mass and charge transfer in the membrane are compared within the framework of the membrane electrode assembly (MEA), which represents the electrochemical unit. Second, reliable values for effective diffusivities in the GDLs which are vital for the simulation of gaseous mass transport are measured. Therefore, a method is developed that allows measuring this quantity both as a function of compression and direction as this is a prerequisite of sophisticated more-dimensional numerical PEFC-models. Besides the cross section of the catalyst layer (CL) mass transfer under channels and ribs is considered as a major source of losses in particular under high load operation. As up to now there have been solely non-validated theoretical investigations, in Chapter 4 an experimental method is developed that is for the first time capable of resolving the current density distribution on the this scale. For this, the electron conductors in the cell are considered as 2-dimensional shunt

  20. Advances in Studies of Electrode Kinetics and Mass Transport in AMTEC Cells (abstract)

    Science.gov (United States)

    Williams, R. M.; Jeffries-Nakamura, B.; Ryan, M. A.; Underwood, M. L.; Kisor, A.; O'Connor, D.; Kikkert, S.

    1993-01-01

    Previous work reported from JPL has included characterization of electrode kinetics and alkali atom transport from electrodes including Mo, W, WRh(sub x), WPt(sub x)(Mn), in sodium AMTEC cells and vapor exposure cells, and Mo in potassium vapor exposure cells. These studies were generally performed in cells with small area electrodes (about 1 to 5 cm(sup 2)), and device geometry had little effect on transport. Alkali diffusion coefficients through these electrodes have been characterized, and approximate surface diffusion coefficients derived in cases of activated transport. A basic model of electrode kinetic at the alkali metal vapor/porous metal electrode/alkali beta'-alumina solid electrolyte three phase boundary has been proposed which accounts for electrochemical reaction rates with a collision frequency near the three phase boundary and tunneling from the porous electrode partially covered with adsorbed alkali metal atoms. The small electrode effect in AMTEC cells has been discussed in several papers, but quantitative investigations have described only the overall effect and the important contribution of electrolyte resistance. The quantitative characterization of transport losses in cells with large area electrodes has been limited to simulations of large area electrode effects, or characterization of transport losses from large area electrodes with significant longitudinal temperature gradients. This paper describes new investigations of electrochemical kinetics and transport, particularily with WPt(sub 3.5) electrodes, including the influence of electrode size on the mass transport loss in the AMTEC cell. These electrodes possess excellent sodium transport properties making verification of device limitations on transport much more readily attained.

  1. Primary angiitis of the central nervous system with diffuse cerebral mass effect and giant cells.

    LENUS (Irish Health Repository)

    Kinsella, J A

    2012-02-01

    Primary angiitis of the central nervous system (PACNS), also called primary CNS vasculitis, is an idiopathic inflammatory condition affecting only intracranial and spinal cord vessels, particularly medium-sized and smaller arteries and arterioles. Angiography and histopathology typically do not reveal evidence of systemic vasculitis.(1,2) Histopathology usually reveals granulomatous inflammation affecting arterioles and small arteries of the parenchyma and\\/or leptomeninges, similar to that seen in Takayasu\\'s or giant cell arteritis.(1-3) We report a patient with biopsy-proven PACNS with giant cells and cerebral mass effect on MRI. Magnetic resonance angiography and cerebral angiography appeared normal and there was no evidence of extracranial vasculitis.

  2. PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

    Science.gov (United States)

    Venkateswaran, Kasthuri J.; Stam, Christina N.; Andersen, Gary L.; DeSantis, Todd

    2011-01-01

    Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated

  3. Selenium Attenuates HPV-18 Associated Apoptosis in Embryo-Derived Trophoblastic Cells but Not Inner Cell Mass In Vitro

    Directory of Open Access Journals (Sweden)

    Jennifer A. Tolen

    2015-01-01

    Full Text Available Objectives. Human papillomaviruses (HPV are associated with cell cycle arrest. This study focused on antioxidant selenomethionine (SeMet inhibition of HPV-mediated necrosis. The objectives were to determine HPV-18 effects on embryonic cells and to evaluate SeMet in blocking HPV-18 effects. Methods. Fertilized mouse embryos were cultured for 5 days to implanted trophoblasts and exposed to either control medium (group 1, HPV-18 (group 2, combined HPV-18 and 0.5 µM SeMet (group 3, or combined HPV-18 and 5.0 µM SeMet (group 4. After 48 hrs, trophoblast integrity and, apoptosis/necrosis were assessed using morphometric and dual-stain fluorescence assays, respectively. Results. HPV-18 exposed trophoblasts nuclei (253.8 ± 28.5 sq·µ were 29% smaller than controls (355.6 ± 35.9 sq·µ. Supplementation with 0.5 and 5.0 µM SeMet prevented nuclear shrinkage after HPV-18 exposure. HPV-18 infected trophoblasts remained larger with SeMet supplementation. HPV-18 decreased cell viability by 44% but SeMet supplementation sustained cell viability. Apoptosis was lower when SeMet was present. HPV-18 decreased inner cell mass (ICM viability by over 60%. Conclusions. HPV-18 decreased nuclear size and trophoblast viability but these effects were attenuated by the antioxidant SeMet. SeMet blocked HPV-18 associated apoptosis process in trophoblasts but not ICM cells suggesting involvement of different oxidative stress pathways.

  4. Fate of viable but non-culturable Listeria monocytogenes in pig manure microcosms

    Directory of Open Access Journals (Sweden)

    Jeremy eDesneux

    2016-03-01

    Full Text Available The fate of two strains of L. monocytogenes and their ability to become viable but non-culturable (VBNC was investigated in microcosms containing piggery effluents (two raw manures and two biologically treated manures stored for two months at 8°C and 20°C. Levels of L. monocytogenes were estimated using the culture method, qPCR, and propidium monoazide treatment combined with qPCR (qPCRPMA. The chemical composition and the microbial community structure of the manures were also analysed. The strains showed similar decline rates and persisted up to 63 days. At day zero, the percentage of VBNC cells among viable cells was higher in raw manures (81.5-94.8% than in treated manures (67.8-79.2%. The changes in their proportion over time depended on the temperature and on the type of effluent: the biggest increase was observed in treated manures at 20°C and the smallest increase in raw manures at 8°C. The chemical parameters had no influence on the behaviour of the strains, but decrease of the persistence of viable cells was associated with an increase in the microbial richness of the manures. This study demonstrated that storing manure altered the culturability of L. monocytogenes, which rapidly entered the VBNC state, and underlines the importance of including VBNC cells when estimating the persistence of the pathogens in farm effluents.

  5. Glioma Surgical Aspirate: A Viable Source of Tumor Tissue for Experimental Research

    Directory of Open Access Journals (Sweden)

    Perry F. Bartlett

    2013-04-01

    Full Text Available Brain cancer research has been hampered by a paucity of viable clinical tissue of sufficient quality and quantity for experimental research. This has driven researchers to rely heavily on long term cultured cells which no longer represent the cancers from which they were derived. Resection of brain tumors, particularly at the interface between normal and tumorigenic tissue, can be carried out using an ultrasonic surgical aspirator (CUSA that deposits liquid (blood and irrigation fluid and resected tissue into a sterile bottle for disposal. To determine the utility of CUSA-derived glioma tissue for experimental research, we collected 48 CUSA specimen bottles from glioma patients and analyzed both the solid tissue fragments and dissociated tumor cells suspended in the liquid waste fraction. We investigated if these fractions would be useful for analyzing tumor heterogeneity, using IHC and multi-parameter flow cytometry; we also assessed culture generation and orthotopic xenograft potential. Both cell sources proved to be an abundant, highly viable source of live tumor cells for cytometric analysis, animal studies and in-vitro studies. Our findings demonstrate that CUSA tissue represents an abundant viable source to conduct experimental research and to carry out diagnostic analyses by flow cytometry or other molecular diagnostic procedures.

  6. Body cell mass evaluation in critically ill patients: killing two birds with one stone.

    Science.gov (United States)

    Fiaccadori, Enrico; Morabito, Santo; Cabassi, Aderville; Regolisti, Giuseppe

    2014-05-01

    Body cell mass (BCM) is the metabolically active cell mass involved in O₂ consumption, CO₂ production and energy expenditure. BCM measurement has been suggested as a tool for the evaluation of nutritional status. Since BCM is closely related to energy expenditure, it could also represent a good reference value for the calculation of nutrient needs. In a recent issue of Critical Care, Ismael and colleagues used bioelectrical impedance analysis parameters and anthropometric variables to evaluate BCM in patients with acute kidney injury, before and after a hemodialysis session. The results of this study suggest that BCM is relatively insensitive to major body fluid shifts, a well known factor interfering with nutritional evaluation/monitoring and energy need calculations in the ICU. Thus, BCM seems to be a more 'stable' nutritional variable, as it is apparently less influenced by non-nutritional factors. The results of this paper emphasize the need to identify biologically sound parameters for nutritional status evaluation and energy need calculation in critically ill patients; in this regard, BCM could fulfill these expectations.

  7. Clear Cell Carcinoma Presented as a Large Polypoid Mass Expanding the Vaginal Fornix: Report of Two Cases

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Young; Cho, Jae Ho [Dept. of Radiology, College of Medicine, Yeungnam University, Daegu (Korea, Republic of)

    2012-11-15

    Primary clear cell carcinoma of the vagina or uterine cervix is a very rare tumor. We report radiologic findings of two cases of clear cell carcinoma, arising in the vagina and uterine cervix in a 16-year-old and a 26-year-old female. These were presented as a large polypoid mass with a stalk and expanding the vaginal fornix. One case with ultrasonography showed relatively homogeneous echoic solid mass; the other case with a CT showed heterogeneously and strongly enhancing mass. All of the two cases showed non-specific signal intensity with heterogeneous and strong enhancement on MRI.

  8. Glucagon Couples Hepatic Amino Acid Catabolism to mTOR-Dependent Regulation of α-Cell Mass

    Directory of Open Access Journals (Sweden)

    Mark J. Solloway

    2015-07-01

    Full Text Available Understanding the regulation of islet cell mass has important implications for the discovery of regenerative therapies for diabetes. The liver plays a central role in metabolism and the regulation of endocrine cell number, but liver-derived factors that regulate α-cell and β-cell mass remain unidentified. We propose a nutrient-sensing circuit between liver and pancreas in which glucagon-dependent control of hepatic amino acid metabolism regulates α-cell mass. We found that glucagon receptor inhibition reduced hepatic amino acid catabolism, increased serum amino acids, and induced α-cell proliferation in an mTOR-dependent manner. In addition, mTOR inhibition blocked amino-acid-dependent α-cell replication ex vivo and enabled conversion of α-cells into β-like cells in vivo. Serum amino acids and α-cell proliferation were increased in neonatal mice but fell throughout postnatal development in a glucagon-dependent manner. These data reveal that amino acids act as sensors of glucagon signaling and can function as growth factors that increase α-cell proliferation.

  9. The induction of tumour suppressor protein P53 limits the entry of cells into the pluripotent inner cell mass lineage in the mouse embryo.

    Science.gov (United States)

    Ganeshan, L; Jin, X L; O'Neill, C

    2017-09-15

    The early preimplantation embryo is susceptible to a range of exogenous stresses which result in their reduced long-term developmental potential. The P53 tumour suppressor protein is normally held at low levels in the preimplantation embryo and we show that culture stress induces the expression of a range of canonical P53-response genes (Mdm2, Bax and Cdkn1a). Culture stress caused a P53-dependent loss of cells from resulting blastocysts, and this was most evident within the inner cell mass population. Culture stress increased the proportion of cells expressing active caspase-3 and undergoing apoptosis, while inhibition of caspase-3 increased the number of cells within the inner cell mass. The P53-dependent loss of cells from the inner cell mass was accompanied by a loss of NANOG-positive epiblast progenitors. Pharmacological activation of P53 by the MDM2 inhibitor, Nutlin-3, also caused increased P53-dependent transcription and the loss of cells from the inner cell mass. This loss of cells could be ameliorated by simultaneous treatment with the P53 inhibitor, Pifithrin-α. Culture stress causes reduced signalling via the phosphatidylinositol-3-kinase signalling pathway, and blocking this pathway caused P53-dependent loss of cells from the inner cell mass. These results point to P53 acting to limit the accumulation and survival of cells within the pluripotent lineage of the blastocyst and provide a molecular framework for the further investigation of the factors determining the effects of stressors on the embryo's developmental potential. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  10. Mitochondrial respiration in human viable platelets-Methodology and influence of gender, age and storage

    DEFF Research Database (Denmark)

    Sjövall, Fredrik; Ehinger, Johannes K H; Marelsson, Sigurður E

    2013-01-01

    Studying whole cell preparations with intact mitochondria and respiratory complexes has a clear benefit compared to isolated or disrupted mitochondria due to the dynamic interplay between mitochondria and other cellular compartments. Platelet mitochondria have a potential to serve as a source...... of human viable mitochondria when studying mitochondrial physiology and pathogenic mechanisms, as well as for the diagnostics of mitochondrial diseases. The objective of the present study was to perform a detailed evaluation of platelet mitochondrial respiration using high-resolution respirometry. Further...

  11. Increased mitochondrial mass in cells with functionally compromised mitochondria after exposure to both direct gamma radiation and bystander factors.

    LENUS (Irish Health Repository)

    Nugent, Sharon M E

    2007-07-01

    The bystander effect describes radiation-like damage in unirradiated cells either in the vicinity of irradiated cells or exposed to medium from irradiated cells. This study aimed to further characterize the poorly understood mitochondrial response to both direct irradiation and bystander factor(s) in human keratinocytes (HPV-G) and Chinese hamster ovarian cells (CHO-K1). Oxygen consumption rates were determined during periods of state 4, state 3 and uncoupled respiration. Mitochondrial mass was determined using MitoTracker FM. CHO-K1 cells showed significantly reduced oxygen consumption rates 4 h after exposure to 5 Gy direct radiation and irradiated cell conditioned medium (ICCM) and an apparent recovery 12-24 h later. The apparent recovery was likely due to the substantial increase in mitochondrial mass observed in these cells as soon as 4 h after exposure. HPV-G cells, on the other hand, showed a sustained increase in oxygen consumption rates after ICCM exposure and a transient increase 4 h after exposure to 5 Gy direct radiation. A significant increase in mitochondrial mass per HPV-G cell was observed after exposure to both direct radiation and ICCM. These findings are indicative of a stress response to mitochondrial dysfunction that increases the number of mitochondria per cell.

  12. Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology

    Science.gov (United States)

    Tanner, Scott D.; Ornatsky, Olga; Bandura, Dmitry R.; Baranov, Vladimir I.

    2007-03-01

    Recent progress in the development of massively multiplexed bioanalytical assays using element tags with inductively coupled plasma mass spectrometry detection is reviewed. Feasibility results using commercially available secondary immunolabeling reagents for leukemic cell lines are presented. Multiplex analysis of higher order is shown with first generation tag reagents based on functionalized carriers that bind lanthanide ions. DNA quantification using metallointercalation allows for cell enumeration or mitotic state differentiation. In situ hybridization permits the determination of cellular RNA. The results provide a feasibility basis for the development of a multivariate assay tool for individual cell analysis based on inductively coupled plasma mass spectrometry in a cytometer configuration.

  13. Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology

    Energy Technology Data Exchange (ETDEWEB)

    Tanner, Scott D. [Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 164 College Street, Toronto, Ontario, M5S 3G9 (Canada)], E-mail: sd.tanner@utoronto.ca; Ornatsky, Olga; Bandura, Dmitry R.; Baranov, Vladimir I. [Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 164 College Street, Toronto, Ontario, M5S 3G9 (Canada)

    2007-03-15

    Recent progress in the development of massively multiplexed bioanalytical assays using element tags with inductively coupled plasma mass spectrometry detection is reviewed. Feasibility results using commercially available secondary immunolabeling reagents for leukemic cell lines are presented. Multiplex analysis of higher order is shown with first generation tag reagents based on functionalized carriers that bind lanthanide ions. DNA quantification using metallointercalation allows for cell enumeration or mitotic state differentiation. In situ hybridization permits the determination of cellular RNA. The results provide a feasibility basis for the development of a multivariate assay tool for individual cell analysis based on inductively coupled plasma mass spectrometry in a cytometer configuration.

  14. Non-Breeding Eusocial Mole-Rats Produce Viable Sperm--Spermiogram and Functional Testicular Morphology of Fukomys anselli.

    Directory of Open Access Journals (Sweden)

    Angelica Garcia Montero

    Full Text Available Ansell's mole-rats (Fukomys anselli are subterranean rodents living in families composed of about 20 members with a single breeding pair and their non-breeding offspring. Most of them remain with their parents for their lifetime and help to maintain and defend the natal burrow system, forage, and care for younger siblings. Since incest avoidance is based on individual recognition (and not on social suppression we expect that non-breeders produce viable sperm spontaneously. We compared the sperm of breeding and non-breeding males, obtained by electroejaculation and found no significant differences in sperm parameters between both groups. Here, we used electroejaculation to obtain semen for the first time in a subterranean mammal. Spermiogram analysis revealed no significant differences in sperm parameters between breeders and non-breeders. We found significantly larger testes (measured on autopsies and on living animals per ultrasonography of breeders compared to non-breeders (with body mass having a significant effect. There were no marked histological differences between breeding and non-breeding males, and the relative area occupied by Leydig cells and seminiferous tubules on histological sections, respectively, was not significantly different between both groups. The seminiferous epithelium and to a lesser degree the interstitial testicular tissue are characterized by lesions (vacuolar degenerations, however, this feature does not hinder fertilization even in advanced stages of life. The continuous production of viable sperm also in sexually abstinent non-breeders might be best understood in light of the mating and social system of Fukomys anselli, and the potential to found a new family following an unpredictable and rare encounter with an unfamiliar female ("provoked or induced dispersal". Apparently, the non-breeders do not reproduce because they do not copulate but not because they would be physiologically infertile. The significantly

  15. Non-Breeding Eusocial Mole-Rats Produce Viable Sperm—Spermiogram and Functional Testicular Morphology of Fukomys anselli

    Science.gov (United States)

    Garcia Montero, Angelica; Vole, Christiane; Burda, Hynek; Malkemper, Erich Pascal; Holtze, Susanne; Morhart, Michaela; Saragusty, Joseph; Hildebrandt, Thomas B.; Begall, Sabine

    2016-01-01

    Ansell’s mole-rats (Fukomys anselli) are subterranean rodents living in families composed of about 20 members with a single breeding pair and their non-breeding offspring. Most of them remain with their parents for their lifetime and help to maintain and defend the natal burrow system, forage, and care for younger siblings. Since incest avoidance is based on individual recognition (and not on social suppression) we expect that non-breeders produce viable sperm spontaneously. We compared the sperm of breeding and non-breeding males, obtained by electroejaculation and found no significant differences in sperm parameters between both groups. Here, we used electroejaculation to obtain semen for the first time in a subterranean mammal. Spermiogram analysis revealed no significant differences in sperm parameters between breeders and non-breeders. We found significantly larger testes (measured on autopsies and on living animals per ultrasonography) of breeders compared to non-breeders (with body mass having a significant effect). There were no marked histological differences between breeding and non-breeding males, and the relative area occupied by Leydig cells and seminiferous tubules on histological sections, respectively, was not significantly different between both groups. The seminiferous epithelium and to a lesser degree the interstitial testicular tissue are characterized by lesions (vacuolar degenerations), however, this feature does not hinder fertilization even in advanced stages of life. The continuous production of viable sperm also in sexually abstinent non-breeders might be best understood in light of the mating and social system of Fukomys anselli, and the potential to found a new family following an unpredictable and rare encounter with an unfamiliar female (“provoked or induced dispersal”). Apparently, the non-breeders do not reproduce because they do not copulate but not because they would be physiologically infertile. The significantly increased

  16. On the Mass and Heat Transfer in the Porous Electrode of a Fuel Cell

    Energy Technology Data Exchange (ETDEWEB)

    Revuelta Bayod, A.

    2004-07-01

    In the first part of this report a reduced model of the mass transport in the PEMFC cathode gas diffusion layer is formulated ro an interrogated flow field design of the cathode bipolar plate. The non-dimensional formulation of the problem allows to identify the leading parameters which determines the fundamental species distribution and flow field structure. The effect of the forced convection of the gases into the porous electrode, caused by the interrogated flow field, is quantified through the Peclet numbers of the active species, and the non-dimensional polarization curves are obtained. In the second part, the diffusion-thermal instability is analyzed in a porous gas diffusion layer (GDL) of a fuel cell. The investigation presented provides an initial guideline to future theoretical and experimental investigations on one aspect of the fuel cell performance not previously considered, with impact on the fuel cell life-time. Starting from the simples possible 1D-model of the flow into the porous electrode, the steady solution of the model is presented an analyzed depending on a minimum number of non-dimensional parameters. From this steady solution, a linear stability analysis is formulated, taking into account the temporal-spatial perturbations transversal to the gas flow direction, and the marginal stability regions are determined from the corresponding dispersion relation. (Author) 33 refs.

  17. Fatty acid profiling of blood cell membranes by gas chromatography with mass spectrometry.

    Science.gov (United States)

    Liu, Zequn; Weng, Rui; Feng, Yu; Li, Zehao; Wang, Lei; Su, Xin; Yu, Changyuan

    2016-10-01

    Fatty acids, which are well-known for their influence on human metabolism and signal transduction, are also a substantial component of cellular membranes and regulate the basic properties and functions of membranes. Owing to their multiple functions, fatty acid profiles of cell membranes are of great interest to those who are studying the relationship between membrane biochemical compositions and functions. A HCl-catalyzed derivation method and a gas chromatography with mass spectrometry analysis method were developed to accurately profile the fatty acids in cell membranes of erythrocytes, leukocytes, and platelets. The detection limits of all 35 fatty acids ranged from 0.58 to 22 ng/mL and the limits of quantitation were between 2.1 and 72 ng/mL. Finally, the established method was used to profile the membrane fatty acids of 44 healthy volunteers from the north and south of China. Results revealed significant differences in the fatty acid profiles from the two regions, particularly those of the erythrocytes. This technique may be applied to cell membrane studies to generate new biological hypotheses concerning fatty acid composition and membrane functions as well as to construct related disease profiles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Modulation of red cell mass by neocytolysis in space and on Earth

    Science.gov (United States)

    Rice, L.; Alfrey, C. P.

    2000-01-01

    Astronauts predictably experience anemia after return from space. Upon entering microgravity, the blood volume in the extremities pools centrally and plasma volume decreases, causing plethora and erythropoietin suppression. There ensues neocytolysis, selective hemolysis of the youngest circulating red cells, allowing rapid adaptation to the space environment but becoming maladaptive on re-entry to a gravitational field. The existence of this physiologic control process was confirmed in polycythemic high-altitude dwellers transported to sea level. Pathologic neocytolysis contributes to the anemia of renal failure. Understanding the process has implications for optimizing erythropoietin-dosing schedules and the therapy of other human disorders. Human and rodent models of neocytolysis are being created to help find out how interactions between endothelial cells, reticuloendothelial phagocytes and young erythrocytes are altered, and to shed light on the expression of surface adhesion molecules underlying this process. Thus, unraveling a problem for space travelers has uncovered a physiologic process controlling the red cell mass that can be applied to human disorders on Earth.

  19. Molecule-Specific Imaging Analysis of Carcinogens in Breast Cancer Cells Using Time-of-Flight Secondary Ion Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Quong, J N; Knize, M G; Kulp, K S; Wu, K J

    2003-08-19

    Imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) is used to study the localization of heterocyclic amines in MCF7 line of human breast cancer cells. The detection sensitivities of a model rodent mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were determined. Following an established criteria for the determination of status of freeze-fracture cells, the distribution of PhIP in the MCF7 cells are reported.

  20. Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR.

    Science.gov (United States)

    Liang, Ningjian; Dong, Jin; Luo, Laixin; Li, Yong

    2011-05-01

    Contamination of lettuce by Salmonella has caused serious public health problems. Polymerase chain reaction (PCR) allows rapid detection of pathogenic bacteria in food, but it is inaccurate as it might amplify DNA from dead target cells as well. This study aimed to investigate the stability of DNA of dead Salmonella cells in lettuce and to develop an approach to detecting viable Salmonella in lettuce. Salmonella-free lettuce was inoculated with heat-killed Salmonella Typhimurium cells and stored at 4 °C. Bacterial DNA extracted from the sample was amplified by real-time PCR targeting the invA gene. Our results indicate that DNA from the dead cells remained stable in lettuce for at least 8 d. To overcome this limitation, propidium monoazide (PMA), a dye that can selectively penetrate dead bacterial cells and cross-link their DNA upon light exposure, was combined with real-time PCR. Lettuce samples inoculated with different levels of dead or viable S. Typhimurium cells were treated or untreated with PMA before DNA extraction. Real-time PCR suggests that PMA treatment effectively prevented PCR amplification from as high as 10(8) CFU/g dead S. Typhimurium cells in lettuce. The PMA real-time PCR assay could detect viable Salmonella at as low as 10(2) CFU/mL in pure culture and 10(3) CFU/g in lettuce. With 12-h enrichment, S. Typhimurium of 10(1) CFU/g in lettuce was detectable. In conclusion, the PMA real-time PCR assay provides an alternative to real-time PCR assay for accurate detection of Salmonella in food. © 2011 Institute of Food Technologists®

  1. Quantitation of Human Metallothionein Isoforms in Cells, Tissues, and Cerebrospinal Fluid by Mass Spectrometry.

    Science.gov (United States)

    Shabb, J B; Muhonen, W W; Mehus, A A

    2017-01-01

    Metallothioneins (MTs) are a family of small, highly conserved, cysteine-rich metal-binding proteins that are important for zinc and copper homeostasis, protection against oxidative stress, and buffering against toxic heavy metals. Individual human MT isoforms are candidate biomarkers for heavy metal toxicity, and selected cancers and neurodegenerative diseases. The similar antigenicity of human MT-1 and MT-2 isoforms precludes development of antibody-based assays for their individual quantitation. Metal-based MT quantitation methods do not directly measure MT isoforms. A bottom-up mass spectrometry-based approach solves these problems by exploiting the unique masses and chromatographic properties of the acetylated N-terminal tryptic peptides of MT isoforms. These unusually hydrophilic 20- to 21-residue peptides contain five invariant cysteines. Strong cation exchange chromatography separates them from bulk internal tryptic peptides. Reversed-phase chromatography further separates them from more hydrophobic peptides of similar mass. Absolute quantitation is obtained by adding MT peptide standards alkylated with (15)N-iodoacetamide to biological samples alkylated with (14)N-iodoacetamide. Accurate quantitation is enhanced by dimethyl sulfide treatment to reverse oxidation of the N-terminal methionine. Originally optimized for measuring MT isoforms in cell lines, the method has been adapted to quantify MT isoforms in brain tissue and cerebrospinal fluid. The method can also be adapted for relative quantitation of MT isoforms between matched biological samples. It cannot be used to measure human MT-4 because of an arginine at position 4. Except for this type of limitation, the method is applicable to MT quantitation in many other species. © 2017 Elsevier Inc. All rights reserved.

  2. GLP-1 derivative liraglutide in rats with beta-cell deficiencies: influence of metabolic state on beta-cell mass dynamics

    DEFF Research Database (Denmark)

    Sturis, Jeppe; Gotfredsen, Carsten F; Rømer, John

    2003-01-01

    (1) Liraglutide is a long-acting GLP-1 derivative, designed for once daily administration in type II diabetic patients. To investigate the effects of liraglutide on glycemic control and beta-cell mass in rat models of beta-cell deficiencies, studies were performed in male Zucker diabetic fatty (ZDF...

  3. Association of pretreatment body mass index and survival in human papillomavirus positive oropharyngeal squamous cell carcinoma.

    Science.gov (United States)

    Albergotti, William G; Davis, Kara S; Abberbock, Shira; Bauman, Julie E; Ohr, James; Clump, David A; Heron, Dwight E; Duvvuri, Umamaheswar; Kim, Seungwon; Johnson, Jonas T; Ferris, Robert L

    2016-09-01

    Pretreatment body mass index (BMI) >25kg/m(2) is a positive prognostic factor in patients with head and neck cancer. Previous studies have not been adequately stratified by human papilloma virus (HPV) status or subsite. Our objective is to determine prognostic significance of pretreatment BMI on overall survival in HPV+ oropharyngeal squamous cell carcinoma (OPSCC). This is a retrospective review of patients with HPV+ OPSCC treated between 8/1/2006 and 8/31/2014. Patients were stratified by BMI status (>/25kg/m(2) had a longer overall survival (HR=0.49, P=0.01) as well as a longer disease-specific survival (HR=0.43, P=0.02). Overall survival remained significantly associated with high BMI on multivariate analysis (HR=0.54, P=0.04). Pre-treatment normal or underweight BMI status is associated with worse overall survival in HPV+ OPSCC. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Mass transport aspects of polymer electrolyte fuel cells under two-phase flow conditions

    Energy Technology Data Exchange (ETDEWEB)

    Kramer, D.

    2007-03-27

    This work deals with selected aspects of mass transport phenomena in PEFCs and DMFCs. Emphasis is placed on the implications originating from the occurrence of two-phase flow within these devices. Optimality of supply, distribution, and removal of the fuel, the oxidant, and the reaction products is of utmost importance for the stability, efficiency, and durability of the devices. Being a prerequisite for high current densities while maintaining sufficient voltage, mass transport optimization contributes to the development of cost effective as well as compact designs and hence competitive fuel cells. [German] Die Visualisierung und Quantifizierung von Fluessigwasseransammlungen in Polymerelektrolytmembran-Brennstoffzellen konnte mittels Neutronenradiographie erreicht werden. Dank dieser neuartigen diagnostischen Methode konnte erstmals die Fluessigwasseransammlung in den poroesen Gasdiffusionsschichten direkt nachgewiesen und quantifiziert werden. Die Kombination von Neutronenradiographie mit ortsaufgeloesten Stromdichtemessungen bzw. lokaler Impedanzspektroskopie erlaubte die Korrelation des inhomogenen Fluessigwasseranfalls mit dem lokalen elektrochemischen Leistungsverhalten. Systematische Untersuchungen an Polymerelektrolyt- und Direkt-Methanol-Brennstoffzellen verdeutlichen sowohl den Einfluss von Betriebsbedingungen als auch die Auswirkung von Materialeigenschaften auf die Ausbildung zweiphasiger Stroemungen.

  5. An advanced PCR method for the specific detection of viable total coliform bacteria in pasteurized milk.

    Science.gov (United States)

    Soejima, Takashi; Minami, Jun-ichi; Yaeshima, Tomoko; Iwatsuki, Keiji

    2012-07-01

    Pasteurized milk is a complex food that contains various inhibitors of polymerase chain reaction (PCR) and may contain a large number of dead bacteria, depending on the milking conditions and environment. Ethidium monoazide bromide (EMA)-PCR is occasionally used to distinguish between viable and dead bacteria in foods other than pasteurized milk. EMA is a DNA-intercalating dye that selectively permeates the compromised cell membranes of dead bacteria and cleaves DNA. Usually, EMA-PCR techniques reduce the detection of dead bacteria by up to 3.5 logs compared with techniques that do not use EMA. However, this difference may still be insufficient to suppress the amplification of DNA from dead Gram-negative bacteria (e.g., total coliform bacteria) if they are present in pasteurized milk in large numbers. Thus, false positives may result. We developed a new method that uses real-time PCR targeting of a long DNA template (16S-23S rRNA gene, principally 2,451 bp) following EMA treatment to completely suppress the amplification of DNA of up to 7 logs (10(7) cells) of dead total coliforms. Furthermore, we found that a low dose of proteinase K (25 U/ml) removed PCR inhibitors and simultaneously increased the signal from viable coliform bacteria. In conclusion, our simple protocol specifically detects viable total coliforms in pasteurized milk at an initial count of ≥1 colony forming unit (CFU)/2.22 ml within 7.5 h of total testing time. This detection limit for viable cells complies with the requirements for the analysis of total coliforms in pasteurized milk set by the Japanese Sanitation Act (which specifies <1 CFU/2.22 ml).

  6. Monosodium Glutamate Dietary Consumption Decreases Pancreatic β-Cell Mass in Adult Wistar Rats.

    Directory of Open Access Journals (Sweden)

    Piyanard Boonnate

    Full Text Available The amount of dietary monosodium glutamate (MSG is increasing worldwide, in parallel with the epidemics of metabolic syndrome. Parenteral administration of MSG to rodents induces obesity, hyperglycemia, hyperlipidemia, insulin resistance, and type 2 diabetes. However, the impact of dietary MSG is still being debated. We investigated the morphological and functional effects of prolonged MSG consumption on rat glucose metabolism and on pancreatic islet histology.Eighty adult male Wistar rats were randomly subdivided into 4 groups, and test rats in each group were supplemented with MSG for a different duration (1, 3, 6, or 9 months, n=20 for each group. All rats were fed ad libitum with a standard rat chow and water. Ten test rats in each group were provided MSG 2 mg/g body weight/day in drinking water and the 10 remaining rats in each group served as non-MSG treated controls. Oral glucose tolerance tests (OGTT were performed and serum insulin measured at 9 months. Animals were sacrificed at 1, 3, 6, or 9 months to examine the histopathology of pancreatic islets.MSG-treated rats had significantly lower pancreatic β-cell mass at 1, 6 and 9 months of study. Islet hemorrhages increased with age in all groups and fibrosis was significantly more frequent in MSG-treated rats at 1 and 3 months. Serum insulin levels and glucose tolerance in MSG-treated and untreated rats were similar at all time points we investigated.Daily MSG dietary consumption was associated with reduced pancreatic β-cell mass and enhanced hemorrhages and fibrosis, but did not affect glucose homeostasis. We speculate that high dietary MSG intake may exert a negative effect on the pancreas and such effect might become functionally significant in the presence or susceptibility to diabetes or NaCl; future experiments will take these crucial cofactors into account.

  7. Early peroxisome proliferator-activated receptor gamma regulated genes involved in expansion of pancreatic beta cell mass

    Directory of Open Access Journals (Sweden)

    Vivas Yurena

    2011-12-01

    Full Text Available Abstract Background The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansion Results Here we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell. Conclusions Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation.

  8. Dual-labelled antibodies for flow and mass cytometry: A new tool for cross-platform comparison and enrichment of target cells for mass cytometry.

    Science.gov (United States)

    Baumgart, Sabine; Schulz, Axel R; Peddinghaus, Anette; Stanislawiak, Silke; Gillert, Sarah; Hirseland, Heike; Krauthäuser, Susanne; Dose, Christian; Mei, Henrik E; Grützkau, Andreas

    2017-08-01

    Antibody conjugates applicable in both conventional flow and mass cytometry would offer interesting options for cross-platform comparison, as well as the enrichment of rare target cells by conventional flow cytometry (FC) sorting prior to deep phenotyping by mass cytometry (MC). Here, we introduce a simple method to generate dual fluorochrome/metal-labelled antibodies by consecutive orthogonal labelling. First, we compared different fluorochrome-conjugated antibodies specific for CD4, such as FITC, Vio667, VioGreen or VioBlue for their compatibility with the conventional secondary MAXPAR® labelling protocol. After labelling with 141 Pr, the fluorescence emission spectra of all fluorochromes investigated retained their characteristics, and CD4 dual conjugates (DCs) provided consistent results in immune phenotyping assays performed by FC and MC. The phenotypical composition of CD4+ T-cells was maintained after enrichment by FC sorting using different CD4 DCs. Finally, magnetic cell depletion was combined with FC sorting using CD19-VioBlue-142 Nd, CD20-VioGreen-147 Sm, CD27-Cy5-167 Er and CD38-Alexa488-143 Nd DC to enrich rare human plasmablasts to purities >80%, which allowed a subsequent deep phenotyping by MC. In conclusion, DCs have been successfully established for direct assay comparison between FC and MC, and help to minimise MC data acquisition time for deep phenotyping of rare cell subsets. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Microfiltration of enzyme treated egg whites for accelerated detection of viable Salmonella.

    Science.gov (United States)

    Ku, Seockmo; Ximenes, Eduardo; Kreke, Thomas; Foster, Kirk; Deering, Amanda J; Ladisch, Michael R

    2016-11-01

    We report detection of egg white within 7 h by concentrating the bacteria using microfiltration through 0.2-μm cutoff polyethersulfone hollow fiber membranes. A combination of enzyme treatment, controlled cross-flow on both sides of the hollow fibers, and media selection were key to controlling membrane fouling so that rapid concentration and the subsequent detection of low numbers of microbial cells were achieved. We leveraged the protective effect of egg white proteins and peptone so that the proteolytic enzymes did not attack the living cells while hydrolyzing the egg white proteins responsible for fouling. The molecular weight of egg white proteins was reduced from about 70 kDa to 15 kDa during hydrolysis. This enabled a 50-fold concentration of the cells when a volume of 525 mL of peptone and egg white, containing 13 CFU of Salmonella, was decreased to a 10 mL volume in 50 min. A 10-min microcentrifugation step further concentrated the viable Salmonella cells by 10×. The final cell recovery exceeded 100%, indicating that microbial growth occurred during the 3-h processing time. The experiments leading to rapid concentration, recovery, and detection provided further insights on the nature of membrane fouling enabling fouling effects to be mitigated. Unlike most membrane processes where protein recovery is the goal, recovery of viable microorganisms for pathogen detection is the key measure of success, with modification of cell-free proteins being both acceptable and required to achieve rapid microfiltration of viable microorganisms. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1464-1471, 2016. © 2016 American Institute of Chemical Engineers.

  10. A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

    Science.gov (United States)

    Yu, F. P.; Pyle, B. H.; McFeters, G. A.

    1993-01-01

    This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.

  11. The search for viable local government system in Nigeria: an ...

    African Journals Online (AJOL)

    The history of the Nigerian local government system has been one long episode of trails and errors aimed at achieving viable local government institution without much success. Local government in the country began its long series of reforms from the colonial period when the colonial government attempted to ...

  12. Comment: Towards a Viable Local Government Structure in Nigeria ...

    African Journals Online (AJOL)

    Local governments are principally established for development at the grassroots and they must be structured in a manner that makes them viable and capable of achieving this purpose. The objective of this comment is to appraise the current local government structure under the Nigerian constitutional framework with a view ...

  13. Cultivation and multiplication of viable axenic Trypanosoma vivax in ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-01

    Sep 1, 2009 ... Cultivation and multiplication of viable axenic. Trypanosoma vivax in vitro and in vivo. O. A. Idowu, A. B. Idowu, C. F. Mafiana and S. O. Sam-Wobo*. Parasitology Laboratory, Department of Biological Sciences, University of Agriculture, Abeokuta, Nigeria. Accepted 13 April, 2006. Trypanosoma vivax was ...

  14. Tuberculosis in postchemotherapy residual masses in germ cell tumor of the testis

    Directory of Open Access Journals (Sweden)

    Rajesh Bansal

    2011-01-01

    Full Text Available Residual masses following chemotherapy in testicular tumors have been characterized as necrosis, mature or immature teratoma, and malignant tumors. Twenty four patients had retroperitoneal lymph node dissection for postchemotherapy residual masses between January 2000 and December 2008. We report two patients; one with late relapse and other with postchemotherapy residual mass, who had tuberculosis. Tumor markers were normal, and PET scan showed increased uptake in residual mass. There are no previous reports of tuberculosis in postchemotherapy residual masses.

  15. Molecular approaches for viable bacterial population and transcriptional analyses in a rodent model of dental caries.

    Science.gov (United States)

    Klein, M I; Scott-Anne, K M; Gregoire, S; Rosalen, P L; Koo, H

    2012-10-01

    Culturing methods are the primary approach for microbiological analysis of plaque biofilms in rodent models of dental caries. In this study, we developed strategies for the isolation of DNA and RNA from plaque biofilms formed in vivo to analyse the viable bacterial population and gene expression. Plaque biofilm samples from rats were treated with propidium monoazide to isolate DNA from viable cells, and the purified DNA was used to quantify total bacteria and the Streptococcus mutans population via quantitative polymerase chain reaction (qPCR) and specific primers; the same samples were also analysed by counting colony-forming units (CFU). In parallel, RNA was isolated from plaque-biofilm samples (from the same animals) and used for transcriptional analyses via reverse transcription-qPCR. The viable populations of both S. mutans and total bacteria assessed by qPCR were positively correlated with the CFU data (P  0.8). However, the qPCR data showed higher bacterial cell counts, particularly for total bacteria (vs. CFU). Moreover, S. mutans proportion in the plaque biofilm determined by qPCR analysis showed strong correlation with incidence of smooth-surface caries (P = 0.0022, r = 0.71). The purified RNAs presented high RNA integrity numbers (> 7), which allowed measurement of the expression of genes that are critical for S. mutans virulence (e.g. gtfB and gtfC). Our data show that the viable microbial population and the gene expression can be analysed simultaneously, providing a global assessment of the infectious aspect of dental caries. Our approach could enhance the value of the current rodent model in further understanding the pathophysiology of this disease and facilitating the exploration of novel anti-caries therapies. © 2012 John Wiley & Sons A/S.

  16. Concurrent detection of other respiratory viruses in children shedding viable human respiratory syncytial virus.

    Science.gov (United States)

    Gagliardi, T B; Paula, F E; Iwamoto, M A; Proença-Modena, J L; Santos, A E; Camara, A A; Cervi, M C; Cintra, O A L; Arruda, E

    2013-10-01

    Human respiratory syncytial virus (HRSV) is an important cause of respiratory disease. The majority of studies addressing the importance of virus co-infections to the HRSV-disease have been based on the detection of HRSV by RT-PCR, which may not distinguish current replication from prolonged shedding of remnant RNA from previous HRSV infections. To assess whether co-detections of other common respiratory viruses are associated with increased severity of HRSV illnesses from patients who were shedding viable-HRSV, nasopharyngeal aspirates from children younger than 5 years who sought medical care for respiratory infections in Ribeirão Preto (Brazil) were tested for HRSV by immunofluorescence, RT-PCR and virus isolation in cell culture. All samples with viable-HRSV were tested further by PCR for other respiratory viruses. HRSV-disease severity was assessed by a clinical score scale. A total of 266 samples from 247 children were collected and 111 (42%) were HRSV-positive. HRSV was isolated from 70 (63%), and 52 (74%) of them were positive for at least one additional virus. HRSV-positive diseases were more severe than HRSV-negative ones, but there was no difference in disease severity between patients with viable-HRSV and those HRSV-positives by RT-PCR. Co-detection of other viruses did not correlate with increased disease severity. HRSV isolation in cell culture does not seem to be superior to RT-PCR to distinguish infections associated with HRSV replication in studies of clinical impact of HRSV. A high rate of co-detection of other respiratory viruses was found in samples with viable-HRSV, but this was not associated with more severe HRSV infection. Copyright © 2013 Wiley Periodicals, Inc.

  17. Comprehensive Mass Cytometry Analysis of Cell Cycle, Activation, and Coinhibitory Receptors Expression in CD4 T Cells from Healthy and HIV-Infected Individuals.

    Science.gov (United States)

    Corneau, Aurélien; Cosma, Antonio; Even, Sophie; Katlama, Christine; Le Grand, Roger; Frachet, Véronique; Blanc, Catherine; Autran, Brigitte

    2017-01-01

    Mass cytometry allows large multiplex analysis of cell cycle stages together with differentiation, activation, and exhaustion markers, allowing further assessment of the quiescence status of resting CD4 T cells. Peripheral blood CD4 T lymphocytes from 8 individuals, 4 healthy donors, and 4 HIV-infected on antiretroviral treatment (T) were stained with the same 26 monoclonal antibodies and dyes targeting surface and intracellular markers of differentiation, activation, exhaustion, and cell cycle stages. Samples were run on a CYTOF-2. Patterns of naïve [TN] CD4 T cells strongly differed from all other memory subsets central-memory (CM), transitional-memory (TM), effector-memory (EM), and terminally differentiated RA-expressing (TEMRA) subsets, while stem-cell memory (SCM) and T follicular-helper cells (TfH) were close to CM and TM cells with the highest percentages in cell cycle. EM and TEMRA were the most altered by HIV infection, with an increased frequency of activated and cycling cells. Activation markers and coinhibitory receptor expression differed among cell cycle stages, with HLA-DR fitting better than CD25 or CD38 with cycle, and opposite PD-1 gradients along differentiation and cell cycle. "Resting" DR-CD25- CD4+ T cells contained similar amounts of cells in G1 than the activated DR ± CD25± ones but three fold lower cells in S-G2-M. This broad multiplex mass cytometry analysis demonstrates some subsets of the so-called "resting" CD25-DR- CD4+ T cells contain noticeable amounts of cells into cycle or expressing coinhibitory receptors, opening new avenues for a redefinition of resting peripheral blood CD4 T cells harboring the HIV reservoirs. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  18. Selective detection of viable seed-borne Acidovorax citrulli by real-time PCR with propidium monoazide

    Science.gov (United States)

    Tian, Qian; Feng, Jian-jun; Hu, Jie; Zhao, Wen-jun

    2016-01-01

    In recent years, use of the DNA-intercalating dye propidium monoazide (PMA) in real-time PCR has been reported as a novel method to detect viable bacteria in different types of samples, such as food, environmental, and microbiological samples. In this study, viable cells of Acidovorax citrulli, the causal agent of bacterial seedling blight and fruit blotch, were selectively detected and differentiated from dead cells by real-time fluorescent polymerase chain reaction amplification after the bacterial solution was treated with the DNA-binding dye PMA. The primers and TaqMan probe were based on the A. citrulli genome (Aave_1909, Gene ID: 4669443) and were highly specific for A. citrulli. The detection threshold of this assay was 103 colony-forming units per mL (CFU/mL) in pure cell suspensions containing viable and dead cells and infected watermelon seeds. Application of this assay enables the selective detection of viable cells of A. citrulli and facilitates monitoring of the pathogen in watermelon and melon seeds. PMID:27739469

  19. Maternal Body-Mass Index and Cord Blood Circulating Endothelial Colony-Forming Cells

    Science.gov (United States)

    Lin, Ruei-Zeng; Miranda, Maria L.; Vallejo-Vaz, Antonio J.; Stiefel, Pablo; Praena-Fernández, Juan M.; Bernal-Bermejo, Jose; Jimenez-Jimenez, Luis M.; Villar, Jose; Melero-Martin, Juan M.

    2013-01-01

    Objective Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that are particularly abundant in umbilical cord blood. We sought to determine whether ECFC abundance in cord blood is associated with maternal body-mass index (BMI) in non-pathological pregnancies. Study design We measured the level of ECFCs in the cord blood of neonates (n=27) born from non-obese healthy mothers with non-pathological pregnancies and examined whether ECFC abundance correlated with maternal BMI. We also examined the effect of maternal BMI on ECFC phenotype and function using angiogenic and vasculogenic assays. Results We observed variation in ECFC abundance among subjects and found a positive correlation between pre-pregnancy maternal BMI and ECFC content (r=0.51, P=0.007), which was independent of other obstetric factors. Despite this variation, ECFC phenotype and functionality were deemed normal and highly similar between subjects with maternal BMI <25 kg/m2 and BMI between 25–30 kg/m2, including the ability to form vascular networks in vivo. Conclusions This study underlines the need to consider maternal BMI as a potential confounding factor for cord blood levels of ECFCs in future comparative studies between healthy and pathological pregnancies. Endothelial colony-forming cells (ECFCs) are a subset of progenitor cells that circulate in peripheral blood and can give rise to endothelial cells (1,2), contributing to the formation of new vasculature and the maintenance of vascular integrity (3–5). The mechanisms that regulate the abundance of these cells in vivo remain poorly understood. ECFCs are rare in adult peripheral blood (1,2,10). In contrast, there is an elevated number of these cells in fetal blood during the third trimester of pregnancy (11–13). Emerging evidence indicates that deleterious conditions during fetal life can impair ECFC content and function. For instance, offspring of diabetic mothers have been shown to have

  20. Biofilms in Full-Scale Drinking Water Ozone Contactors Contribute Viable Bacteria to Ozonated Water.

    Science.gov (United States)

    Kotlarz, Nadine; Rockey, Nicole; Olson, Terese M; Haig, Sarah-Jane; Sanford, Larry; LiPuma, John J; Raskin, Lutgarde

    2018-02-13

    Concentrations of viable microbial cells were monitored using culture-based and culture-independent methods across multichamber ozone contactors in a full-scale drinking water treatment plant. Membrane-intact and culturable cell concentrations in ozone contactor effluents ranged from 1200 to 3750 cells/mL and from 200 to 3850 colony forming units/mL, respectively. Viable cell concentrations decreased significantly in the first ozone contact chamber, but rose, even as ozone exposure increased, in subsequent chambers. Our results implicate microbial detachment from biofilms on contactor surfaces, and from biomass present within lime softening sediments in a hydraulic dead zone, as a possible reason for increasing cell concentrations in water samples from sequential ozone chambers. Biofilm community structures on baffle walls upstream and downstream from the dead zone were significantly different from each other (p = 0.017). The biofilms downstream of the dead zone contained a significantly (p = 0.036) higher relative abundance of bacteria of the genera Mycobacterium and Legionella than the upstream biofilms. These results have important implications as the effluent from ozone contactors is often treated further in biologically active filters and bacteria in ozonated water continuously seed filter microbial communities.

  1. Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

    Science.gov (United States)

    Ordax, Mónica; Marco-Noales, Ester; López, María M.; Biosca, Elena G.

    2006-01-01

    Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu2+ was inoculated with 107 CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper. PMID:16672494

  2. Determination of viable wine yeast using DNA binding dyes and quantitative PCR.

    Science.gov (United States)

    Andorrà, Imma; Esteve-Zarzoso, Braulio; Guillamón, José M; Mas, Albert

    2010-12-15

    The detection and quantification of wine yeast can be misleading due to under or overestimation of these microorganisms. Underestimation may be caused by variable growing rates of different microorganisms in culture media or the presence of viable but non-cultivable microorganisms. Overestimation may be caused by the lack of discrimination between live and dead microorganisms if quantitative PCR is used to quantify with DNA as the template. However, culture-independent methods that use dyes have been described to remove the DNA from dead cells and then quantify the live microorganisms. Two dyes have been studied in this paper: ethidium monoazide bromide (EMA) and propidium monoazide bromide (PMA). The technique was applied to grape must fermentation and ageing wines. Both dyes presented similar results on yeast monitoring. Membrane cell recovery was necessary when yeasts were originated from ethanol-containing media. When applied to grape must fermentation, differences of up to 1 log unit were seen between the QPCR estimation with or without the dye during the stationary phase. In ageing wines, good agreement was found between plating techniques and QPCR. Most of the viable cells were also culturable and no differences were observed with the methods, except for Zygosaccharomyces bailii and Dekkera bruxellensis where much higher counts were occasionally detected by QPCR. The presence of excess dead cells did not interfere with the quantification of live cells with either of the dyes. Copyright © 2010 Elsevier B.V. All rights reserved.

  3. A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells*

    Science.gov (United States)

    Kaake, Robyn M.; Wang, Xiaorong; Burke, Anthony; Yu, Clinton; Kandur, Wynne; Yang, Yingying; Novtisky, Eric J.; Second, Tonya; Duan, Jicheng; Kao, Athit; Guan, Shenheng; Vellucci, Danielle; Rychnovsky, Scott D.; Huang, Lan

    2014-01-01

    Protein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems. PMID:25253489

  4. Killing of targets by CD8 T cells in the mouse spleen follows the law of mass action.

    Directory of Open Access Journals (Sweden)

    Vitaly V Ganusov

    2011-01-01

    Full Text Available It has been difficult to correlate the quality of CD8 T cell responses with protection against viral infections. To investigate the relationship between efficacy and magnitude of T cell responses, we quantify the rate at which individual CD8 effector and memory T cells kill target cells in the mouse spleen. Using mathematical modeling, we analyze recent data on the loss of target cells pulsed with three different peptides from the mouse lymphocytic choriomeningitis virus (LCMV in mouse spleens with varying numbers of epitope-specific CD8 T cells. We find that the killing of targets follows the law of mass-action, i.e., the death rate of individual target cells remains proportional to the frequency (or the total number of specific CD8 T cells in the spleen despite the fact that effector cell densities and effector to target ratios vary about a 1000-fold. The killing rate of LCMV-specific CD8 T cells is largely independent of T cell specificity and differentiation stage. Our results thus allow one to calculate the critical T cell concentration at which growth of a virus with a given replication rate can be prevented from the start of infection by memory CD8 T cell response.

  5. Killing of targets by CD8 T cells in the mouse spleen follows the law of mass action.

    Science.gov (United States)

    Ganusov, Vitaly V; Barber, Daniel L; De Boer, Rob J

    2011-01-24

    It has been difficult to correlate the quality of CD8 T cell responses with protection against viral infections. To investigate the relationship between efficacy and magnitude of T cell responses, we quantify the rate at which individual CD8 effector and memory T cells kill target cells in the mouse spleen. Using mathematical modeling, we analyze recent data on the loss of target cells pulsed with three different peptides from the mouse lymphocytic choriomeningitis virus (LCMV) in mouse spleens with varying numbers of epitope-specific CD8 T cells. We find that the killing of targets follows the law of mass-action, i.e., the death rate of individual target cells remains proportional to the frequency (or the total number) of specific CD8 T cells in the spleen despite the fact that effector cell densities and effector to target ratios vary about a 1000-fold. The killing rate of LCMV-specific CD8 T cells is largely independent of T cell specificity and differentiation stage. Our results thus allow one to calculate the critical T cell concentration at which growth of a virus with a given replication rate can be prevented from the start of infection by memory CD8 T cell response.

  6. Strategy for signaling molecule detection by using an integrated microfluidic device coupled with mass spectrometry to study cell-to-cell communication.

    Science.gov (United States)

    Mao, Sifeng; Zhang, Jie; Li, Haifang; Lin, Jin-Ming

    2013-01-15

    Cell-to-cell communication is a very important physiological behavior in life entity, and most of human behaviors are related to it. Although cell-to-cell communications are attracting much attention and financial support, rare methods have been successfully developed for in vitro cell-to-cell communication study. In this work, we developed a novel method for cell-to-cell communication study on an integrated microdevice, and signaling molecule and metabolites were online-detected by an electrospray ionization-quadrupole-time-of-flight-mass spectrometer (ESI-Q-TOF-MS) after on-chip solid-phase extraction. Moreover, we presented a "Surface Tension Plug" on a microchip to control cell-to-cell communication. The microdevice consists of three functional sections: cell coculture channel, targets pretreatment, and targets detection sections. To verify the feasibility of cell-to-cell communications on the integrated microdevice, we studied the communication between the 293 and the L-02 cells. Epinephrine and glucose were successfully detected using an ESI-Q-TOF-MS with short analysis time (communication study.

  7. Viable group A streptococci in macrophages during acute soft tissue infection.

    Directory of Open Access Journals (Sweden)

    Pontus Thulin

    2006-03-01

    Full Text Available Group A streptococcal severe soft tissue infections, such as necrotizing fasciitis, are rapidly progressive infections associated with high mortality. Group A streptococcus is typically considered an extracellular pathogen, but has been shown to reside intracellularly in host cells.We characterized in vivo interactions between group A streptococci (GAS and cells involved in innate immune responses, using human biopsies (n = 70 collected from 17 patients with soft tissue infections. Immunostaining and in situ image analysis revealed high amounts of bacteria in the biopsies, even in those collected after prolonged antibiotic therapy. Viability of the streptococci was assessed by use of a bacterial viability stain, which demonstrated viable bacteria in 74% of the biopsies. GAS were present both extracellularly and intracellularly within phagocytic cells, primarily within macrophages. Intracellular GAS were predominantly noted in biopsies from newly involved tissue characterized by lower inflammation and bacterial load, whereas purely extracellular GAS or a combination of intra- and extracellular GAS dominated in severely inflamed tissue. The latter tissue was also associated with a significantly increased amount of the cysteine protease streptococcal pyrogenic exotoxin SpeB. In vitro studies confirmed that macrophages serve as reservoirs for viable GAS, and infection with a speB-deletion mutant produced significantly lower frequencies of cells with viable GAS following infection as compared to the wild-type bacteria.This is the first study to demonstrate that GAS survive intracellularly in macrophages during acute invasive infections. This intracellular presence may have evolved as a mechanism to avoid antibiotic eradication, which may explain our finding that high bacterial load is present even in tissue collected after prolonged intravenous antibiotic therapy. This new insight into the pathogenesis of streptococcal soft tissue infections

  8. Viable Group A Streptococci in Macrophages during Acute Soft Tissue Infection.

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available BACKGROUND: Group A streptococcal severe soft tissue infections, such as necrotizing fasciitis, are rapidly progressive infections associated with high mortality. Group A streptococcus is typically considered an extracellular pathogen, but has been shown to reside intracellularly in host cells. METHODS AND FINDINGS: We characterized in vivo interactions between group A streptococci (GAS and cells involved in innate immune responses, using human biopsies (n = 70 collected from 17 patients with soft tissue infections. Immunostaining and in situ image analysis revealed high amounts of bacteria in the biopsies, even in those collected after prolonged antibiotic therapy. Viability of the streptococci was assessed by use of a bacterial viability stain, which demonstrated viable bacteria in 74% of the biopsies. GAS were present both extracellularly and intracellularly within phagocytic cells, primarily within macrophages. Intracellular GAS were predominantly noted in biopsies from newly involved tissue characterized by lower inflammation and bacterial load, whereas purely extracellular GAS or a combination of intra- and extracellular GAS dominated in severely inflamed tissue. The latter tissue was also associated with a significantly increased amount of the cysteine protease streptococcal pyrogenic exotoxin SpeB. In vitro studies confirmed that macrophages serve as reservoirs for viable GAS, and infection with a speB-deletion mutant produced significantly lower frequencies of cells with viable GAS following infection as compared to the wild-type bacteria. CONCLUSIONS: This is the first study to demonstrate that GAS survive intracellularly in macrophages during acute invasive infections. This intracellular presence may have evolved as a mechanism to avoid antibiotic eradication, which may explain our finding that high bacterial load is present even in tissue collected after prolonged intravenous antibiotic therapy. This new insight into the pathogenesis

  9. Mass transport of direct methanol fuel cell species in sulfonated poly(ether ether ketone) membranes

    Energy Technology Data Exchange (ETDEWEB)

    Silva, V.S.; Boaventura, M.; Mendes, A.M.; Madeira, L.M. [LEPAE, Chemical Engineering Department, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto (Portugal); Ruffmann, B.; Vetter, S.; Nunes, S.P. [GKSS Research Centre, Max-Planck Str., 21502 Geesthacht (Germany)

    2006-05-05

    Homogeneous membranes based on sulfonated poly(ether ether ketone) (sPEEK) with different sulfonation degrees (SD) were prepared and characterized. In order to perform a critical analysis of the SD effect on the polymer barrier and mass transport properties towards direct methanol fuel cell species, proton conductivity, water/methanol pervaporation and nitrogen/oxygen/carbon dioxide pressure rise method experiments are proposed. This procedure allows the evaluation of the individual permeability coefficients in hydrated sPEEK membranes with different sulfonation degrees. Nafion{sup (R)} 112 was used as reference material. DMFC tests were also performed at 50{sup o}C. It was observed that the proton conductivity and the permeability towards water, methanol, oxygen and carbon dioxide increase with the sPEEK sulfonation degree. In contrast, the SD seems to not affect the nitrogen permeability coefficient. In terms of selectivity, it was observed that the carbon dioxide/oxygen selectivity increases with the sPEEK SD. In contrast, the nitrogen/oxygen selectivity decreases. In terms of barrier properties for preventing the DMFC reactants loss, the polymer electrolyte membrane based on the sulfonated poly(ether ether ketone) with SD lower or equal to 71%, although having slightly lower proton conductivity, presented much better characteristics for fuel cell applications compared with the well known Nafion{sup (R)} 112. In terms of the DMFC tests of the studied membranes at low temperature, the sPEEK membrane with SD=71% showed to have similar performance, or even better, as that of Nafion{sup (R)} 112. However, the highest DMFC overall efficiency was achieved using sPEEK membrane with SD=52%. (author)

  10. Acupuntura un tratamiento viable para las adicciones en Colombia

    Directory of Open Access Journals (Sweden)

    Hernán López Seuscún

    2013-07-01

    Los tratamientos con auriculoterapia, como el protocolo NADA (National Acupuncture Detoxification Association, son los métodos más usados para las adicciones en el mundo, y aunque no se ha logrado evidenciar su efectividad, por su costo, facilidad y el poco riesgo de efectos adversos se hace viable en un país con pocos recursos económicos como Colombia.

  11. Academic Pediatric Dentistry is a Rewarding, Financially Viable Career Path.

    Science.gov (United States)

    Townsend, Janice A; Chi, Donald L

    2017-09-15

    Newly graduated pediatric dentists have unprecedented levels of debt. High levels of student debt may be perceived as an obstacle to pursue an academic career. However, opportunities exist through faculty compensation models and loan repayment programs that make an academic career financially viable. The purpose of this paper is to outline the benefits of a career in academic dentistry and provide examples of young pediatric dentistry faculty members who have been able to manage student debt while pursuing meaningful and rewarding careers.

  12. How Can We Prevent Violence Becoming a Viable Political Strategy?

    OpenAIRE

    Patricia Justino

    2009-01-01

    A basic issue that conflict analysis investigates is how non-peaceful ways of living and governing become viable political strategies. Macro-level studies provide some important insights but micro-level analysis is vital to understand the mechanisms that make violence possible. This briefing outlines some preliminary findings in this respect from MICROCON, a major research programme analysing violent conflict at the micro level. It also discusses their implications for policies aimed at preve...

  13. Immunomagnetic separation combined with inductively coupled plasma mass spectrometry for the detection of tumor cells using gold nanoparticle labeling.

    Science.gov (United States)

    Zhang, Yuan; Chen, Beibei; He, Man; Yang, Bin; Zhang, Jing; Hu, Bin

    2014-08-19

    This work reports an efficient, specific, and sensitive immunoassay protocol for detection of tumor cells by using inductively coupled plasma mass spectrometry (ICP-MS) with two probes. Magnetic nanobeads modified with anti-CD3 were used as capture probes for efficient and fast magnetic separation of Jurkat T cells from a mixture of cells, and gold nanoparticles (Au NPs) conjugated with anti-CD2 were used as detection probes for ICP-MS measurement. The capture and detection probes target the Jurkat T cells with high affinity and specificity, while they do not target other CD2/CD3-negative cells such as 97L cells and A549 cells. On the basis of these results, we proposed a new immunoassay for specific detection of Jurkat T cells. The conditions for this immunoassay were carefully optimized, including the incubation time and temperature, the concentration of the labeling probe, and the elution conditions. Under the optimized conditions, the linear range of 300-30,000 and the limit of detection of 86 Jurkat T cells were obtained, and the relative standard deviation for seven replicate detection of Jurkat T cells was 5.2% (3000 Jurkat T cells). This method has numerous advantages, including ease of preparation, low sample consumption, and high sensitivity and selectivity. Importantly, the methodology could be extended to the simultaneous detection of other cells based on their cellular biomarkers.

  14. Combination of direct infusion mass spectrometry and gas chromatography mass spectrometry for toxicometabolomic study of red blood cells and serum of mice Mus musculus after mercury exposure.

    Science.gov (United States)

    García-Sevillano, M A; García-Barrera, T; Navarro, F; Abril, N; Pueyo, C; López-Barea, J; Gómez-Ariza, J L

    2015-03-15

    Although mercury (Hg) is an important environmental and occupational pollutant, its toxicological effects, especially in serum and red blood cells (RBCs), have been scarcely studied. A toxicometabolomics workflow based on high resolution mass spectrometry approaches has been applied to investigate the toxicological effects of Hg in Mus musculus mice after subcutaneous injection for 10 days, which produced inflammation and vacuolization, steatosis and karyolysis in the hepatic tissue. To this end, direct infusion mass spectrometry (DIMS) of polar and lipophilic extracts from serum and RBCs, using positive and negative mode of acquisition (ESI+/ESI-), and gas chromatography-mass spectrometry were used. A quantitative analysis of reversible oxidized thiols in serum proteins demonstrated a strong oxidative stress induction in the liver of Hg-exposed mice. Endogenous metabolites alterations were identified by partial least squares-discriminant analysis (PLS-DA). Mercury-exposed mice show perturbations in energy metabolism, amino acid metabolism, membrane phospholipid breakdown and oxidative stress-related metabolites in serum along the exposure. This work reports for the first time the effects of Hg-exposure on RBCs metabolic pathways, and reveals disturbances in glycolysis, membrane turnover, glutathione and ascorbate metabolisms. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Incidental solid renal mass in a cadaveric donor kidney

    Directory of Open Access Journals (Sweden)

    R M Meyyappan

    2012-01-01

    Full Text Available The number of patients living with end-stage renal disease (ESRD is increasing in our country and demand for renal grafts is ever increasing. Cadaver renal transplantation is being established as a viable supplement to live transplantation. We present a case where a mass lesion was encountered in the donor kidney from a cadaver. Enucleation of the lesion was done and we proceeded with the grafting. Histopathological examination showed a ′Renomedullary interstitial cell tumour′, a rare benign lesion. Post transplant, the renal function recovered well and the patient is asymptomatic. Such incidental renal masses present an ethical dilemma to the operating surgeon.

  16. Quantitative analysis of antiretroviral drugs in lysates of peripheral blood mononuclear cells using MALDI-triple quadrupole mass spectrometry.

    NARCIS (Netherlands)

    Kampen, JJ van; Burgers, P.C.; Gruters, R.A.; Osterhaus, A.D.; Groot, R. de; Luider, T.M.; Volmer, D.A.

    2008-01-01

    We report here on the use of a prototype matrix-assisted laser desorption/ionization (MALDI)-triple quadrupole mass spectrometer for quantitative analysis of six antiretroviral drugs in lysates of peripheral blood mononuclear cells (PBMC). Of the five investigated MALDI matrixes,

  17. Mass-produced multi-walled carbon nanotubes as catalyst supports for direct methanol fuel cells.

    Science.gov (United States)

    Jang, In Young; Park, Ki Chul; Jung, Yong Chae; Lee, Sun Hyung; Song, Sung Moo; Muramatsu, Hiroyuki; Kim, Yong Jung; Endo, Morinobu

    2011-01-01

    Commercially mass-produced multi-walled carbon nanotubes, i.e., VGNF (Showa Denko Co.), were applied to support materials for platinum-ruthenium (PtRu) nanoparticles as anode catalysts for direct methanol fuel cells. The original VGNFs are composed of high-crystalline graphitic shells, which hinder the favorable surface deposition of the PtRu nanoparticles that are formed via borohydride reduction. The chemical treatment of VGNFs with potassium hydroxide (KOH), however, enables highly dispersed and dense deposition of PtRu nanoparticles on the VGNF surface. This capability becomes more remarkable depending on the KOH amount. The electrochemical evaluation of the PtRu-deposited VGNF catalysts showed enhanced active surface areas and methanol oxidation, due to the high dispersion and dense deposition of the PtRu nanoparticles. The improvement of the surface deposition states of the PtRu nanoparticles was significantly due to the high surface area and mesorporous surface structure of the KOH-activated VGNFs.

  18. Profiling the effects of process changes on residual host cell proteins in biotherapeutics by mass spectrometry.

    Science.gov (United States)

    Schenauer, Matthew R; Flynn, Gregory C; Goetze, Andrew M

    2013-01-01

    An advanced liquid chromatography/mass spectrometry (MS) platform was used to identify and quantify residual Escherichia coli host cell proteins (HCPs) in the drug substance (DS) of several peptibodies (Pbs). Significantly different HCP impurity profiles were observed among different biotherapeutic Pbs as well as one Pb purified via multiple processes. The results can be rationally interpreted in terms of differences among the purification processes, and demonstrate the power of this technique to sensitively monitor both the quantity and composition of residual HCPs in DS, where these may represent a safety risk to patients. The breadth of information obtained using MS is compared to traditional multiproduct enzyme-linked immunosorbent assay (ELISA) values for total HCP in the same samples and shows that, in this case, the ELISA failed to detect multiple HCPs. The HCP composition of two upstream samples was also analyzed and used to demonstrate that HCPs that carry through purification processes to be detectable in DS are not always among those that are the most abundant upstream. Compared to ELISA, we demonstrate that MS can provide a more comprehensive, and accurate, characterization of DS HCPs, thereby facilitating process development as well as more rationally assessing potential safety risks posed by individual, identified HCPs. © 2013 American Institute of Chemical Engineers.

  19. Hybrid Markov-mass action law for cell activation by rare binding events

    CERN Document Server

    Holcman, C Guerrier D

    2016-01-01

    The binding of molecules, ions or proteins to specific target sites is a generic step for cell activation. However, this step relies on rare events where stochastic particles located in a large bulk are searching for small and often hidden targets and thus remains difficult to study. We present here a hybrid discrete-continuum model where the large ensemble of particles is described by mass-action laws. The rare discrete binding events are modeled by a Markov chain for the encounter of a finite number of small targets by few Brownian particles, for which the arrival time is Poissonian. This model is applied for predicting the time distribution of vesicular release at neuronal synapses that remains elusive. This release is triggered by the binding of few calcium ions that can originate either from the synaptic bulk or from the transient entry through calcium channels. We report that the distribution of release time is bimodal although triggered by a single fast action potential: while the first peak follows a ...

  20. Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry.

    Science.gov (United States)

    Bandura, Dmitry R; Baranov, Vladimir I; Ornatsky, Olga I; Antonov, Alexei; Kinach, Robert; Lou, Xudong; Pavlov, Serguei; Vorobiev, Sergey; Dick, John E; Tanner, Scott D

    2009-08-15

    A novel instrument for real time analysis of individual biological cells or other microparticles is described. The instrument is based on inductively coupled plasma time-of-flight mass spectrometry and comprises a three-aperture plasma-vacuum interface, a dc quadrupole turning optics for decoupling ions from neutral components, an rf quadrupole ion guide discriminating against low-mass dominant plasma ions, a point-to-parallel focusing dc quadrupole doublet, an orthogonal acceleration reflectron analyzer, a discrete dynode fast ion detector, and an 8-bit 1 GHz digitizer. A high spectrum generation frequency of 76.8 kHz provides capability for collecting multiple spectra from each particle-induced transient ion cloud, typically of 200-300 micros duration. It is shown that the transients can be resolved and characterized individually at a peak frequency of 1100 particles per second. Design considerations and optimization data are presented. The figures of merit of the instrument are measured under standard inductively coupled plasma (ICP) operating conditions ( 900 for m/z = 159, the sensitivity with a standard sample introduction system of >1.4 x 10(8) ion counts per second per mg L(-1) of Tb and an abundance sensitivity of (6 x 10(-4))-(1.4 x 10(-3)) (trailing and leading masses, respectively) are shown. The mass range (m/z = 125-215) and abundance sensitivity are sufficient for elemental immunoassay with up to 60 distinct available elemental tags. When 500) can be used, which provides >2.4 x 10(8) cps per mg L(-1) of Tb, at (1.5 x 10(-3))-(5.0 x 10(-3)) abundance sensitivity. The real-time simultaneous detection of multiple isotopes from individual 1.8 microm polystyrene beads labeled with lanthanides is shown. A real time single cell 20 antigen expression assay of model cell lines and leukemia patient samples immuno-labeled with lanthanide-tagged antibodies is presented.

  1. Multivariate Calibration Approach for Quantitative Determination of Cell-Line Cross Contamination by Intact Cell Mass Spectrometry and Artificial Neural Networks.

    Directory of Open Access Journals (Sweden)

    Elisa Valletta

    Full Text Available Cross-contamination of eukaryotic cell lines used in biomedical research represents a highly relevant problem. Analysis of repetitive DNA sequences, such as Short Tandem Repeats (STR, or Simple Sequence Repeats (SSR, is a widely accepted, simple, and commercially available technique to authenticate cell lines. However, it provides only qualitative information that depends on the extent of reference databases for interpretation. In this work, we developed and validated a rapid and routinely applicable method for evaluation of cell culture cross-contamination levels based on mass spectrometric fingerprints of intact mammalian cells coupled with artificial neural networks (ANNs. We used human embryonic stem cells (hESCs contaminated by either mouse embryonic stem cells (mESCs or mouse embryonic fibroblasts (MEFs as a model. We determined the contamination level using a mass spectra database of known calibration mixtures that served as training input for an ANN. The ANN was then capable of correct quantification of the level of contamination of hESCs by mESCs or MEFs. We demonstrate that MS analysis, when linked to proper mathematical instruments, is a tangible tool for unraveling and quantifying heterogeneity in cell cultures. The analysis is applicable in routine scenarios for cell authentication and/or cell phenotyping in general.

  2. Assessing CMT cell line stability by two dimensional polyacrylamide gel electrophoresis and mass spectrometry based proteome analysis

    DEFF Research Database (Denmark)

    Zhang, Kelan; Wrzesinski, Krzysztof; Fey, Stephen J

    2008-01-01

    Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by mass spectrometric identification of the proteins in the protein spots has become a central tool in proteomics. CMT167(H), CMT64(M) and CMT170(L) cell lines, selected from a spontaneous mouse lung adenocarcinoma, with high-...... to be a useful tool for assessing differences in cell line stability. This approach provided a tool to select the best cell line and optimal subculture period for studies of cancer related phenomena and for testing the effect of potential anticancer drugs....

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

    Energy Technology Data Exchange (ETDEWEB)

    James, Brian David [Strategic Analysis Inc., Arlington, VA (United States); Huya-Kouadio, Jennie Moton [Strategic Analysis Inc., Arlington, VA (United States); Houchins, Cassidy [Strategic Analysis Inc., Arlington, VA (United States); DeSantis, Daniel Allen [Strategic Analysis Inc., Arlington, VA (United States)

    2016-09-01

    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.

  4. Investigation of nanostructured electrocatalysts and mass transport phenomena in polymer electrolyte fuel cells

    Science.gov (United States)

    Goenaga, Gabriel A.

    Proton exchange membrane (PEM) fuel cells (FC) are promising devices in the search of clean and efficient technologies to reduce the use of fossil fuels. However, their poor performance in dynamic applications and high cost of platinum group metal (PGM) catalysts, have prevented them from becoming an affordable solution. This dissertation comprehend three research projects that study the mass transport phenomena in modified PEMs, the reduction of the amount of PGM catalyst used for oxygen reduction reaction (ORR) and the use of non-PGM catalysts as alternative catalyst to Pt for ORR. Nafion is the most used PEM for FC applications. Nafion proton conductivity is proportional to its degree of hydration, what imposes low temperature operation to maintain appropriate water content. In this research, Nafion composite membranes doped with hydrophilic metal inorganic particles have been studied using pulse field gradient (PFG) nuclear magnetic resonance (NMR). The Nafion composite membranes were found to have higher water uptake, higher water retention, higher water diffusion and, in some cases, lower methanol diffusion (crossover) than the filler free Nafion membrane. The amount of Pt and PGM catalysts supported on carbon used in the electrodes, has a great impact in the PEMFC cost. In particular, it is of high relevance to reduce the amount of Pt in the cathode electrode, in which the sluggish ORR demands four to five times more Pt catalyst than in the anode. In this thesis is shown that the use of aligned carbon nanotubes (ACNTs) as Pt support, allows a more uniform distribution of the Pt nanoparticles, what in addition to their high hydrophobicity and high corrosive resistance, lead to improved mass transport and stability of the membrane electrode assembly (MEA), when compared to a benchmark MEA that uses Pt catalyst supported on carbon black. The improvement was accomplished using less Pt than in the benchmark MEA. Replacing Pt with non-PGM catalyst can lead to an

  5. Sorption and precipitation of Mn2+ by viable and autoclaved Shewanella putrefaciens: Effect of contact time

    KAUST Repository

    Chubar, Natalia

    2013-01-01

    The sorption of Mn(II) by viable and inactivated cells of Shewanella putrefaciens, a non-pathogenic, facultative anaerobic, gram-negative bacterium characterised as a Mn(IV) and Fe(III) reducer, was studied under aerobic conditions, as a function of pH, bacterial density and metal loading. During a short contact time (3-24h), the adsorptive behaviour of live and dead bacteria toward Mn(II) was sufficiently similar, an observation that was reflected in the studies on adsorption kinetics at various metal loadings, effects of pH, bacteria density, isotherms and drifting of pH during adsorption. Continuing the experiment for an additional 2-30days demonstrated that the Mn(II) sorption by suspensions of viable and autoclaved cells differed significantly from one another. The sorption to dead cells was characterised by a rapid equilibration and was described by an isotherm. In contrast, the sorption (uptake) to live bacteria exhibited a complex time-dependent uptake. This uptake began as adsorption and ion exchange processes followed by bioprecipitation, and it was accompanied by the formation of polymeric sugars (EPS) and the release of dissolved organic substances. FTIR, EXAFS/XANES and XPS demonstrated that manganese(II) phosphate was the main precipitate formed in 125ml batches, which is the first evidence of the ability of microbes to synthesise manganese phosphates. XPS and XANES spectra did not detect Mn(II) oxidation. Although the release of protein-like compounds by the viable bacteria increased in the presence of Mn2+ (and, by contrast, the release of carbohydrates did not change), electrochemical analyses did not indicate any aqueous complexation of Mn(II) by the organic ligands. © 2012 Elsevier Ltd.

  6. Fully Automated Islet Cell Counter (ICC) for the Assessment of Islet Mass, Purity, and Size Distribution by Digital Image Analysis.

    Science.gov (United States)

    Buchwald, Peter; Bernal, Andres; Echeverri, Felipe; Tamayo-Garcia, Alejandro; Linetsky, Elina; Ricordi, Camillo

    2016-10-01

    For isolated pancreatic islet cell preparations, it is important to be able to reliably assess their mass and quality, and for clinical applications, it is part of the regulatory requirement. Accurate assessment, however, is difficult because islets are spheroid-like cell aggregates of different sizes (<50 to 500 μm) resulting in possible thousandfold differences between the mass contribution of individual particles. The current standard manual counting method that uses size-based group classification is known to be error prone and operator dependent. Digital image analysis (DIA)-based methods can provide less subjective, more reproducible, and better-documented islet cell mass (IEQ) estimates; however, so far, none has become widely accepted or used. Here we present results obtained using a compact, self-contained islet cell counter (ICC3) that includes both the hardware and software needed for automated islet counting and requires minimal operator training and input; hence, it can be easily adapted at any center and could provide a convenient standardized cGMP-compliant IEQ assessment. Using cross-validated sample counting, we found that for most human islet cell preparations, ICC3 provides islet mass (IEQ) estimates that correlate well with those obtained by trained operators using the current manual SOP method ( r2 = 0.78, slope = 1.02). Variability and reproducibility are also improved compared to the manual method, and most of the remaining variability (CV = 8.9%) results from the rearrangement of the islet particles due to movement of the sample between counts. Characterization of the size distribution is also important, and the present digitally collected data allow more detailed analysis and coverage of a wider size range. We found again that for human islet cell preparations, a Weibull distribution function provides good description of the particle size.

  7. Killing of targets by effector CD8 T cells in the mouse spleen follows the law of mass action

    Energy Technology Data Exchange (ETDEWEB)

    Ganusov, Vitaly V [Los Alamos National Laboratory

    2009-01-01

    In contrast with antibody-based vaccines, it has been difficult to measure the efficacy of T cell-based vaccines and to correlate the efficacy of CD8 T cell responses with protection again viral infections. In part, this difficulty is due to poor understanding of the in vivo efficacy of CD8 T cells produced by vaccination. Using a: recently developed experimental method of in vivo cytotoxicity we have investigated quantitative aspects of killing of peptide-pulsed targets by effector and memory CD8 T cells, specific to three epitopes of lymphocytic choriomeningitis virus (LCMV), in the mouse spleen. By analyzing data on killing of targets with varying number of epitope-specific effector and memory CD8 T cells, we find that killing of targets by effectors follows the law of mass-action, that is the death rate of peptide-pulsed targets is proportional to the frequency of CTLs in the spleen. In contrast, killing of targets by memory CD8 T cells does not follow the mass action law because the death rate of targets saturates at high frequencies of memory CD8 T cells. For both effector and memory cells, we also find little support for the killing term that includes the decrease of the death rate of targets with target cell density. Interestingly, our analysis suggests that at low CD8 T cell frequencies, memory CD8 T cells on the per capita basis are more efficient at killing peptide-pulsed targets than effectors, but at high frequencies, effectors are more efficient killers than memory T cells. Comparison of the estimated killing efficacy of effector T cells with the value that is predicted from theoretical physics and based on motility of T cells in lymphoid tissues, suggests that limiting step in the killing of peptide-pulsed targets is delivering the lethal hit and not finding the target. Our results thus form a basis for quantitative understanding of the process of killing of virus-infected cells by T cell responses in tissues and can be used to correlate the

  8. Liquid biopsy in cancer patients: advances in capturing viable CTCs for functional studies using the EPISPOT assay.

    Science.gov (United States)

    Alix-Panabières, Catherine; Pantel, Klaus

    2015-01-01

    Circulating tumor cells (CTCs) in the blood of cancer patients have received increasing attention as new diagnostic tool enabling 'liquid biopsies'. In contrast to the wealth of descriptive studies demonstrating the clinical relevance of CTCs as biomarkers, the extremely low concentration of CTCs in the peripheral blood of most cancer patients challenges further functional studies. This article discusses the current possibilities to enrich and, in particular, detect viable CTCs with emphasis on the EPithelial ImmunoSPOT technology. This functional assay detects viable CTCs at the single-cell level and has been used on hundreds of patients with different tumor types including epithelial tumors (breast, prostate and colon cancer) and melanomas. Moreover, the article summarizes recent advances in the in vitro and in vivo expansion of CTCs from cancer patients. These functional analyses will contribute to identifying the biological properties of metastatic cells and reveal new therapeutic targets against disseminating cancer cells.

  9. Modulation of the endocannabinoid system in viable and non-viable first trimester pregnancies by pregnancy-related hormones

    Directory of Open Access Journals (Sweden)

    Taylor Anthony H

    2011-11-01

    Full Text Available Abstract Background In early pregnancy, increased plasma levels of the endocannabinoid anandamide (AEA are associated with miscarriage through mechanisms that might affect the developing placenta or maternal decidua. Methods In this study, we compare AEA levels in failed and viable pregnancies with the levels of the trophoblastic hormones (beta-human chorionic gonadotrophin (beta-hCG, progesterone (P4 and (pregnancy-associated placental protein-A (PAPP-A essential for early pregnancy success and relate that to the expression of the cannabinoid receptors and enzymes that modulate AEA levels. Results The median plasma AEA level in non-viable pregnancies (1.48 nM; n = 20 was higher than in viable pregnancies (1.21 nM; n = 25; P = 0.013, as were progesterone and beta-hCG levels (41.0 vs 51.5 ng/mL; P = 0.052 for P4 and 28,650 vs 6,560 mIU/L; P = 0.144 for beta-hCG, respectively, but were not statistically significant. Serum PAPP-A levels in the viable group were approximately 6.8 times lower than those in the non-viable group (1.82 vs 12.25 mg/L; P = 0.071, but again these differences were statistically insignificant. In the spontaneous miscarriage group, significant correlations between P4 and beta-hCG, P4 and PAPP-A and AEA and PAPP-A levels were observed. Simultaneously, immunohistochemical distributions of the two main cannabinoid receptors and the AEA-modifying enzymes, fatty acid amide hydrolase (FAAH and N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD, changed within both the decidua and trophoblast. Conclusions The association of higher AEA levels with early pregnancy failure and with beta-hCG and PAPP-A, but not with progesterone concentrations suggest that plasma AEA levels and pregnancy failure are linked via a mechanism that may involve trophoblastic beta-hCG, and PAPP-A, but not, progesterone production. Although the trophoblast, decidua and embryo contain receptors for AEA, the main AEA target in early pregnancy failure

  10. High Efficiency, High Mass Specific Power Two-Terminal Solar Cells Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Future NASA missions will require significant improvements in photovoltaic energy conversion efficiency (>30%) and mass specific power (>600 W/kg), and...

  11. arXiv Cosmologically Viable Low-energy Supersymmetry Breaking

    CERN Document Server

    Hook, Anson; Murayama, Hitoshi

    A recent cosmological bound on the gravitino mass, $m_{3/2}<4.7$ eV, together with LHC results on the Higgs mass and direct searches, excludes minimal gauge mediation with high reheating temperatures. We discuss a minimal, vector-mediated model which incorporates the seesaw mechanism for neutrino masses, allows for thermal leptogenesis, ameliorates the $\\mu$ problem, and achieves the observed Higgs mass and a gravitino as light as $1$-$2$ eV.

  12. Genotoxic effects of daily personal exposure to particle mass and number concentrations on buccal cells

    Science.gov (United States)

    de Almeida, Daniela S.; da Costa, Silvano César; Ribeiro, Marcos; Moreira, Camila A. B.; Beal, Alexandra; Squizzato, Rafaela; Rudke, Anderson Paulo; Rafee, Sameh Adib Abou; Martins, Jorge A.; Palioto, Graciana Freitas; Kumar, Prashant; Martins, Leila D.

    2018-03-01

    The aim of this study is to assess personal exposure to Particle Number Concentrations (PNC) in four size ranges between 0.3 and 10 μm, and particulate matter (PM1; PM2.5; PM4; PM10) in order to evaluate possible genotoxic effects through a comet assay in buccal cells. A convenience cohort of 30 individuals from a Brazilian medium-sized city was selected. These individuals aged between 20 and 61 and worked in typical job categories (i.e., administrative, commerce, education, general services and transport). They were recruited to perform personal exposure measurements during their typical daily routine activities, totaling 240 h of sampling. The 8-h average mass concentrations in air for volunteers ranged from 2.4 to 31.8 μg m-3 for PM1, 4.2-45.1 μg m-3 for PM2.5, 7.9-66.1 μg m-3 for PM4 and from 23.1 to 131.7 μg m-3 for PM10. The highest PNC variation was found for 0.3-0.5 range, between 14 and 181 particles cm-3, 1 to 14 particles cm-3 for the 0.5-1.0 range, 0.2 to 2 particles cm-3 for the 1.0-2.5 range, and 0.06 to 0.7 particles cm-3 for the 2.5-10 range. Volunteers in the 'education' category experienced the lowest inhaled dose of PM2.5, as opposed to those involved in 'commercial' activities with the highest doses for PM10 (1.63 μg kg-1 h-1) and PM2.5 (0.61 μg kg-1 h-1). The predominant cause for these high doses was associated with the proximity of the workplace to the street and vehicle traffic. The comet assay performed in buccal cells indicated that the volunteers in 'commerce' category experienced the highest damage to their DeoxyriboNucleic Acid (DNA) compared with the control category (i.e. 'education'). These results indicate the variability in personal exposure of the volunteers in different groups, and the potential damage to DNA was much higher for those spending time in close proximity to the vehicle sources (e.g. commercial services) leading to exposure to a higher fraction of fine particles. This study builds understanding on the exposure

  13. Removal of viable bioaerosol particles with a low-efficiency HVAC filter enhanced by continuous emission of unipolar air ions.

    Science.gov (United States)

    Huang, R; Agranovski, I; Pyankov, O; Grinshpun, S

    2008-04-01

    Continuous emission of unipolar ions has been shown to improve the performance of respirators and stationary filters challenged with non-biological particles. In this study, we investigated the ion-induced enhancement effect while challenging a low-efficiency heating, ventilation and air-conditioning (HVAC) filter with viable bacterial cells, bacterial and fungal spores, and viruses. The aerosol concentration was measured in real time. Samples were also collected with a bioaerosol sampler for viable microbial analysis. The removal efficiency of the filter was determined, respectively, with and without an ion emitter. The ionization was found to significantly enhance the filter efficiency in removing viable biological particles from the airflow. For example, when challenged with viable bacteria, the filter efficiency increased as much as four- to fivefold. For viable fungal spores, the ion-induced enhancement improved the efficiency by a factor of approximately 2. When testing with virus-carrying liquid droplets, the original removal efficiency provided by the filter was rather low: 9.09 +/- 4.84%. While the ion emission increased collection about fourfold, the efficiency did not reach 75-100% observed with bacteria and fungi. These findings, together with our previously published results for non-biological particles, demonstrate the feasibility of a new approach for reducing aerosol particles in HVAC systems used for indoor air quality control. Recirculated air in HVAC systems used for indoor air quality control in buildings often contains considerable number of viable bioaerosol particles because of limited efficiency of the filters installed in these systems. In the present study, we investigated - using aerosolized bacterial cells, bacterial and fungal spores, and virus-carrying particles - a novel idea of enhancing the performance of a low-efficiency HVAC filter utilizing continuous emission of unipolar ions in the filter vicinity. The findings described in

  14. Initial Presentation of Renal Cell Carcinoma as a Metastatic Mass within the Masseter Muscle: A Case Report and Literature Review

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Kyung Eun; Lee, Han Bee; Cho, Woo Ho; Kim, Jae Hyung; Lee, Ji Hae; Kang, Min Jin [Dept. of Radiology, Sanggye Paik Hospital, Inje University College of Medicine, Seoul (Korea, Republic of); Kim, Hyun Jung [Dept. of Pathology, Sanggye Paik Hospital, Inje University College of Medicine, Seoul (Korea, Republic of)

    2012-02-15

    Renal cell carcinoma (RCC) is often concomitant with distant metastasis, and these metastases are the first sign of an otherwise occult primary. Whereas metastasis of RCC to the head and neck has been reported, metastasis to the masseter muscle, which is composed of skeletal muscle, is quite rare. We now report the case of a 66-year-old man who had a past history of pulmonary tuberculosis, with RCC metastasis of a well-defined intensely enhancing hypervascular mass in the masseter muscle as the initial presentation. We present the imaging findings of this case and a literature review about radiologic differential diagnosis of intramasseteric masses.

  15. Mass Transfer Studies with Submerged Impinging Jets in Closed Cylindrical Electrolytic Cell in the Presence of Solids

    Directory of Open Access Journals (Sweden)

    S. Feroz

    2008-12-01

    Full Text Available An experimental study of mass transfer in forced convective flow of fluid electrolyte through submerged jets impinging normal to the target surface in a closed cylindrical cell in the presence of solids (Porcelain beads is reported. The pertinent dynamic and geometric variables of this study are flow rate, diameter of the nozzle, height of the nozzle from the target surface and solids fraction. The mass transfer measurements, made by the electrochemical method propose empirical correlations in the impingement and decreasing coefficient regions.

  16. Mandibular Mass as an Only Presentation of Metatatic Renal Cell Carcinoma For Four Years: A Case Report.

    Science.gov (United States)

    Gooran, Shahram; Fakhr Yasseri, Alimohammad; Behtash, Negar; Karimi, Arash; Khalili, Masoud; Asadi, Mahboobeh

    2017-01-18

    Renal cell carcinoma is one of the most common tumors of the urinary tract. This tumor may appear as Para neoplasticsyndromes or distant metastasis. Metastases in uncommon areas are one of the characteristics of renal tumors.One of the uncommon metastatic renal masses areas is the mandible. In different studies, patient survival aftermetastasis diagnosis is usually one year or less. In this study we introduce a patient with mass of the right mandiblewhich existed four years before his referral, and in examinations it was diagnosed as metastasis with renal origin.

  17. Unacylated ghrelin and obestatin increase islet cell mass and prevent diabetes in streptozotocin-treated newborn rats.

    Science.gov (United States)

    Granata, Riccarda; Volante, Marco; Settanni, Fabio; Gauna, Carlotta; Ghé, Corrado; Annunziata, Marta; Deidda, Barbara; Gesmundo, Iacopo; Abribat, Thierry; van der Lely, Aart-Jan; Muccioli, Giampiero; Ghigo, Ezio; Papotti, Mauro

    2010-07-01

    The ghrelin gene products, namely acylated ghrelin (AG), unacylated ghrelin (UAG), and obestatin (Ob), were shown to prevent pancreatic beta-cell death and to improve beta-cell function under treatment with cytokines, which are major cause of beta-cell destruction in diabetes. Moreover, AG had been described previously to prevent streptozotocin (STZ)-induced diabetes in rats; however, the effect of either UAG or Ob has never been examined in this context. In the present study, we investigated the potential of UAG and Ob to increase islet beta-cell mass and to reduce diabetes at adult age in STZ-treated neonatal rats. One-day-old rats were injected with STZ and subsequently administered with either AG, UAG or Ob for 7 days. On day 70, plasma glucose levels, plasma and pancreatic insulin levels, pancreatic islet area and number, insulin and pancreatic/duodenal homeobox-1 (Pdx1) gene expression, and antiapoptotic BCL2 protein expression were determined. Similarly to AG, both UAG and Ob counteracted STZ-induced high glucose levels and improved plasma and pancreatic insulin levels, which were reduced by the diabetogenic compound. UAG and Ob increased islet area, islet number, and beta-cell mass with respect to STZ treatment alone. Finally, in STZ-treated animals, UAG and Ob up-regulated insulin and Pdx1 mRNA and increased the expression of BCL2 similarly to AG. Taken together, our results suggest that in STZ-treated newborn rats, UAG and Ob improve glucose metabolism and preserve islet cell mass, granting a therapeutic potential in medical conditions associated with impaired beta-cell function.

  18. Molar Pregnancy with a Co-Existing Viable Fetus

    Directory of Open Access Journals (Sweden)

    Ruya Deveer

    2014-03-01

    Full Text Available     The aim of this study was to report the clinical features, management, and outcome of a case of molar pregnancy with a coexisting viable fetus and to review the literature. In this article, we report a case of pregnancy with diffuse placental molar change and a normal fetus which presented with hyperemesis gravidarum and hyperthyroidism. Genetic amniocentesis showed normal fetal karyotype. A healthy full-term live male infant was delivered by cesarean section. In molar pregnancies with a normal karyotype fetus, with intensive maternal follow-up, continuation of pregnancy can be suggested.

  19. Use of sodium lauroyl sarcosinate (sarkosyl) in viable real-time PCR for enumeration of Escherichia coli.

    Science.gov (United States)

    Wang, Hui; Gill, Colin O; Yang, Xianqin

    2014-03-01

    The cell membranes of inactivated Escherichia coli are not always permeable to propidium monoazide (PMA). This limits the use of PMA real-time PCR (PMA-qPCR) for quantification of DNA from only viable cells for enumeration of E. coli. The aim of this study was to develop PMA-qPCR procedures for E. coli with improved selectivity for viable cells. E. coli inactivated by incubation at 52°C were treated with 12 detergents before PMA treatment, and DNA was quantified by real-time PCR. Treatment with each of the 12 detergents and PMA increased the cycle threshold (Ct) values for heat inactivated E. coli suspensions. The greatest increase, of 10.68 Ct was obtained with sarkosyl. Treatment with sodium deoxycholate (NaDC) increased the Ct value by 8.99 Ct. Treatment with sarkosyl or NaDC of 16 heat treated 5-strain cocktails of verotoxigenic E. coli (VTEC) increased the mean Ct values by 8.15 or 6.82 Ct, respectively. Those mean values were significantly (pnumbers of viable E. coli were 2.24 and 2.47, respectively, with regression coefficient values ≥0.85. The findings show that sarkosyl was more effective than NaDC for dissipation of PMA-barrier properties of membranes of inactivated E. coli cells. Viable E. coli in mixtures of viable E. coli and E. coli inactivated by heat, lactic acid or peroxyacetic acid could be reliably enumerated by sarkosyl PMA-qPCR. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Overexpression of PPARγ specifically in pancreatic β-cells exacerbates obesity-induced glucose intolerance, reduces β-cell mass, and alters islet lipid metabolism in male mice.

    Science.gov (United States)

    Hogh, K-Lynn N; Craig, Michael N; Uy, Christopher E; Nygren, Heli; Asadi, Ali; Speck, Madeline; Fraser, Jordie D; Rudecki, Alexander P; Baker, Robert K; Orešič, Matej; Gray, Sarah L

    2014-10-01

    The contribution of peroxisomal proliferator-activated receptor (PPAR)-γ agonism in pancreatic β-cells to the antidiabetic actions of thiazolidinediones has not been clearly elucidated. Genetic models of pancreatic β-cell PPARγ ablation have revealed a potential role for PPARγ in β-cell expansion in obesity but a limited role in normal β-cell physiology. Here we overexpressed PPARγ1 or PPARγ2 specifically in pancreatic β-cells of mice subjected to high-fat feeding using an associated adenovirus (β-PPARγ1-HFD and β-PPARγ2-HFD mice). We show β-cell-specific PPARγ1 or PPARγ2 overexpression in diet-induced obese mice exacerbated obesity-induced glucose intolerance with decreased β-cell mass, increased islet cell apoptosis, and decreased plasma insulin compared with obese control mice (β-eGFP-HFD mice). Analysis of islet lipid composition in β-PPARγ2-HFD mice revealed no significant changes in islet triglyceride content and an increase in only one of eight ceramide species measured. Interestingly β-PPARγ2-HFD islets had significantly lower levels of lysophosphatidylcholines, lipid species shown to enhance insulin secretion in β-cells. Gene expression profiling revealed increased expression of uncoupling protein 2 and genes involved in fatty acid transport and β-oxidation. In summary, transgenic overexpression of PPARγ in β-cells in diet-induced obesity negatively impacts whole-animal carbohydrate metabolism associated with altered islet lipid content, increased expression of β-oxidative genes, and reduced β-cell mass.

  1. Viable mass production method for cotton pink bollworm, Pectinophora gossypiella (Saunders

    Directory of Open Access Journals (Sweden)

    B. Dhara Jothi

    2016-01-01

    Full Text Available Cotton seed based artificial diet has been standardized for continuous rearing of pink bollworm Pectinophora gossypiella (Saunders at the Central Institute for Cotton Research, Regional Station, Coimbatore. The ingredients of the diet are easily available and are cost effective. Basic ingredients of the diet are cotton seed flour (processed and chick pea flour, Carbohydrate, Protein, Fat sources, multi vitamin, antimicrobial agents and agar as thickening agent are used as other ingredients. Micro centrifuge tubes with lid were used as rearing containers. Individual neonate larvae were released on each piece of the diet inside the micro centrifuge tube and the lids were closed. This prevented larval escape, retaining them inside the tubes and also prevented diet dehydration. The recovery of insect reared on diet was recorded as 95.56%. Egg hatchability and adult emergence were 100% while pupal malformation was nil. Eggs, larval and pupal periods were recorded as 4.8 ± 0.632, 25.10 ± 0.994 and 7.9 ± 0.88 days, respectively. Larval and pupal weights were recorded as 21.40 mg ± 3.63, 18.00 mg ± 2.73, respectively.

  2. Propidium monoazide combined with real-time quantitative PCR to quantify viable Alternaria spp. contamination in tomato products.

    Science.gov (United States)

    Crespo-Sempere, Ana; Estiarte, Núria; Marín, Sonia; Sanchis, Vicente; Ramos, Antonio J

    2013-08-01

    Alternaria is a common contaminating genus of fungi in fruits, grains, and vegetables that causes severe economic losses to farmers and the food industry. Furthermore, it is claimed that Alternaria spp. are able to produce phytotoxic metabolites, and mycotoxins that are unsafe for human and animal health. DNA amplification techniques are being increasingly applied to detect, identify, and quantify mycotoxigenic fungi in foodstuffs, but the inability of these methods to distinguish between viable and nonviable cells might lead to an overestimation of mycotoxin-producing living cells. A promising technique to overcome this problem is the pre-treatment of samples with nucleic acid intercalating dyes, such as propidium monoazide (PMA), prior to quantitative PCR (qPCR). PMA selectively penetrates cells with a damaged membrane inhibiting DNA amplification during qPCRs. In our study, a primer pair (Alt4-Alt5) to specifically amplify and quantify Alternaria spp. by qPCR was designed. Quantification data of qPCR achieved a detection limit of 10(2)conidia/g of tomato. Here, we have optimized for the first time a DNA amplification-based PMA sample pre-treatment protocol for detecting viable Alternaria spp. cells. Artificially inoculated tomato samples treated with 65μM of PMA, showed a reduction in the signal by almost 7cycles in qPCR between live and heat-killed Alternaria spp. conidia. The tomato matrix had a protective effect on the cells against PMA toxicity, reducing the efficiency to distinguish between viable and nonviable cells. The results reported here indicate that the PMA-qPCR method is a suitable tool for quantifying viable Alternaria cells, which could be useful for estimating potential risks of mycotoxin contamination. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. COUP-TFII controls mouse pancreatic β-cell mass through GLP-1-β-catenin signaling pathways.

    Directory of Open Access Journals (Sweden)

    Marie Boutant

    Full Text Available BACKGROUND: The control of the functional pancreatic β-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how β-cell mass is determined. METHODOLOGY/PRINCIPAL FINDINGS: Conditional chicken ovalbumin upstream promoter transcription factor II (COUP-TFII-deficient mice were generated and crossed with mice expressing Cre under the control of pancreatic duodenal homeobox 1 (pdx1 gene promoter. Ablation of COUP-TFII in pancreas resulted in glucose intolerance. Beta-cell number was reduced at 1 day and 3 weeks postnatal. Together with a reduced number of insulin-containing cells in the ductal epithelium and normal β-cell proliferation and apoptosis, this suggests decreased β-cell differentiation in the neonatal period. By testing islets isolated from these mice and cultured β-cells with loss and gain of COUP-TFII function, we found that COUP-TFII induces the expression of the β-catenin gene and its target genes such as cyclin D1 and axin 2. Moreover, induction of these genes by glucagon-like peptide 1 (GLP-1 via β-catenin was impaired in absence of COUP-TFII. The expression of two other target genes of GLP-1 signaling, GLP-1R and PDX-1 was significantly lower in mutant islets compared to control islets, possibly contributing to reduced β-cell mass. Finally, we demonstrated that COUP-TFII expression was activated by the Wnt signaling-associated transcription factor TCF7L2 (T-cell factor 7-like 2 in human islets and rat β-cells providing a feedback loop. CONCLUSIONS/SIGNIFICANCE: Our findings show that COUP-TFII is a novel component of the GLP-1 signaling cascade that increases β-cell number during the neonatal period. COUP-TFII is required for GLP-1 activation of the β-catenin-dependent pathway and its expression is under the control of TCF7L2.

  4. A High-Sensitivity Lanthanide Nanoparticle Reporter for Mass Cytometry: Tests on Microgels as a Proxy for Cells

    Science.gov (United States)

    2015-01-01

    This paper addresses the question of whether one can use lanthanide nanoparticles (e.g., NaHoF4) to detect surface biomarkers expressed at low levels by mass cytometry. To avoid many of the complications of experiments on live or fixed cells, we carried out proof-of-concept experiments using aqueous microgels with a diameter on the order of 700 nm as a proxy for cells. These microgels were used to test whether nanoparticle (NP) reagents would allow the detection of as few as 100 proteins per “cell” in cell-by-cell assays. Streptavidin (SAv), which served as the model biomarker, was attached to the microgel in two different ways. Covalent coupling to surface carboxyls of the microgel led to large numbers (>104) of proteins per microgel, whereas biotinylation of the microgel followed by exposure to SAv led to much smaller numbers of SAv per microgel. Using mass cytometry, we compared two biotin-containing reagents, which recognized and bound to the SAvs on the microgel. One was a metal chelating polymer (MCP), a biotin end-capped polyaspartamide containing 50 Tb3+ ions per probe. The other was a biotinylated NaHoF4 NP containing 15 000 Ho atoms per probe. Nonspecific binding was determined with bovine serum albumin (BSA) conjugated microgels. The MCP was effective at detecting and quantifying SAvs on the microgel with covalently bound SAv (20 000 SAvs per microgel) but was unable to give a meaningful signal above that of the BSA-coated microgel for the samples with low levels of SAv. Here the NP reagent gave a signal 2 orders of magnitude stronger than that of the MCP and allowed detection of NPs ranging from 100 to 500 per microgel. Sensitivity was limited by the level of nonspecific adsorption. This proof of concept experiment demonstrates the enhanced sensitivity possible with NP reagents in cell-by-cell assays by mass cytometry. PMID:24617504

  5. High-temperature, Knudsen cell-mass spectroscopic studies on lanthanum oxide/uranium dioxide solid solutions

    Energy Technology Data Exchange (ETDEWEB)

    Sunder, S.; McEachern, R.; LeBlanc, J.C

    2001-07-01

    Knudsen cell-mass spectroscopic experiments were carried out with lanthanum oxide/uranium oxide solid solutions (1%, 2% and 5% (metal at.% basis)) to assess the volatilization characteristics of rare earths present in irradiated nuclear fuel. The oxidation state of each sample used was conditioned to the 'uranium dioxide stage' by heating in the Knudsen cell under an atmosphere of 10% C0{sub 2} in CO. The mass spectra were analyzed to obtain the vapour pressures of the lanthanum and uranium species. It was found that the vapour pressure of lanthanum oxide follows Henry's law, i.e., its value is directly proportional to its concentration in the solid phase. Also, the vapour pressure of lanthanum oxide over the solid solution, after correction for its concentration in the solid phase, is similar to that of uranium dioxide. (author)

  6. Metglas-Elgiloy bi-layer, stent cell resonators for wireless monitoring of viscosity and mass loading

    KAUST Repository

    Viswanath, Anupam

    2012-12-21

    This paper presents the design and evaluation of magnetoelastic sensors intended for wireless monitoring of tissue accumulation in peripheral artery stents. The sensors are fabricated from 28 μm thick foils of magnetoelastic 2826MB Metglas™, an amorphous Ni-Fe alloy. The sensor layer consists of a frame and an active resonator portion. The frame consists of 150 μm wide struts that are patterned in the same wishbone array pattern as a 12 mm × 1.46 mm Elgiloy stent cell. The active portion is a 10 mm long symmetric leaf shape and is anchored to the frame at mid length. The active portion nests within the stent cell, with a uniform gap separating the two. A gold-indium eutectic bonding process is used to bond Metglas™ and Elgiloy foils, which are subsequently patterned to form bi-layer resonators. The response of the sensor to viscosity changes and mass loading that precede and accompany artery occlusion is tested in vitro. The typical sensitivity to viscosity of the fundamental, longitudinal resonant frequency at 361 kHz is 427 ppm cP -1 over a 1.1-8.6 cP range. The sensitivity to mass loading is typically between 63000 and 65000 ppm mg-1 with the resonant frequency showing a reduction of 8.1% for an applied mass that is 15% of the unloaded mass of the sensor. This is in good agreement with the theoretical response. © 2013 IOP Publishing Ltd.

  7. Cell-Free Transmission of Human Adenovirus by Passive Mass Transfer in Cell Culture Simulated in a Computer Model

    Science.gov (United States)

    Yakimovich, Artur; Gumpert, Heidi; Burckhardt, Christoph J.; Lütschg, Verena A.; Jurgeit, Andreas; Sbalzarini, Ivo F.

    2012-01-01

    Viruses spread between cells, tissues, and organisms by cell-free and cell-cell transmissions. Both mechanisms enhance disease development, but it is difficult to distinguish between them. Here, we analyzed the transmission mode of human adenovirus (HAdV) in monolayers of epithelial cells by wet laboratory experimentation and a computer simulation. Using live-cell fluorescence microscopy and replication-competent HAdV2 expressing green fluorescent protein, we found that the spread of infection invariably occurred after cell lysis. It was affected by convection and blocked by neutralizing antibodies but was independent of second-round infections. If cells were overlaid with agarose, convection was blocked and round plaques developed around lytic infected cells. Infected cells that did not lyse did not give rise to plaques, highlighting the importance of cell-free transmission. Key parameters for cell-free virus transmission were the time from infection to lysis, the dose of free viruses determining infection probability, and the diffusion of single HAdV particles in aqueous medium. With these parameters, we developed an in silico model using multiscale hybrid dynamics, cellular automata, and particle strength exchange. This so-called white box model is based on experimentally determined parameters and reproduces viral infection spreading as a function of the local concentration of free viruses. These analyses imply that the extent of lytic infections can be determined by either direct plaque assays or can be predicted by calculations of virus diffusion constants and modeling. PMID:22787215

  8. Expression analysis of pluripotency factors in the undifferentiated porcine inner cell mass and epiblast during in vitro culture.

    Science.gov (United States)

    Blomberg, Le Ann; Schreier, Lori L; Talbot, Neil C

    2008-03-01

    Limited understanding of the importance of known pluripotency factors in pig embryonic stem cells (ESC) impedes the establishment and validation of porcine ESC lines. This study evaluated the expression of known mouse ESC and human ESC (hESC) pluripotency markers in in vivo inner cell mass (ICM) and in vitro-cultured undifferentiated porcine epiblast cells isolated from 8-day porcine blastocysts, primary cultures of epiblast-derived neuroprogenitor cells, and endoderm cells. The expression profile of common pluripotency markers (POU domain 5 transcript factor 1, SRY-box containing gene 2, and Nanog homeobox), species-specific markers, ESC-associated factors, and differentiation markers was evaluated. The mRNA of uncultured ICMs, cultured epiblast cells, epiblast-derived neuroprogenitor cells, and endoderm cells was amplified prior to expression analysis of candidate genes by real-time RT-PCR. ESC factors whose expression correlated best with the undifferentiated epiblast state were identified by comparative mRNA expression analysis between porcine epiblast-derived somatic cell lines, fetal fibroblasts, and adult tissues. Across tissue types Nanog homeobox exhibited ubiquitous expression, whereas POU domain 5 transcript factor 1, teratocarcinoma-derived growth factor 1, and RNA exonuclease homolog 1 transcript expression was restricted primarily to undifferentiated epiblasts. Our results suggested that expression of pluripotency markers in undifferentiated pig epiblast cells more closely resembled that observed in hESC. Expression alterations of ESC-associated factors in epiblast cells were also observed during in vitro culture. Our data demonstrate the potential use of some pluripotency factors as markers of porcine epiblast stem cells and indicate that the in vitro environment may influence the cultured epiblast's developmental state. Copyright 2007 Wiley-Liss, Inc.

  9. Primary hyperparathyroidism diagnosed after surgical ablation of a costal mass mistaken for giant-cell bone tumor: a case report

    Directory of Open Access Journals (Sweden)

    Vera Lara

    2011-12-01

    Full Text Available Abstract Introduction Primary hyperparathyroidism is a common endocrine disorder characterized by elevated parathyroid hormone levels, which cause continuous osteoclastic bone resorption. Giant cell tumor of bone is an expansile osteolytic tumor that contains numerous osteoclast-like giant cells. There are many similarities in the radiological and histological features of giant cell tumor of bone and brown tumor. This is a rare benign focal osteolytic process most commonly caused by hyperparathyroidism. Case presentation We report the unusual case of a 40-year-old Caucasian woman in which primary hyperparathyroidism was diagnosed after surgical ablation of a costal mass. The mass was suspected of being neoplastic and histopathology was compatible with a giant cell tumor of bone. On the basis of the biochemical results (including serum calcium, phosphorous and intact parathyroid hormone levels primary hyperparathyroidism was suspected and a brown tumor secondary to refractory hyperparathyroidism was diagnosed. Conclusions Since giant cell tumor is a bone neoplasm that has major implications for the patient, the standard laboratory tests in patients with bone lesions are important for a correct diagnosis.

  10. Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

    OpenAIRE

    Doneanu, Catalin E; Xenopoulos, Alex; Fadgen, Keith; Murphy, Jim; Skilton, St. John; Prentice, Holly; Stapels, Martha; Chen, Weibin

    2012-01-01

    Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a “discovery” assa...

  11. Real-time quantification of viable bacteria in liquid medium using infrared thermography

    Science.gov (United States)

    Salaimeh, Ahmad A.; Campion, Jeffrey J.; Gharaibeh, Belal Y.; Evans, Martin E.; Saito, Kozo

    2011-11-01

    Quantifying viable bacteria in liquids is important in environmental, food processing, manufacturing, and medical applications. Since vegetative bacteria generate heat as a result of biochemical reactions associated with cellular functions, thermal sensing techniques, including infrared thermography (IRT), have been used to detect viable cells in biologic samples. We developed a novel method that extends the dynamic range and improves the sensitivity of bacterial quantification by IRT. The approach uses IRT video, thermodynamics laws, and heat transfer mechanisms to directly measure, in real-time, the amount of energy lost as heat from the surface of a liquid sample containing bacteria when the specimen cools to a lower temperature over 2 min. We show that the Energy Content ( EC) of liquid media containing as few as 120 colony-forming units (CFU) of Escherichia coli per ml was significantly higher than that of sterile media ( P method that provides real-time bacterial enumeration over a wide dynamic range without the need for sample concentration, modification, or destruction. The approach could be adapted to quantify other living cells in a liquid milieu and has the potential for automation and high throughput.

  12. Laser ablation-inductively coupled plasma mass spectrometry: an emerging technology for detecting rare cells in tissue sections.

    Science.gov (United States)

    Managh, Amy J; Hutchinson, Robert W; Riquelme, Paloma; Broichhausen, Christiane; Wege, Anja K; Ritter, Uwe; Ahrens, Norbert; Koehl, Gudrun E; Walter, Lisa; Florian, Christian; Schlitt, Hans J; Reid, Helen J; Geissler, Edward K; Sharp, Barry L; Hutchinson, James A

    2014-09-01

    Administering immunoregulatory cells to patients as medicinal agents is a potentially revolutionary approach to the treatment of immunologically mediated diseases. Presently, there are no satisfactory, clinically applicable methods of tracking human cells in patients with adequate spatial resolution and target cell specificity over a sufficient period of time. Laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) represents a potential solution to the problem of detecting very rare cells in tissues. In this article, this exquisitely sensitive technique is applied to the tracking of gold-labeled human regulatory macrophages (Mregs) in immunodeficient mice. Optimal conditions for labeling Mregs with 50-nm gold particles were investigated by exposing Mregs in culture to variable concentrations of label: Mregs incubated with 3.5 × 10(9) particles/ml for 1 h incorporated an average of 3.39 × 10(8) Au atoms/cell without loss of cell viability. Analysis of single, gold-labeled Mregs by LA-ICP-MS registered an average of 1.9 × 10(5) counts/cell. Under these conditions, 100% labeling efficiency was achieved, and label was retained by Mregs for ≥36 h. Gold-labeled Mregs adhered to glass surfaces; after 24 h of culture, it was possible to colabel these cells with human-specific (154)Sm-tagged anti-HLA-DR or (174)Yb-tagged anti-CD45 mAbs. Following injection into immunodeficient mice, signals from gold-labeled human Mregs could be detected in mouse lung, liver, and spleen for at least 7 d by solution-based inductively coupled plasma mass spectrometry and LA-ICP-MS. These promising results indicate that LA-ICP-MS tissue imaging has great potential as an analytical technique in immunology. Copyright © 2014 by The American Association of Immunologists, Inc.

  13. Allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture

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    Restu Syamsul Hadi

    2014-08-01

    Full Text Available Background Transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. Their potential for regenerative therapy of chronic wounds should be evaluated. The aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (PBMC. Methods In this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. We used one intervention group of allogeneic fibroblasts co-cultured with PBMC and 2 control groups of separate fibroblast and PBMC cultures.Fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (OCT4 expression. Viability was evaluated using water soluble tetrazolium-1 (WST-1 proliferation assay. Absorbances were measured at 450 nm. Data analysis was performed by student’s paired t-test. Results Dermal fibroblasts were shown to secrete collagen, express OCT4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with PBMC. Co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05. Calculated viable cell numbers increased 1.8 and 5.1-fold, respectively, at days 2 and 4 in vitro. Both groups showed comparable doubling times at days 2 and 4 in vitro. PBMC did not interfere with allogeneic fibroblast viability and proliferative capacity Conclusions Allogeneic fibroblasts remain viable and proliferate in the presence of host PBMC. Future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. Dermal fibroblasts are a potential source for cell therapy in chronic wound management.

  14. Allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture

    Directory of Open Access Journals (Sweden)

    Restu Syamsul Hadi

    2015-12-01

    Full Text Available BACKGROUND Transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. Their potential for regenerative therapy of chronic wounds should be evaluated. The aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (PBMC. METHODS In this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. We used one intervention group of allogeneic fibroblasts co-cultured with PBMC and 2 control groups of separate fibroblast and PBMC cultures.Fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (OCT4 expression. Viability was evaluated using water soluble tetrazolium-1 (WST-1 proliferation assay. Absorbances were measured at 450 nm. Data analysis was performed by student’s paired t-test. RESULTS Dermal fibroblasts were shown to secrete collagen, express OCT4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with PBMC. Co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05. Calculated viable cell numbers increased 1.8 and 5.1- fold, respectively, at days 2 and 4 in vitro. Both groups showed comparable doubling times at days 2 and 4 in vitro. PBMC did not interfere with allogeneic fibroblast viability and proliferative capacity CONCLUSIONS Allogeneic fibroblasts remain viable and proliferate in the presence of host PBMC. Future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. Dermal fibroblasts are a potential source for cell therapy in chronic wound management.

  15. Differentiation of oncocytoma and renal cell carcinoma in small renal masses (<4 cm): the role of 4-phase computerized tomography.

    Science.gov (United States)

    Bird, Vincent G; Kanagarajah, Prashanth; Morillo, Gaston; Caruso, Daniel J; Ayyathurai, Rajinikanth; Leveillee, Raymond; Jorda, Merce

    2011-12-01

    We investigate the use of 4-phase computerized tomography with intravenous contrast to help distinguish oncocytoma from renal cell carcinoma (RCC) in tumors renal tumors renal mass protocol and had confirmed pathological diagnosis of either oncocytoma or RCC were included in the study. Enhancement readings were obtained for the tumor and the renal cortex using the same slice simultaneously. Our cohort involved 69 patients (46 men, 23 women; mean age 66) who presented with 79 renal masses. Histopathologically 40 were clear cell, 22 papillary, 5 chromophobe RCC and 12 oncocytoma. On the arterial, venous and delayed phase images, oncocytoma showed the highest mean enhancement change, i.e.,546, 396 and 239% followed by clear cell RCC 261, 261 and 174%, chromophobe RCC 147, 127 and 66% and papillary RCC 137, 184 and 118%, respectively. The enhancement pattern differed significantly on comparing oncocytoma with RCC (P oncocytoma, respectively. The enhancement and washout values in Hounsfield units obtained by multiphasic CT scan aid in distinguishing oncocytoma from the commonly seen subtypes of RCC in renal masses renal oncocytomas.

  16. Body cell mass measured by bioimpedance spectroscopy as a nutritional marker.

    Directory of Open Access Journals (Sweden)

    Aleksandra Rymarz

    2012-06-01

    Full Text Available Body Cell Mass (BCM is a sum of all metabolically active cells of the body. Aim of the study was to compare BCM with other nutritional and inflammatory markers in patients with chronic kidney disease (CKD stage 4-5 (NKF without dialysis treatment and in hemodialysis patients(HD. We included 45 adult patients with CKD and eGFR<30 ml/min not treated with dialysis (26 male, age: 59,7±16,8 and 39 adults treated with HD three times a week, for more than three months (26 male, 5 diabetics, age: 59,8 ±16. Body composition was measured using multifrequency biopimpedance spectroscopy: Body Composition Monitor - FMC. We used BCM index (BCMI defined as BCM divided by height to the power of 2. To measure hand grip strength (HGS we used dynamometr Jamar. In statistics analysis we used Pearson correlations (SPSS v18. Predialysis group: BCMI: 7,1 ±1,6 kg/m², Lean Tissue Index (LTI: 12,9 ±2,4 kg/m², Fat Tissue Index (FTI: 14,7 ±5,4 kg/m², BMI: 28,2 ±5 kg/m², serum creatinine level (SCr: 3,9 ±2,1 mg/dl, eGFR: 18,3 ±7,0034 ml/min/1,73 m², albumin (SA: 3,9 ±0,3 g/dl, prealbumin (PA: 32,8 ±8,8 mg/dl, CRP: 0,5 ±0,3 mg/dl. A positive correlation was found with BCMI and HGS (r = 0,55; p=0,001, PA (r = 0,41; p=0,004 and SCr (r =0,37; p=0,012. A negative correlation was found between BCMI and age (r = -0,48; p=0,006, CRP (r = -0,33; p=0,028. We do not observed correlation with BMI and SA. HD group: BCMI: 6,4±1,7 kg/m², LTI: 12,1±2,3 kg/m², FTI: 12 ±6 kg/m², BMI: 24,8 ±4,8, SCr: 8,9 ±2,6 mg/dl, TP: 6,7 ±0,6 g/dl, SA: 3,9 ±0,47 g/dl, PA 33,8 ±11,4 g/dl, CRP: 1,1 ±1,4 mg/dl. A positive, significant correlation was found between BCMI and HGS (r = 0,47; p=0,003. A negative correlation was found with BCMI and age (r = -0,55; p=0,0005 and with CRP (r = -0,31, but not statistically significant. We do not observed correlation between BCMI and BMI, SCr, TP, SA, PA, hemodialysis vintage, Kt/V. Assessment of body compartments is

  17. Cost Analysis of Direct Methanol Fuel Cell Stacks for Mass Production

    OpenAIRE

    Mauro Francesco Sgroi; Furio Zedde; Orazio Barbera; Alessandro Stassi; David Sebastián; Francesco Lufrano; Vincenzo Baglio; Antonino Salvatore Aricò; Jacob Linder Bonde; Michael Schuster

    2016-01-01

    Fuel cells are very promising technologies for efficient electrical energy generation. The development of enhanced system components and new engineering solutions is fundamental for the large-scale deployment of these devices. Besides automotive and stationary applications, fuel cells can be widely used as auxiliary power units (APUs). The concept of a direct methanol fuel cell (DMFC) is based on the direct feed of a methanol solution to the fuel cell anode, thus simplifying safety, delivery,...

  18. Characterization of thin film tandem solar cells by radiofrequency pulsed glow discharge - Time of flight mass spectrometry.

    Science.gov (United States)

    Fernandez, Beatriz; Lobo, Lara; Reininghaus, Nies; Pereiro, Rosario; Sanz-Medel, Alfredo

    2017-04-01

    Beside low production costs and the use of nontoxic and abundant raw materials, silicon based thin-film solar cells have the advantage to be built up as multi junction devices like tandem or triple junction solar cells. Silicon thin film modules made of tandem cells with hydrogenated amorphous silicon (a-Si:H) top cell and microcrystalline (μc) Si:H bottom cell are available on the market. In this work, the analytical potential of state-of-the art radiofrequency (rf) pulsed glow discharge (PGD) time of flight mass spectrometry (TOFMS) commercial instrumentation is investigated for depth profiling analysis of tandem-junctions solar cells on 2mm thick glass substrate with 1µm thick ZnO:Al. Depth profile characterization of two thin film tandem photovoltaic devices was compared using millisecond and sub-millisecond rf-PGD regimes, as well as the so-called "low mass mode" available in the commercial instrument used. Two procedures for sample preparation, namely using flat or rough cell substrates, were compared and the distribution of dopant elements (phosphorous, boron and germanium) was investigated in both cases. Experimental results obtained by rf-PGD-TOFMS as well as electrical measurements of the samples showed that a worse depth resolution of dopant elements in the silicon layers (e.g. distribution of boron in a thicker region that suggests a diffusion of this dopant in the coating of the sample) found using a rough sample substrate was related to a higher power conversion efficiency. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Social Networking and Smart Technology: Viable Environmental Communication Tools…?

    Science.gov (United States)

    Montain, J.; Byrne, J. M.

    2010-12-01

    To what extent do popular social networking channels represent a viable means for disseminating information regarding environmental change to the general public? Are new forms of communication such as YouTube™, Facebook™, MySpace™ and Twitter™ and smart devices such as iPhone™ and BlackBerry™ useful and effective in terms motivating people into social action and behavioural modification; or do they simply pay ‘lip service’ to these pressing environmental issues? This project will explore the background connections between social networking and environmental communication and education; and outline why such tools might be an appropriate way to connect to a broad audience in an efficient and unconventional manner. Further, research will survey the current prevalence of reliable environmental change information on social networking Internet-based media; and finally, suggestions for improved strategies and new directions will be provided.

  20. Dissolvable tattoo sensors: from science fiction to a viable technology

    Science.gov (United States)

    Cheng, Huanyu; Yi, Ning

    2017-01-01

    Early surrealistic painting and science fiction movies have envisioned dissolvable tattoo electronic devices. In this paper, we will review the recent advances that transform that vision into a viable technology, with extended capabilities even beyond the early vision. Specifically, we focus on the discussion of a stretchable design for tattoo sensors and degradable materials for dissolvable sensors, in the form of inorganic devices with a performance comparable to modern electronics. Integration of these two technologies as well as the future developments of bio-integrated devices is also discussed. Many of the appealing ideas behind developments of these devices are drawn from nature and especially biological systems. Thus, bio-inspiration is believed to continue playing a key role in future devices for bio-integration and beyond.

  1. A viable logarithmic f(R) model for inflation

    Energy Technology Data Exchange (ETDEWEB)

    Amin, M.; Khalil, S. [Center for Fundamental Physics, Zewail City of Science and Technology,6 October City, Giza (Egypt); Salah, M. [Center for Fundamental Physics, Zewail City of Science and Technology,6 October City, Giza (Egypt); Department of Mathematics, Faculty of Science, Cairo University,Giza (Egypt)

    2016-08-18

    Inflation in the framework of f(R) modified gravity is revisited. We study the conditions that f(R) should satisfy in order to lead to a viable inflationary model in the original form and in the Einstein frame. Based on these criteria we propose a new logarithmic model as a potential candidate for f(R) theories aiming to describe inflation consistent with observations from Planck satellite (2015). The model predicts scalar spectral index 0.9615

  2. Simultaneous pyometra and viable puppies’ gestation in a bitch

    Directory of Open Access Journals (Sweden)

    A. Risso

    2014-08-01

    Full Text Available Here we describe a case of pyometra coexisting with gestation in a 4.5 year-old miniature short-haired Dachshund. The dog exhibited depression, vaginal discharge, polydipsia and dehydration. Ultrasound examination revealed the presence of low to moderate anechoic fluid collection in the left uterine horn. Blood analysis revealed mild neutrophilia with a left shift. Based on these findings a presumptive diagnosis of pyometra was made and the bitch was treated using amoxicillin-clavulanate with dopaminergic agonist (cabergoline. A second ultrasound scan revealed the presence of two gestational vesicles in the right uterine horn that were successfully carried to term. Unusually, while pyometra persisted in the left uterine horn, two viable puppies were delivered by caesarean section from the right uterine horn.

  3. The viable but non-culturable state in pathogenic Escherichia coli: A general review

    Directory of Open Access Journals (Sweden)

    Jennifer A. Pienaar

    2016-05-01

    Objectives: This review discusses various general aspects of the VBNC state, the mechanisms and possible public health impact of indicator and pathogenic E. coli entering into the VBNC state. Method: A literature review was conducted to ascertain the possibleimpact of E. coli entering into the VBNC state. Results: Escherichia coli enter into the VBNC state by means of several induction mechanisms. Various authors have found that E. coli can be resuscitated post-VBNC. Certain strains of pathogenic E. coli are still able to produce toxins in the VBNC state, whilst others are avirulent during the VBNC state but are able to regain virulence after resuscitation. Conclusion: Pathogenic and indicator E. coli entering into the VBNC state could have an adverse effect on public health if conventional detection methods are used, where the number of viable cells could be underestimated and the VBNC cells still produce toxins or could, at anytime, be resuscitated and become virulent again.

  4. Analysis of heat and mass transport in a miniature passive and semi passive liquid-feed direct methanol fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Bin; Bahrami, Hafez; Faghri, Amir [Department of Mechanical Engineering, University of Connecticut, 261 Glenbrook Road, Unit 2337, Storrs, CT 06169 (United States)

    2010-04-15

    A two-dimensional, transient, multi-phase, multi-component, and non-isothermal model has been developed to solve the heat and mass transport in a passive and semi passive liquid-feed direct methanol fuel cell (DMFC). A semi passive DMFC uses channel at the cathode side to facilitate the oxidant transport. The transient characteristics of the temperature, methanol concentration, methanol crossover, useful current density and methanol evaporation are investigated. The results indicate that the temperature in the fuel cell increases during operation as much as 10 C, due to the heat generation by internal phase change and the electrochemical reactions. However, it is revealed that the temperature distribution is nearly uniform at any time through all porous layers including the fuel cell and fuel delivery system. The effect of using an active feeding system in the cathode and passive methanol feeding in the anode (semi passive system) on the performance of a fuel cell is also studied. The active oxidant feeding to the cathode catalyst layer in the semi passive cell improved the fuel cell performance compared to that in a passive one. However, in general, the performance of passive cell is better than that in a semi passive one because of more temperature increase in the passive system. (author)

  5. Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns.

    Science.gov (United States)

    Bengsch, Bertram; Ohtani, Takuya; Herati, Ramin Sedaghat; Bovenschen, Niels; Chang, Kyong-Mi; Wherry, E John

    2018-02-01

    The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56 hi versus CD56 dim defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation

  6. Mass entrapment and lysis of Mesodinium rubrum cells in mucus threads observed in cultures with Dinophysis

    DEFF Research Database (Denmark)

    Ojamäe, Karin; Hansen, Per Juel; Lips, Inga

    2016-01-01

    and aggregate formation of the ciliates and the feeding behaviour of Dinophysis were carried out. In cultures of either Dinophysis species, the ciliates became entrapped in the mucus, which led to the formation of immobile aggregates of M. rubrum and subsequent cell lysis. The proportion of entrapped ciliates...... was influenced by the concentration of Dinophysis and the ratio of predator and prey in the cultures. At high cell concentrations of prey (136 cells mL−1) and predator (100 cells mL−1), a maximum of 17% of M. rubrum cells became immobile and went through cell lysis. Ciliates were observed trapped in the mucus...... even when a single D. acuminata cell was present in a 3.4 mL growth medium. Both Dinophysis species were able to detect immobile or partly immobile ciliates at a distance and circled around the prey prior to the capture with a stretched out peduncle. Relatively high entrapment and lysis of M. rubrum...

  7. Validation of contrast-enhanced magnetic resonance imaging to monitor regenerative efficacy after cell therapy in a porcine model of convalescent myocardial infarction.

    Science.gov (United States)

    Malliaras, Konstantinos; Smith, Rachel R; Kanazawa, Hideaki; Yee, Kristine; Seinfeld, Jeffrey; Tseliou, Eleni; Dawkins, James F; Kreke, Michelle; Cheng, Ke; Luthringer, Daniel; Ho, Chak-Sum; Blusztajn, Agnieszka; Valle, Ileana; Chowdhury, Supurna; Makkar, Raj R; Dharmakumar, Rohan; Li, Debiao; Marbán, Linda; Marbán, Eduardo

    2013-12-24

    Magnetic resonance imaging (MRI) in the CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction (CADUCEUS) trial revealed that cardiosphere-derived cells (CDCs) decrease scar size and increase viable myocardium after myocardial infarction (MI), but MRI has not been validated as an index of regeneration after cell therapy. We tested the validity of contrast-enhanced MRI in quantifying scarred and viable myocardium after cell therapy in a porcine model of convalescent MI. Yucatan minipigs underwent induction of MI and 2-3 weeks later were randomized to receive intracoronary infusion of 12.5×10(6) mismatched allogeneic CDCs or vehicle. Allogeneic CDCs induced mild local mononuclear infiltration but no systemic immunogenicity. MRI revealed that allogeneic CDCs attenuated remodeling, improved global and regional function, decreased scar size, and increased viable myocardium compared with placebo 2 months post-treatment. Extensive histological analysis validated quantitatively the MRI measurements of scar size, scar mass, and viable mass. CDCs neither altered gadolinium contrast myocardial kinetics nor induced changes in vascular density or architecture in viable and scarred myocardium. Histology demonstrated that CDCs lead to cardiomyocyte hyperplasia in the border zone, consistent with the observed stimulation of endogenous regenerative mechanisms (cardiomyocyte cycling, upregulation of endogenous progenitors, angiogenesis). Contrast-enhanced MRI accurately measures scarred and viable myocardium after cell therapy in a porcine model of convalescent MI. MRI represents a useful tool for assessing dynamic changes in the infarct and monitoring regenerative efficacy.

  8. Profiling Total Viable Bacteria in a Hemodialysis Water Treatment System.

    Science.gov (United States)

    Chen, Lihua; Zhu, Xuan; Zhang, Menglu; Wang, Yuxin; Lv, Tianyu; Zhang, Shenghua; Yu, Xin

    2017-05-28

    Culture-dependent methods, such as heterotrophic plate counting (HPC), are usually applied to evaluate the bacteriological quality of hemodialysis water. However, these methods cannot detect the uncultured or viable but non-culturable (VBNC) bacteria, both of which may be quantitatively predominant throughout the hemodialysis water treatment system. Therefore, propidium monoazide (PMA)-qPCR associated with HPC was used together to profile the distribution of the total viable bacteria in such a system. Moreover, high-throughput sequencing of 16S rRNA gene amplicons was utilized to analyze the microbial community structure and diversity. The HPC results indicated that the total bacterial counts conformed to the standards, yet the bacteria amounts were abruptly enhanced after carbon filter treatment. Nevertheless, the bacterial counts detected by PMA-qPCR, with the highest levels of 2.14 × 10 7 copies/100 ml in softener water, were much higher than the corresponding HPC results, which demonstrated the occurrence of numerous uncultured or VBNC bacteria among the entire system before reverse osmosis (RO). In addition, the microbial community structure was very different and the diversity was enhanced after the carbon filter. Although the diversity was minimized after RO treatment, pathogens such as Escherichia could still be detected in the RO effluent. In general, both the amounts of bacteria and the complexity of microbial community in the hemodialysis water treatment system revealed by molecular approaches were much higher than by traditional method. These results suggested the higher health risk potential for hemodialysis patients from the up-to-standard water. The treatment process could also be optimized, based on the results of this study.

  9. Tailored liquid chromatography-mass spectrometry analysis improves the coverage of the intracellular metabolome of HepaRG cells.

    Science.gov (United States)

    Cuykx, Matthias; Negreira, Noelia; Beirnaert, Charlie; Van den Eede, Nele; Rodrigues, Robim; Vanhaecke, Tamara; Laukens, Kris; Covaci, Adrian

    2017-03-03

    Metabolomics protocols are often combined with Liquid Chromatography-Mass Spectrometry (LC-MS) using mostly reversed phase chromatography coupled to accurate mass spectrometry, e.g. quadrupole time-of-flight (QTOF) mass spectrometers to measure as many metabolites as possible. In this study, we optimised the LC-MS separation of cell extracts after fractionation in polar and non-polar fractions. Both phases were analysed separately in a tailored approach in four different runs (two for the non-polar and two for the polar-fraction), each of them specifically adapted to improve the separation of the metabolites present in the extract. This approach improves the coverage of a broad range of the metabolome of the HepaRG cells and the separation of intra-class metabolites. The non-polar fraction was analysed using a C18-column with end-capping, mobile phase compositions were specifically adapted for each ionisation mode using different co-solvents and buffers. The polar extracts were analysed with a mixed mode Hydrophilic Interaction Liquid Chromatography (HILIC) system. Acidic metabolites from glycolysis and the Krebs cycle, together with phosphorylated compounds, were best detected with a method using ion pairing (IP) with tributylamine and separation on a phenyl-hexyl column. Accurate mass detection was performed with the QTOF in MS-mode only using an extended dynamic range to improve the quality of the dataset. Parameters with the greatest impact on the detection were the balance between mass accuracy and linear range, the fragmentor voltage, the capillary voltage, the nozzle voltage, and the nebuliser pressure. By using a tailored approach for the intracellular HepaRG metabolome, consisting of three different LC techniques, over 2200 metabolites can be measured with a high precision and acceptable linear range. The developed method is suited for qualitative untargeted LC-MS metabolomics studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Optical quantification of cellular mass, volume and density of circulating tumor cells identified in an ovarian cancer patient

    Directory of Open Access Journals (Sweden)

    Kevin Gregory Phillips

    2012-07-01

    Full Text Available Clinical studies have demonstrated that circulating tumor cells (CTCs are present in the blood of cancer patients with known metastatic disease across the major types of epithelial malignancies. Recent studies have shown that the concentration of CTCs in the blood is prognostic of overall survival in breast, prostate, colorectal and non-small cell lung cancer. This study characterizes CTCs identified using the high-definition (HD-CTC assay in an ovarian cancer patient with stage IIIC disease. We characterized the physical properties of 31 HD-CTCs and 50 normal leukocytes from a single blood draw taken just prior to the initial debulking surgery. We utilized a non-interferometric quantitative phase microscopy technique using brightfield imagery to measure cellular dry mass. Next we used a quantitative differential interference contrast microscopy technique to measure cellular volume. These techniques were combined to determine cellular dry mass density. We found that HD-CTCs were more massive than leukocytes: 33.6 ± 3.2 pg (HD-CTC compared to 18.7 ± 0.6 pg (leukocytes, p < 0.001; had greater volumes: 518.3 ± 24.5 fL (HD-CTC compared to 230.9 ± 78.5 fL (leukocyte, p<0.001; and possessed a decreased dry mass density with respect to leukocytes: 0.065 ± 0.006 pg/fL (HD-CTC compared to 0.085 ± 0.004 pg/fL (leukocyte, p < 0.006. Quantification of HD-CTC dry mass content and volume provide key insights into the fluid dynamics of cancer, and may provide the rationale for strategies to isolate, monitor or target CTCs based on their physical properties. The parameters reported here can also be incorporated into blood cell flow models to better understand metastasis.

  11. Effects of Obesity and Diabetes on α- and β-Cell Mass in Surgically Resected Human Pancreas.

    Science.gov (United States)

    Inaishi, Jun; Saisho, Yoshifumi; Sato, Seiji; Kou, Kinsei; Murakami, Rie; Watanabe, Yuusuke; Kitago, Minoru; Kitagawa, Yuko; Yamada, Taketo; Itoh, Hiroshi

    2016-07-01

    The ethnic difference in β-cell regenerative capacity in response to obesity may be attributable to different phenotypes of type 2 diabetes among ethnicities. This study aimed to clarify the effects of diabetes and obesity on β- (BCM) and α-cell mass (ACM) in the Japanese population. We obtained the pancreases of 99 individuals who underwent pancreatic surgery and whose resected pancreas sample contained adequate normal pancreas for histological analysis. Questionnaires on a family history of diabetes and history of obesity were conducted in 59 patients. Pancreatic sections were stained for insulin or glucagon, and fractional β- and α-cell area were measured. Islet size and density as well as β-cell turnover were also quantified. In patients with diabetes, BCM was decreased by 46% compared with age- and body mass index-matched nondiabetic patients (1.48% ± 1.08% vs 0.80% ± 0.54%, P obesity or history of obesity on BCM and ACM irrespective of the presence or absence of diabetes. There was a negative correlation between BCM, but not ACM, and glycated hemoglobin before and after pancreatic surgery. In addition, reduced BCM was observed in patients with pancreatic cancer compared with those with other pancreatic tumors. These findings suggest that the increase in BCM in the face of insulin resistance is extremely limited in the Japanese, and BCM rather than ACM has a major role in regulating blood glucose level in humans.

  12. A new in vivo cross-linking mass spectrometry platform to define protein-protein interactions in living cells.

    Science.gov (United States)

    Kaake, Robyn M; Wang, Xiaorong; Burke, Anthony; Yu, Clinton; Kandur, Wynne; Yang, Yingying; Novtisky, Eric J; Second, Tonya; Duan, Jicheng; Kao, Athit; Guan, Shenheng; Vellucci, Danielle; Rychnovsky, Scott D; Huang, Lan

    2014-12-01

    Protein-protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Combined use of atomic force microscopy, X-ray photoelectron spectroscopy, and secondary ion mass spectrometry for cell surface analysis.

    Science.gov (United States)

    Dague, Etienne; Delcorte, Arnaud; Latgé, Jean-Paul; Dufrêne, Yves F

    2008-04-01

    Understanding the surface properties of microbial cells is a major challenge of current microbiological research and a key to efficiently exploit them in biotechnology. Here, we used three advanced surface analysis techniques with different sensitivity, probing depth, and lateral resolution, that is, in situ atomic force microscopy, X-ray photoelectron spectroscopy, and secondary ion mass spectrometry, to gain insight into the surface properties of the conidia of the human fungal pathogen Aspergillus fumigatus. We show that the native ultrastructure, surface protein and polysaccharide concentrations, and amino acid composition of three mutants affected in hydrophobin production are markedly different from those of the wild-type, thereby providing novel insight into the cell wall architecture of A. fumigatus. The results demonstrate the power of using multiple complementary techniques for probing microbial cell surfaces.

  14. PID Testing Method Suitable for Process Control of Solar Cells Mass Production

    Directory of Open Access Journals (Sweden)

    Xianfang Gou

    2015-01-01

    Full Text Available Voltage bias of several hundred volts which are applied between solar cells and module frames may lead to significant power losses, so-called potential-induced degradation (PID, in normal photovoltaic (PV installations system. Modules and minimodules are used to conduct PID test of solar cells. The test procedure is time consuming and of high cost, which cannot be used as process monitoring method during solar cells fabrication. In this paper, three kinds of test including minimodule, Rsh, and V-Q test are conducted on solar cells or wafers with SiNx of different refractive index. All comparisons between test results of Rsh, V-Q, and minimodule tests have shown equal results. It is shown that Rsh test can be used as quality inspection of solar cells and V-Q test of coated wafer can be used as process control of solar cells.

  15. Mass-charge-heat coupled transfers in a single cell of a proton exchange membrane fuel cell; Transferts couples masse-charge-chaleur dans une cellule de pile a combustible a membrane polymere

    Energy Technology Data Exchange (ETDEWEB)

    Ramousse, J.

    2005-11-15

    Understanding and modelling of coupled mass, charges and heat transfers phenomena are fundamental to analyze the electrical behaviour of the system. The aim of the present model is to describe electrical performances of a PEFMC according to the fluidic and thermal operating conditions. The water content of the membrane and the water distribution in the single cell are estimated according to the coupled simulations of mass transport in the thickness of the single cell and in the feeding channels of the bipolar plates. A microscopic model of a Gas Diffusion Electrode is built up to describe charges transfer phenomena occurring at the electrodes. Completed by a study of heat transfer in the Membrane Electrode Assembly, conditions and preferential sites of water vapor condensation can be highlighted. A set of measurements of the effective thermal conductivity of carbon felts used in fuel cells as porous backing layers have also been performed. Although the value of this parameter is essential for the study of heat transfer, it is still under investigation because of the strong thermal anisotropy of the medium. (author)

  16. Picrorhiza kurroa Enhances β-Cell Mass Proliferation and Insulin Secretion in Streptozotocin Evoked β-Cell Damage in Rats

    Directory of Open Access Journals (Sweden)

    Shiv Kumar

    2017-08-01

    Full Text Available Autoimmune destruction of insulin producing pancreatic β-cells leads to insulin insufficiency and hyperglycemia in type 1 diabetes mellitus. Regeneration of β-cells is one of the proposed treatment for type 1 diabetes and insulin insufficiency. Picrorhiza kurroa is a medicinal herb and is traditionally being used for the treatment of various diseases. Previous studies reported the hypoglycemic potential of P. kurroa. However, its potential role in β-cell induction in insulin secretion have not been fully investigated. Here, we characterized the hydro alcoholic extract of P. kurroa rhizome (PKRE and further studied its β-cell regeneration and induction of insulin secretion potential in streptozotocin (STZ induced diabetic rats as well as in insulin producing Rin5f cells. 1H-NMR revealed the presence of more than thirty metabolites including picroside I and II in PKRE. Further, we found that PKRE treatment (100 and 200 mg/kg dose for 30 days significantly (p ≤ 0.05 protected the pancreatic β-cells against streptozotocin (STZ evoked damage and inhibited the glucagon receptor expression (Gcgr in hepatic and renal tissues. It significantly (p ≤ 0.05 enhanced the insulin expression and aids in proliferation of insulin producing Rin5f cells with elevated insulin secretion. Furthermore it significantly (p ≤ 0.05 increased insulin mediated glucose uptake in 3T3L1 and L6 cells. On the contrary, in diabetic rats, PKRE significantly (p ≤ 0.05 decreased high blood glucose and restored the normal levels of serum biochemicals. Altogether, our results showed that PKRE displayed β-cell regeneration with enhanced insulin production and antihyperglycemic effects. PKRE also improves hepatic and renal functions against oxidative damage.

  17. Mass spectrometric study of rhamnolipid biosurfactants and their interactions with cell membrane phospholipids

    Directory of Open Access Journals (Sweden)

    Pashynska V. A.

    2009-12-01

    Full Text Available Aim. To examine the formation of supramolecular complexes of biogenous rhamnolipids with membrane phospholipids that is considered as a molecular mechanism of the biosurfactants antimicrobial action. Method. In the present work rhamnolipid biosurfactant samples produced by Pseudomonas sp. PS-17 strain have been investigated by electrospray ionization mass spectrometry for the first time. Results. As a result of the study, characteristic mass spectra of the rhamnolipid samples were obtained, that can be used as reference spectra for mass spectrometric identification of the compounds in any biological or industrial samples. At the next stage of the experiments the pair systems, containing the biosurfactants and a membrane phospholipid dipalmitoylphosphatidylcholine, have been tested. The cationized noncovalent complexes of the rhamnolipids with the phospholipid were observed in the spectra. Conclusions. The results obtained testify to the consideration that rhamnolipids (similar to other membranotropic agents can form stable supramolecular complexes with membrane phospholipids that are able to evoke the biosurfactants antimicrobial action. A great potential of electrospray ionization mass spectrometry for the biosurfactants identification and study has been demonstrated in the work.

  18. Regulation of pancreatic beta-cell mass and proliferation by SOCS-3

    DEFF Research Database (Denmark)

    Lindberg, K; Rønn, S G; Tornehave, D

    2005-01-01

    Growth hormone and prolactin are important growth factors for pancreatic beta-cells. The effects exerted by these hormones on proliferation and on insulin synthesis and secretion in beta-cells are largely mediated through the Janus kinase (JAK)/signal transducer and activator of transcription (ST......-type littermates following an oral glucose-tolerance test. Together these data suggest that SOCS-3 modulates cytokine signaling in pancreatic beta-cells and therefore potentially could be a candidate target for development of new treatment strategies for diabetes.......Growth hormone and prolactin are important growth factors for pancreatic beta-cells. The effects exerted by these hormones on proliferation and on insulin synthesis and secretion in beta-cells are largely mediated through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT...

  19. Direct detection of diverse metabolic changes in virally transformed and tax-expressing cells by mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Prabhakar Sripadi

    Full Text Available BACKGROUND: Viral transformation of a cell starts at the genetic level, followed by changes in the proteome and the metabolome of the host. There is limited information on the broad metabolic changes in HTLV transformed cells. METHODS AND PRINCIPAL FINDINGS: Here, we report the detection of key changes in metabolites and lipids directly from human T-lymphotropic virus type 1 and type 3 (HTLV1 and HTLV3 transformed, as well as Tax1 and Tax3 expressing cell lines by laser ablation electrospray ionization (LAESI mass spectrometry (MS. Comparing LAESI-MS spectra of non-HTLV1 transformed and HTLV1 transformed cells revealed that glycerophosphocholine (PC lipid components were dominant in the non-HTLV1 transformed cells, and PC(O-32:1 and PC(O-34:1 plasmalogens were displaced by PC(30:0 and PC(32:0 species in the HTLV1 transformed cells. In HTLV1 transformed cells, choline, phosphocholine, spermine and glutathione, among others, were downregulated, whereas creatine, dopamine, arginine and AMP were present at higher levels. When comparing metabolite levels between HTLV3 and Tax3 transfected 293T cells, there were a number of common changes observed, including decreased choline, phosphocholine, spermine, homovanillic acid, and glycerophosphocholine and increased spermidine and N-acetyl aspartic acid. These results indicate that the lipid metabolism pathway as well as the creatine and polyamine biosynthesis pathways are commonly deregulated after expression of HTLV3 and Tax3, indicating that the noted changes are likely due to Tax3 expression. N-acetyl aspartic acid is a novel metabolite that is upregulated in all cell types and all conditions tested. CONCLUSIONS AND SIGNIFICANCE: We demonstrate the high throughput in situ metabolite profiling of HTLV transformed and Tax expressing cells, which facilitates the identification of virus-induced perturbations in the biochemical processes of the host cells. We found virus type-specific (HTLV1 vs. HTLV3

  20. Effect of flow pulsation on mass transport in a cathode channel of polymer electrolyte membrane fuel cell

    Science.gov (United States)

    Han, Hun Sik; Kim, Yun Ho; Kim, Seo Young; Hyun, Jae Min

    2012-09-01

    An experimental and theoretical study on the cathode flow pulsation in a polymer electrolyte membrane (PEM) fuel cell is performed. A 10-cell PEM fuel cell stack with open-air cathode channels is employed to investigate the effects of the cathode flow pulsation on the overall performance. The polarization and corresponding power curves obtained show that both the limiting current density and the maximum power density are substantially enhanced when the pulsating component is added to the cathode mainstream flow. The flow pulsation at Re = 77 provides the maximum increment of 40% and 35.5% in the limiting current density and in the maximum power density, respectively. The enhancement of the overall performance is more pronounced at low Reynolds numbers. Also, the theoretical mass transport analysis in the pulsating cathode flow channel is carried out to verify the present experimental results. The momentum and species conservation equations are analytically solved, and the effective time-averaged dispersion coefficient is defined to account for the enhanced mass transport by the flow pulsation. Comprehensive analytical solutions show that the effect of the relevant parameters is in well accordance with the experimental results.

  1. Extranodal NK/T-cell lymphoma, nasal type, manifesting as rapidly progressive dementia without any mass or enhancing brain lesion.

    Science.gov (United States)

    Shimatani, Yoshimitsu; Nakano, Yuta; Tsuyama, Naoko; Murayama, Shigeo; Oki, Ryosuke; Miyamoto, Ryosuke; Murakami, Nagahisa; Fujita, Koji; Watanabe, Syunsuke; Uehara, Hisanori; Abe, Takashi; Nodera, Hiroyuki; Kawarai, Toshitaka; Izumi, Yuishin; Kaji, Ryuji

    2016-10-01

    Among the many potential etiologies for rapidly progressive dementia (RPD), primary central nervous system extranodal NK/T-cell lymphoma, nasal-type (ENKL) is a rare entity. We present the first reported case of autopsy-proven RPD due to ENKL without any mass or enhancing lesion of the brain. A 54-year-old immunocompetent man presented with RPD, myoclonus and ataxia. The mini-mental state examination (MMSE) score was 22/30. His brain MRI revealed progressive brain atrophy without gadolinium enhancement or mass lesion. Five months after the initial evaluation, cognitive impairment further worsened with an MMSE score of 3/30. At the advanced stage, lumbar MRI showed swollen cauda equina with gadolinium enhancement. The number of Epstein-Barr virus (EBV) DNA in cerebrospinal fluid had gradually increased. Twelve months after onset, the patient died of respiratory failure. Pathological findings revealed that lymphoma cells had diffusely invaded the meninges, parenchyma of the brain, spinal cord and cauda equina. Cells were positive for CD3, CD56 and EBV-encoded small RNAs and negative for CD20. No evidence of malignancy was identified in the visceral organs. This report indicates that ENKL should be recognized as one of the rare causes of RPD. Early testing for EBV-DNA in cerebrospinal fluid and imaging of cauda equina would be useful diagnostic tools. © 2016 Japanese Society of Neuropathology.

  2. Bioprocess development for mass production of size-controlled human pluripotent stem cell aggregates in stirred suspension bioreactor.

    Science.gov (United States)

    Abbasalizadeh, Saeed; Larijani, Mehran Rezaei; Samadian, Azam; Baharvand, Hossein

    2012-11-01

    Current protocols for the scalable suspension culture of human pluripotent stem cells (hPSCs) are limited by multiple biological and technical challenges that need to be addressed before their use in clinical trials. To overcome these challenges, we have developed a novel bioprocess platform for large-scale expansion of human embryonic and induced pluripotent stem cell lines as three-dimensional size-controlled aggregates. This novel bioprocess utilizes the stepwise optimization of both static and dynamic suspension culture conditions. After screening eight xeno-free media in static suspension culture and optimizing single-cell passaging in dynamic conditions, the scale-up from a static to a dynamic suspension culture in the stirred bioreactor resulted in a two- to threefold improvement in expansion rates, as measured by cell counts and metabolic activity. We successfully produced size-specific aggregates through optimization of bioreactor hydrodynamic conditions by using combinations of different agitation rates and shear protectant concentrations. The expansion rates were further improved by controlling oxygen concentration at normoxic conditions, and reached a maximum eightfold increase for both types of hPSCs. Subsequently, we demonstrated a simple and rapid scale-up strategy that produced clinically relevant numbers of hPSCs (∼2×10(9) cells) over a 1-month period by the direct transfer of "suspension-adapted frozen cells" to a stirred suspension bioreactor. We omitted the required preadaptation passages in the static suspension culture. The cells underwent proliferation over multiple passages in the demonstrated xeno-free dynamic suspension culture while maintaining their self-renewal capabilities, as determined by marker expressions and in vitro spontaneous differentiation. In conclusion, suspension culture protocols of hPSCs could be used to mass produce homogenous and pluripotent undifferentiated cells by identification and optimization of key bioprocess

  3. Validation of Contrast-Enhanced MRI to Monitor Regenerative Efficacy after Cell Therapy in a Porcine Model of Convalescent Myocardial Infarction

    Science.gov (United States)

    Malliaras, Konstantinos; Smith, Rachel R.; Kanazawa, Hideaki; Yee, Kristine; Seinfeld, Jeffrey; Tseliou, Eleni; Dawkins, James F.; Kreke, Michelle; Cheng, Ke; Luthringer, Daniel; Ho, Chak-Sum; Blusztajn, Agnieszka; Valle, Ileana; Chowdhury, Supurna; Makkar, Raj R.; Dharmakumar, Rohan; Li, Debiao; Marbán, Linda; Marbán, Eduardo

    2014-01-01

    Background Magnetic Resonance Imaging (MRI) in the CADUCEUS trial revealed that cardiosphere-derived cells (CDCs) decrease scar size and increase viable myocardium post-myocardial infarction (MI), but MRI has not been validated as an index of regeneration after cell therapy. We tested the validity of contrast-enhanced MRI in quantifying scarred and viable myocardium after cell therapy in a porcine model of convalescent MI. Methods and Results Yucatan minipigs underwent induction of MI and 2-3 weeks later were randomized to receive intracoronary infusion of 12.5×106 mismatched allogeneic CDCs or vehicle. Allogeneic CDCs induced mild local mononuclear infiltration but no systemic immunogenicity. MRI revealed that allogeneic CDCs attenuated remodeling, improved global and regional function, decreased scar size and increased viable myocardium compared to placebo 2 months post-treatment. Extensive histological analysis validated quantitatively the MRI measurements of scar size, scar mass and viable mass. CDCs neither altered gadolinium contrast myocardial kinetics, nor induced changes in vascular density or architecture in viable and scarred myocardium. Histology demonstrated that CDCs lead to cardiomyocyte hyperplasia in the border zone, consistent with the observed stimulation of endogenous regenerative mechanisms (cardiomyocyte cycling, upregulation of endogenous progenitors, angiogenesis). Conclusions Contrast-enhanced MRI accurately measures scarred and viable myocardium after cell therapy in a porcine model of convalescent MI. MRI represents a useful tool for assessing dynamic changes in the infarct and monitoring regenerative efficacy. PMID:24061088

  4. Exercise and Amino Acid Anabolic Cell Signaling and the Regulation of Skeletal Muscle Mass

    OpenAIRE

    Pasiakos, Stefan M.

    2012-01-01

    A series of complex intracellular networks influence the regulation of skeletal muscle protein turnover. In recent years, studies have examined how cellular regulators of muscle protein turnover modulate metabolic mechanisms contributing to the loss, gain, or conservation of skeletal muscle mass. Exercise and amino acids both stimulate anabolic signaling potentially through several intracellular pathways including the mammalian target of rapamycin complex 1 and the mitogen activated protein k...

  5. The elusive minimum viable population size for white sturgeon

    Energy Technology Data Exchange (ETDEWEB)

    Jager, Yetta [ORNL; Lepla, Ken B. [Idaho Power Company; Van Winkle, Webb [Van Windle Environmental Consulting; James, Mr Brad [Washington Department of Fish and Wildlife; McAdam, Dr Steve [University of British Columbia, Vancouver

    2010-01-01

    Biological conservation of sturgeon populations is a concern for many species. Those responsible for managing the white sturgeon (Acipenser transmontanus) and similar species are interested in identifying extinction thresholds to avoid. Two thresholds that exist in theory are the minimum viable population size (MVP) and minimum amount of suitable habitat. In this paper, we present both model and empirical estimates of these thresholds. We modified a population viability analysis (PVA) model for white sturgeon to include two new Allee mechanisms. Despite this, PVA-based MVP estimates were unrealistically low compared with empirical estimates unless opportunities for spawning were assumed to be less frequent. PVA results revealed a trade-off between MVP and habitat thresholds; smaller populations persisted in longer river segments and vice versa. Our empirical analyses suggested (1) a MVP range based on population trends from 1,194 to 27,700 individuals, and (2) a MVP estimate of 4,000 individuals based on recruitment. Long-term historical population surveys are needed for more populations to pinpoint an MVP based on trends, whereas the available data were sufficient to estimate MVP based on recruitment. Beyond the MVP, we developed a hierarchical model for population status based on empirical data. Metapopulation support was the most important predictor of population health, followed by the length of free-flowing habitat, with habitat thresholds at 26 and 150 km. Together, these results suggest that habitat and connectivity are important determinants of population status that likely influence the site-specific MVP thresholds.

  6. Towards a viable and just global nursing ethics.

    Science.gov (United States)

    Crigger, Nancy J

    2008-01-01

    Globalization, an outgrowth of technology, while informing us about people throughout the world, also raises our awareness of the extreme economic and social disparities that exist among nations. As part of a global discipline, nurses are vitally interested in reducing and eliminating disparities so that better health is achieved for all people. Recent literature in nursing encourages our discipline to engage more actively with social justice issues. Justice in health care is a major commitment of nursing; thus questions in the larger sphere of globalization, justice and ethics, are our discipline's questions also. Global justice, or fairness, is not an issue for some groups or institutions, but a deeper human rights issue that is a responsibility for everyone. What can we do to help reduce or eliminate the social and economic disparities that are so evident? What kind of ethical milieu is needed to address the threat that globalization imposes on justice and fairness? This article enriches the conceptualization of globalization by investigating recent work by Schweiker and Twiss. In addition, I discuss five qualities or characteristics that will facilitate the development of a viable and just global ethic. A global ethic guides all people in their response to human rights and poverty. Technology and business, two major forces in globalization that are generally considered beneficial, are critiqued as barriers to social justice and the common good.

  7. Keeping checkpoint/restart viable for exascale systems.

    Energy Technology Data Exchange (ETDEWEB)

    Riesen, Rolf E.; Bridges, Patrick G. (IBM Research, Ireland, Mulhuddart, Dublin); Stearley, Jon R.; Laros, James H., III; Oldfield, Ron A.; Arnold, Dorian (University of New Mexico, Albuquerque, NM); Pedretti, Kevin Thomas Tauke; Ferreira, Kurt Brian; Brightwell, Ronald Brian

    2011-09-01

    Next-generation exascale systems, those capable of performing a quintillion (10{sup 18}) operations per second, are expected to be delivered in the next 8-10 years. These systems, which will be 1,000 times faster than current systems, will be of unprecedented scale. As these systems continue to grow in size, faults will become increasingly common, even over the course of small calculations. Therefore, issues such as fault tolerance and reliability will limit application scalability. Current techniques to ensure progress across faults like checkpoint/restart, the dominant fault tolerance mechanism for the last 25 years, are increasingly problematic at the scales of future systems due to their excessive overheads. In this work, we evaluate a number of techniques to decrease the overhead of checkpoint/restart and keep this method viable for future exascale systems. More specifically, this work evaluates state-machine replication to dramatically increase the checkpoint interval (the time between successive checkpoint) and hash-based, probabilistic incremental checkpointing using graphics processing units to decrease the checkpoint commit time (the time to save one checkpoint). Using a combination of empirical analysis, modeling, and simulation, we study the costs and benefits of these approaches on a wide range of parameters. These results, which cover of number of high-performance computing capability workloads, different failure distributions, hardware mean time to failures, and I/O bandwidths, show the potential benefits of these techniques for meeting the reliability demands of future exascale platforms.

  8. Is Greenberg’s “Macro-Carib” viable?

    Directory of Open Access Journals (Sweden)

    Spike Gildea

    2007-08-01

    Full Text Available In his landmark work Language in the Americas, Greenberg (1987 proposed that Macro-Carib was one of the major low-level stocks of South America, which together with Macro-Panoan and Macro-Ge-Bororo were claimed to comprise the putative Ge-Pano-Carib Phylum. His Macro-Carib includes the isolates Andoke and Kukura, and the Witotoan, Peba-Yaguan, and Cariban families. Greenberg’s primary evidence came from person-marking paradigms in individual languages, plus scattered words from individual languages collected into 79 Macro-Carib ‘etymologies’ and another 64 Amerind ‘etymologies’. The goal of this paper is to re-evaluate Greenberg’s Macro-Carib claim in the light of the much more extensive and reliable language data that has become available largely since 1987. Based on full person-marking paradigms for Proto-Cariban, Yagua, Bora and Andoke, we conclude that Greenberg’s morphological claims are unfounded. For our lexical comparison, we created lexical lists for Proto-Cariban, Proto-Witotoan, Yagua and Andoke, for both Greenberg’s 143 putative etymologies and for the Swadesh 100 list. From both lists, a total of 23 potential cognates were found, but no consonantal correspondences were repeated even once. We conclude that our greatly expanded and improved database does not provide sufficient evidence to convince the skeptic that the Macro-Carib hypothesis is viable.

  9. Case-based anatomy teaching: a viable alternative?

    Science.gov (United States)

    Eseonu, Onyedikachi; Carachi, Robert; Brindley, Nicola

    2013-08-01

    Over the last two decades, there has been a decline in the amount of time available for anatomy teaching in the medical undergraduate curriculum, and new methods of anatomy teaching have been adopted for pragmatic reasons, with little evidence base to support their proposed educational benefits. This study seeks to establish the effect of a case-based teaching method on students' confidence in anatomy. Forty-three student volunteers in the clinical phase of the Glasgow medical course were given weekly anatomy teaching sessions based on clinical case presentations over 4 weeks. The students were given an anatomy test, and were asked to rate their confidence in their anatomy knowledge before and after the teaching sessions. There was a two-point increase in students' self-rated confidence, and a 10.9 per cent increase in average test score after the case-based anatomy teaching sessions. Both of these increases were statistically significant (p teaching was also highly rated by students, which may make it a viable option for the teaching of anatomy in the modern medical curriculum. © 2013 John Wiley & Sons Ltd.

  10. Protein design algorithms predict viable resistance to an experimental antifolate.

    Science.gov (United States)

    Reeve, Stephanie M; Gainza, Pablo; Frey, Kathleen M; Georgiev, Ivelin; Donald, Bruce R; Anderson, Amy C

    2015-01-20

    Methods to accurately predict potential drug target mutations in response to early-stage leads could drive the design of more resilient first generation drug candidates. In this study, a structure-based protein design algorithm (K* in the OSPREY suite) was used to prospectively identify single-nucleotide polymorphisms that confer resistance to an experimental inhibitor effective against dihydrofolate reductase (DHFR) from Staphylococcus aureus. Four of the top-ranked mutations in DHFR were found to be catalytically competent and resistant to the inhibitor. Selection of resistant bacteria in vitro reveals that two of the predicted mutations arise in the background of a compensatory mutation. Using enzyme kinetics, microbiology, and crystal structures of the complexes, we determined the fitness of the mutant enzymes and strains, the structural basis of resistance, and the compensatory relationship of the mutations. To our knowledge, this work illustrates the first application of protein design algorithms to prospectively predict viable resistance mutations that arise in bacteria under antibiotic pressure.

  11. Towards viable cosmological models of disformal theories of gravity

    Science.gov (United States)

    Sakstein, Jeremy

    2015-01-01

    The late-time cosmological dynamics of disformal gravity are investigated using dynamical systems methods. It is shown that in the general case there are no stable attractors that screen fifth forces locally and simultaneously describe a dark energy dominated universe. Viable scenarios have late-time properties that are independent of the disformal parameters and are identical to the equivalent conformal quintessence model. Our analysis reveals that configurations where the Jordan frame metric becomes singular are only reached in the infinite future, thus explaining the natural pathology resistance observed numerically by several previous works. The viability of models where this can happen is discussed in terms of both the cosmological dynamics and local phenomena. We identify a special parameter tuning such that there is a new fixed point that can match the presently observed dark energy density and equation of state. This model is unviable when the scalar couples to the visible sector but may provide a good candidate model for theories where only dark matter is disformally coupled.

  12. SMA actuators: a viable practical technology (Presentation Video)

    Science.gov (United States)

    Browne, Alan L.; Brown, Jeffrey; Hodgson, Darel E.

    2015-04-01

    Diverse products either based solely on or incorporating Shape Memory Alloys (SMA) have and are being made in a wide range of industries, and IP is being captured. Why then compared to SE (superelastic) Nitinol, and especially conventional technology, do so few ideas reach production? This presentation delves deeply into this topic in reaching the final assessment that SMA actuators are indeed now a viable practical technology. The presentation begins with an introduction to and description of the fundamental basis of SMA actuator technology. Examples of multiple commercially available geometric forms of SMA actuators are given and the functionalities that they provide are described. This is followed by examples of multiple commercial products incorporating such SMA actuators. Given that there are literally millions of commercial products incorporating conventional actuator technologies, indications are given as to why there are their less than 1000 that utilize SMA. Experience based challenges to the commercial use of SMA actuators are described. Besides having to compete with existing non-SMA technology which is quite mature additional challenges that are unique to SM actuators are indicated these including a wider than expected set of technical engineering problems and challenges and that a broader scope of dynamics is required.

  13. Ambient ionisation mass spectrometry for lipid profiling and structural analysis of mammalian oocytes, preimplantation embryos and stem cells.

    Science.gov (United States)

    Ferreira, Christina R; Jarmusch, Alan K; Pirro, Valentina; Alfaro, Clint M; González-Serrano, Andres F; Niemann, Heiner; Wheeler, Matthew B; Rabel, Rathnaweera A C; Hallett, Judy E; Houser, Rebecca; Kaufman, Annemarie; Cooks, R Graham

    2015-05-01

    Lipids play fundamental roles in mammalian embryo preimplantation development and cell fate. Triacylglycerol accumulates in oocytes and blastomeres as lipid droplets, phospholipids influence membrane functional properties, and essential fatty acid metabolism is important for maintaining the stemness of cells cultured in vitro. The growing impact that lipids have in the field of developmental biology makes analytical approaches to analyse structural information of great interest. This paper describes the concept and presents the results of lipid profiling by mass spectrometry (MS) of oocytes and preimplantation embryos, with special focus on ambient ionisation. Based on our previous experience with oocytes and embryos, we aim to convey that ambient MS is also valuable for stem cell differentiation analysis. Ambient ionisation MS allows the detection of a wide range of lipid classes (e.g. free fatty acids, cholesterol esters, phospholipids) in single oocytes, embryos and cell pellets, which are informative of in vitro culture impact, developmental and differentiation stages. Background on MS principles, the importance of underused MS scan modes for structural analysis of lipids, and statistical approaches used for data analysis are covered. We envisage that MS alone or in combination with other techniques will have a profound impact on the understanding of lipid metabolism, particularly in early embryo development and cell differentiation research.

  14. Association of white blood cell counts with left ventricular mass index in hypertensive patients undergoing anti-hypertensive drug therapy.

    Science.gov (United States)

    Shi, Hongtao; Chu, Hongxia; Lv, Zhiyang; Qi, Guanming; Guo, Junjie; Fu, Wei; Wang, Xiaojing; Guo, Xiangyu; Ge, Junbo; Yin, Chengqian

    2017-04-01

    Although studies using animal models have demonstrated that nonhemodynamic factors, including inflammatory cells and cytokines, contribute to left ventricular hypertrophy (LVH), there is little clinical data to confirm this association. Therefore in the present study, levels of circulating specific types of leukocyte were measured to determine the association between white blood cells and left ventricular mass index (LVMI) in hypertensive patients undergoing anti-hypertensive drug therapy. A total of 144 consecutive hypertensive patients taking anti-hypertensive drug therapy were enrolled in the current study. Subjects were divided into two groups: Those with normal geometry and those with left LVH. Total white blood cells and differentiated subtypes (neutrophils, lymphocytes, monocytes) were counted, and left ventricular end-diastolic diameter, left ventricular posterior wall thickness in diastole and inter-ventricular septal wall thickness in diastole were all measured. Analysis revealed a significant correlation between LVMI and total white blood cell levels (P=0.013). The percentage of LVH in the highest tertile of WBC was increased compared with the middle tertile (P=0.008). Furthermore, a significant correlation between the highest tertile of neutrophil counts and LVH was observed (P=0.039). However, no significant associations between LVMI and monocyte or lymphocyte counts were detected. Therefore, the current study determined that increased total white blood cell and neutrophil subtype counts were associated with LVMI in hypertensive patients undergoing anti-hypertensive drug therapy. They may provide convenient and useful markers for further risk appraisal of LVH caused by nonhemodynamic factors of hypertension.

  15. Fluoride sample matrices and reaction cells — new capabilities for isotope measurements in accelerator mass spectrometry

    Directory of Open Access Journals (Sweden)

    Eliades J.

    2012-04-01

    Full Text Available Two new techniques, which extend the range of elements that can be analyzed by Accelerator Mass Spectrometry (AMS, and which increase its isobar selection capabilities, have been recently introduced. The first consists of embedding the sample material in a fluoride matrix (e.g. PbF2, which facilitates the production, in the ion source, of fluoride molecular anions that include the isotope of interest. In addition to forming anions with large electron binding energies and thereby increasing the range of analysable elements, in many cases by selection of a molecular form with a particular number of fluorine atoms, some isobar discrimination can be obtained. The second technique, for the significant reduction of atomic isobar interferences, is used following mass selection of the rare isotope. It consists of the deceleration, cooling and reaction of the rare mass beam with a gas, selected so that unwanted isobars are greatly attenuated in comparison with the isotope of interest. Proof of principle measurements for the analysis of 36C1 and 41Ca have provided encouraging results and work is proceeding on the integration of these techniques in a new AMS system planned for installation in late 2012 at the University of Ottawa.

  16. MALDI mass spectrometry based molecular phenotyping of CNS glial cells for prediction in mammalian brain tissue

    DEFF Research Database (Denmark)

    Hanrieder, Jørg; Wicher, Grzegorz; Bergquist, Jonas

    2011-01-01

    tracers for prediction of oligodendroglial and astroglial localization in brain tissue. The different cell type specific protein distributions in tissue were validated using immunohistochemistry. ICMS of intact neuroglia is a simple and straightforward approach for characterization and discrimination...

  17. Effect of body mass index on the survival of patients with early-stage small cell lung cancer after surgery

    Directory of Open Access Journals (Sweden)

    A. P. Kolesnik

    2013-04-01

    Full Text Available Lung cancer is an important medico-social problem. It is connected with high level of incidence and mortality of lung cancer. Nowadays we know that obesity has significant influence on cancer development, including lung cancer. Multivariate analysis confirmed that histological type of tumour, kind of operation and body mass index (BMI influence on prognosis in patients with early stages of mall cell lung cancer. So, now we can use BMI along with other significant prognostic criteria for detection of unfavorable prognostic group of patients.

  18. Small (Renal Mass: Differentiation of Oncocytoma From Renal Cell Carcinoma on Biphasic Contrast-Enhanced CT.

    Science.gov (United States)

    Sasaguri, Kohei; Takahashi, Naoki; Gomez-Cardona, Daniel; Leng, Shuai; Schmit, Grant D; Carter, Rickey E; Leibovich, Bradley C; Kawashima, Akira

    2015-11-01

    The purpose of this study was to evaluate whether small (oncocytomas can be differentiated from renal cell carcinomas (RCCs) on biphasic contrast-enhanced CT. Forty-three patients with 53 oncocytomas and 123 patients with 128 RCCs (24 papillary subtype and 104 clear cell and other subtypes) who underwent biphasic contrast-enhanced CT were included in the study. Patient demographics and CT tumor characteristics were evaluated in each case. A multinomial logistic regression model was then constructed for differentiating oncocytoma from clear cell and other subtype RCCs, oncocytoma from papillary RCCs, and clear cell and other subtype RCCs from papillary RCCs. The probability of each group was calculated from the model. Diagnostic performance among three pairwise diagnoses and between oncocytoma and any RCC (clear cell and other subtypes and papillary) were assessed by AUC values. Patient age, tumor CT attenuation values and skewness (i.e., histogram analysis of CT values) in both the corticomedullary and nephrographic phases, and subjective tumor heterogeneity were statistically significant variables in the multinomial logistic regression analysis. The logistic regression model using the variables yielded AUCs of 0.82, 0.95, 0.91, and 0.84 for differentiating oncocytomas from clear cell and other subtype RCCs, oncocytomas from papillary RCCs, clear cell and other subtype RCCs from papillary RCCs, and oncocytomas from any RCC (clear cell and other subtypes and papillary), respectively. A combination of imaging features on biphasic CT, including tumor CT attenuation values and tumor texture (heterogeneity and skewness), can help differentiate oncocytoma from RCC.

  19. Intracellular lysyl oxidase: effect of a specific inhibitor on nuclear mass in proliferating cells.

    Science.gov (United States)

    Saad, Fawzy A; Torres, Marie; Wang, Hao; Graham, Lila

    2010-06-11

    LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN (beta-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation. (c) 2010 Elsevier Inc. All rights reserved.

  20. Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

    Energy Technology Data Exchange (ETDEWEB)

    Saad, Fawzy A. [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Torres, Marie [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Wang, Hao [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Graham, Lila, E-mail: lilagraham@cs.com [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States)

    2010-06-11

    LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN ({beta}-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

  1. Microfluidic Platform with In-Chip Electrophoresis Coupled to Mass Spectrometry for Monitoring Neurochemical Release from Nerve Cells.

    Science.gov (United States)

    Li, Xiangtang; Hu, Hankun; Zhao, Shulin; Liu, Yi-Ming

    2016-05-17

    Chemical stimulus-induced neurotransmitter release from neuronal cells is well-documented. However, the dynamic changes in neurochemical release remain to be fully explored. In this work, a three-layered microfluidic chip was fabricated and evaluated for studying the dynamics of neurotransmitter release from PC-12 cells. The chip features integration of a nanoliter sized chamber for cell perfusion, pneumatic pressure valves for fluidic control, a microfluidic channel for electrophoretic separation, and a nanoelectrospray emitter for ionization in MS detection. Deploying this platform, a microchip electrophoresis-mass spectrometric method (MCE-MS) was developed to simultaneously quantify important neurotransmitters, including dopamine (DA), serotonin (5-HT), aspartic acid (Asp), and glutamic acid (Glu) without need for labeling or enrichment. Monitoring neurotransmitter release from PC-12 cells exposed to KCl (or alcohol) revealed that all four neurotransmitters investigated were released. Two release patterns were observed, one for the two monoamine neurotransmitters (i.e., DA and 5-HT) and another for the two amino acid neurotransmitters. Release dynamics for the two monoamine neurotransmitters was significantly different. The cells released DA most quickly and heavily in response to the stimulation. After exposure to the chemical stimulus for 4 min, the DA level in the perfusate from the cells was 86% lower than that at the beginning. Very interestingly, the cells started to release 5-HT in large quantities when they stopped releasing DA. These results suggest that DA and 5-HT are packaged into different vesicle pools and they are mobilized differently in response to chemical stimuli. The microfluidic platform proposed is proven useful for monitoring cellular release in biological studies.

  2. Anterior Mediastinal Mass in a Young Marijuana Smoker: A Rare Case of Small-Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Jiten P. Kothadia

    2012-01-01

    Full Text Available The use of cannabis is embedded within many societies, mostly used by the young and widely perceived to be safe. Increasing concern regarding the potential for cannabis to cause mental health effects has dominated cannabis research, and the potential adverse respiratory effects have received relatively little attention. We report a rare case of 22-year-old man who presented with bilateral neck lymphadenopathy, fatigue, and sore throat without significant medical or family history. The patient had smoked one marijuana joint three times a week for three years but no cigarettes. Chest CT demonstrated a large anterior mediastinal mass compressing the superior vena cava and mediastinal lymphadenopathy. A final diagnosis of small-cell lung cancer was reached. Although rare, a small-cell lung cancer in this patient should alert the physician that cannabis smoking may be a risk factor for lung cancer.

  3. Langerhans’ cell histiocytosis involving posterior elements of the dorsal spine: An unusual cause of extradural spinal mass in an adult

    Science.gov (United States)

    Tyagi, Devendra K.; Balasubramaniam, Srikant; Savant, Hemant V.

    2011-01-01

    Langerhans cell histiocytosis (LCH) is a clonal proliferation of Langerhans cells occurring as an isolated lesion or as part of a systemic proliferation. It is commoner in children younger than 10 years of age with sparing of the posterior elements in more than 95% of cases. We describe a case of LCH in an adult female presenting with paraplegia. MRI revealed a well-defined extradural contrast enhancing mass at D2-D4 vertebral level involving the posterior elements of spine. D2-5 laminectomy with excision of lesion was performed which lead to marked improvement of patients neurological status. Histopathology was suggestive of eosinophilic granuloma. We describe the case, discuss its uniqueness and review the literature on this rare tumor presentation. PMID:23125497

  4. Langerhans' cell histiocytosis involving posterior elements of the dorsal spine: An unusual cause of extradural spinal mass in an adult.

    Science.gov (United States)

    Tyagi, Devendra K; Balasubramaniam, Srikant; Savant, Hemant V

    2011-07-01

    Langerhans cell histiocytosis (LCH) is a clonal proliferation of Langerhans cells occurring as an isolated lesion or as part of a systemic proliferation. It is commoner in children younger than 10 years of age with sparing of the posterior elements in more than 95% of cases. We describe a case of LCH in an adult female presenting with paraplegia. MRI revealed a well-defined extradural contrast enhancing mass at D2-D4 vertebral level involving the posterior elements of spine. D2-5 laminectomy with excision of lesion was performed which lead to marked improvement of patients neurological status. Histopathology was suggestive of eosinophilic granuloma. We describe the case, discuss its uniqueness and review the literature on this rare tumor presentation.

  5. Mass Cytometry and Topological Data Analysis Reveal Immune Parameters Associated with Complications after Allogeneic Stem Cell Transplantation

    Directory of Open Access Journals (Sweden)

    Tadepally Lakshmikanth

    2017-08-01

    Full Text Available Human immune systems are variable, and immune responses are often unpredictable. Systems-level analyses offer increased power to sort patients on the basis of coordinated changes across immune cells and proteins. Allogeneic stem cell transplantation is a well-established form of immunotherapy whereby a donor immune system induces a graft-versus-leukemia response. This fails when the donor immune system regenerates improperly, leaving the patient susceptible to infections and leukemia relapse. We present a systems-level analysis by mass cytometry and serum profiling in 26 patients sampled 1, 2, 3, 6, and 12 months after transplantation. Using a combination of machine learning and topological data analyses, we show that global immune signatures associated with clinical outcome can be revealed, even when patients are few and heterogeneous. This high-resolution systems immune monitoring approach holds the potential for improving the development and evaluation of immunotherapies in the future.

  6. MHC-I Ligand Discovery Using Targeted Database Searches of Mass Spectrometry Data: Implications for T-Cell Immunotherapies

    DEFF Research Database (Denmark)

    Murphy, J. Patrick; Konda, Prathyusha; Kowalewski, Daniel J.

    2017-01-01

    Class I major histocompatibility complex (MHC-I)-bound peptide ligands dictate the activation and specificity of CD8+ T cells and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these MHC......-I peptides, wherein MS spectra are compared against a reference proteome. Unfortunately, matching these spectra to reference proteome databases is hindered by inflated search spaces attributed to a lack of enzyme restriction in the searches, limiting the efficiency with which MHC ligands are discovered. Here...... we offer a solution to this problem whereby we developed a targeted database search approach and accompanying tool SpectMHC, that is based on a priori-predicted MHC-I peptides. We first validated the approach using MS data from two different allotype-specific immunoprecipitates for the C57BL/6 mouse...

  7. Prediction of renal function (GFR) from cystatin C and creatinine in children: Body cell mass increases accuracy of the estimate

    DEFF Research Database (Denmark)

    Andersen, Trine Borup; Jødal, Lars; Bøgsted, Martin

    AIM: To derive an accurate prediction model for estimating glomerular filtration rate (GFR) in children based primarily on the endogenous renal function marker cystatin C (CysC) and body cell mass (BCM). THEORY: Cystatin C is produced at a constant rate in all cells of the body and is excreted......) aged 2-14 years. GFR was 14-147 mL/min/1.73m2. BCM was estimated using bioimpedance spectroscopy. Log-transformed data on BCM/CysC, serum creatinine (SCr), body-surface-area (BSA), height×BSA/SCr, CysC, weight, sex, age, height, serum urea and albumin were considered possible explanatory variables...

  8. Langerhans′ cell histiocytosis involving posterior elements of the dorsal spine: An unusual cause of extradural spinal mass in an adult

    Directory of Open Access Journals (Sweden)

    Devendra K Tyagi

    2011-01-01

    Full Text Available Langerhans cell histiocytosis (LCH is a clonal proliferation of Langerhans cells occurring as an isolated lesion or as part of a systemic proliferation. It is commoner in children younger than 10 years of age with sparing of the posterior elements in more than 95% of cases. We describe a case of LCH in an adult female presenting with paraplegia. MRI revealed a well-defined extradural contrast enhancing mass at D2-D4 vertebral level involving the posterior elements of spine. D2-5 laminectomy with excision of lesion was performed which lead to marked improvement of patients neurological status. Histopathology was suggestive of eosinophilic granuloma. We describe the case, discuss its uniqueness and review the literature on this rare tumor presentation.

  9. The viable but non-culturable state in pathogenic Escherichia coli: A general review

    Directory of Open Access Journals (Sweden)

    Jennifer A. Pienaar

    2016-02-01

    Full Text Available Background: The persistence and pathogenicity of pathogenic bacteria are dependent on the ability of the species to survive in adverse conditions. During the infectious process, the organism may need to pass through certain hostile anatomical sites, such as the stomach. Under various environmental stresses, many bacteria enter into the viable but non-culturable (VBNC state, where they are ‘alive’ or metabolically active, but will not grow on conventional media. Escherichia coli bacteria encounter several diverse stress factors during their growth, survival and infection and thus may enter into the VBNC state.Objectives: This review discusses various general aspects of the VBNC state, the mechanisms and possible public health impact of indicator and pathogenic E. coli entering into the VBNC state.Method: A literature review was conducted to ascertain the possibleimpact of E. coli entering into the VBNC state.Results: Escherichia coli enter into the VBNC state by means of several induction mechanisms. Various authors have found that E. coli can be resuscitated post-VBNC. Certain strains of pathogenic E. coli are still able to produce toxins in the VBNC state, whilst others are avirulent during the VBNC state but are able to regain virulence after resuscitation.Conclusion: Pathogenic and indicator E. coli entering into the VBNC state could have an adverse effect on public health if conventional detection methods are used, where the number of viable cells could be underestimated and the VBNC cells still produce toxins or could, at anytime, be resuscitated and become virulent again.

  10. Kissing naevus arising from neural crest cells presenting as upper and the lower lid mass

    OpenAIRE

    Gian Chand Rajput; Deepti Mahajan; Kulbhushan Prakash Chaudhary; Deewana, V.

    2015-01-01

    A kissing nevus is a type of congenital compound nevus that affects equal portions of the upper and lower eyelid, and it extends to the lid margins. Congenital divided nevi of the eyelids are a rare melanocytic lesion. Only 30 patients are reported in the literature. We report a 40-year-old female of rural background who presented with a large painless enlarging pigmented mass, involving both upper and lower left eyelid since the past 20 years. Complete excision of the lesion was done, and th...

  11. Patients With High Bone Mass Phenotype Exhibit Enhanced Osteoblast Differentiation and Inhibition of Adipogenesis of Human Mesenchymal Stem Cells

    DEFF Research Database (Denmark)

    Qiu, Weimin; Andersen, Tom; Bollerslev, Jens

    2007-01-01

    Genetic mutations in the LRP5 gene affect Wnt signaling and lead to changes in bone mass in humans. Our in vivo and in vitro results show that activated mutation T253I of LRP5 enhances osteogenesis and inhibits adipogenesis. Inactivating mutation T244M of LRP5 exerts opposite effects. Introduction......: Mutations in the Wnt co-receptor, LRP5, leading to decreased or increased canonical Wnt signaling, result in osteoporosis or a high bone mass (HBM) phenotype, respectively. However, the mechanisms whereby mutated LRP5 causes changes in bone mass are not known. Materials and Methods: We studied bone marrow composition...... in iliac crest bone biopsies from patients with the HBM phenotype and controls. We also used retrovirus-mediated gene transduction to establish three different human mesenchymal stem cell (hMSC) strains stably expressing wildtype LRP5 (hMSC-LRP5WT), LRP5T244 (hMSC-LRP5T244, inactivation mutation leading...

  12. Acupuntura un tratamiento viable para las adicciones en Colombia

    Directory of Open Access Journals (Sweden)

    Hernán López-Suescún

    2013-09-01

    Full Text Available Resumen La acupuntura es una antigua técnica terapéutica desarrollada en China, que ha evidenciado ser efectiva en síntomas como las náuseas, vómito y dolor dentario. A pesar del sustento fisiológico que posibilitaría un uso efectivo en otras patologías, incluyendo el campo de las adicciones, los estudios son contradictorios, posiblemente por la diferencias de visión entre la medicina oriental y la occidental. El consumo de psicoactivos es un problema de salud pública en Colombia y en el mundo que genera grandes costos tangibles e intangibles, los cuales, en países desarrollados, puede llegar hasta el 1,6 % del PIB. En contraste, el beneficio económico del tratamiento de las adicciones, según las Naciones Unidas Contra la Droga y el Delito (UNODC, está entre 1:3 a 1:13; por lo tanto, cualquier esfuerzo que se realice en favor de los consumidores es una ganancia. Con base en estos datos, los organismos internacionales han generado políticas que ayudan a aminorar estos efectos. Colombia, como integrante de estos organismos, ha realizado varios compromisos para llevar a cabo dichas metas. Los tratamientos con auriculoterapia, como el protocolo NADA (National Acupuncture Detoxification Association, son los métodos más usados para las adicciones en el mundo, y aunque no se ha logrado evidenciar su efectividad, por su costo, facilidad y el poco riesgo de efectos adversos se hace viable en un país con pocos recursos económicos como Colombia.

  13. Cosmic constraint on massive neutrinos in viable f(R) gravity with producing ΛCDM background expansion

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Jianbo; Wu, Yabo; Wang, Yan; Yang, Weiqiang [Liaoning Normal University, Department of Physics, Dalian (China); Liu, Molin [Xinyang Normal University, Department of Physics, Xinyang (China)

    2016-12-15

    Tensions between several cosmic observations were found recently, such as the inconsistent values of H{sub 0} (or σ{sub 8}) were indicated by the different cosmic observations. Introducing the massive neutrinos in ΛCDM could potentially solve the tensions. Viable f(R) gravity producing ΛCDM background expansion with massive neutrinos is investigated in this paper. We fit the current observational data: Planck-2015 CMB, RSD, BAO, and SNIa to constrain the mass of neutrinos in viable f(R) theory. The constraint results at 95% confidence level are: Σm{sub ν} < 0.202 eV for the active-neutrino case, m{sub ν,sterile}{sup eff} < 0.757 eV with N{sub eff} < 3.22 for the sterile neutrino case. For the effects due to the mass of the neutrinos, the constraint results on model parameter at 95% confidence level become f{sub R0} x 10{sup -6} > -1.89 and f{sub R0} x 10{sup -6} > -2.02 for two cases, respectively. It is also shown that the fitting values of several parameters much depend on the neutrino properties, such as the cold dark matter density, the cosmological quantities at matter-radiation equality, the neutrino density and the fraction of baryonic mass in helium. Finally, the constraint result shows that the tension between direct and CMB measurements of H{sub 0} gets slightly weaker in the viable f(R) model than that in the base ΛCDM model. (orig.)

  14. An open-source library for the numerical modeling of mass-transfer in solid oxide fuel cells

    Science.gov (United States)

    Novaresio, Valerio; García-Camprubí, María; Izquierdo, Salvador; Asinari, Pietro; Fueyo, Norberto

    2012-01-01

    The generation of direct current electricity using solid oxide fuel cells (SOFCs) involves several interplaying transport phenomena. Their simulation is crucial for the design and optimization of reliable and competitive equipment, and for the eventual market deployment of this technology. An open-source library for the computational modeling of mass-transport phenomena in SOFCs is presented in this article. It includes several multicomponent mass-transport models ( i.e. Fickian, Stefan-Maxwell and Dusty Gas Model), which can be applied both within porous media and in porosity-free domains, and several diffusivity models for gases. The library has been developed for its use with OpenFOAM ®, a widespread open-source code for fluid and continuum mechanics. The library can be used to model any fluid flow configuration involving multicomponent transport phenomena and it is validated in this paper against the analytical solution of one-dimensional test cases. In addition, it is applied for the simulation of a real SOFC and further validated using experimental data. Program summaryProgram title: multiSpeciesTransportModels Catalogue identifier: AEKB_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEKB_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU General Public License No. of lines in distributed program, including test data, etc.: 18 140 No. of bytes in distributed program, including test data, etc.: 64 285 Distribution format: tar.gz Programming language:: C++ Computer: Any x86 (the instructions reported in the paper consider only the 64 bit case for the sake of simplicity) Operating system: Generic Linux (the instructions reported in the paper consider only the open-source Ubuntu distribution for the sake of simplicity) Classification: 12 External routines: OpenFOAM® (version 1.6-ext) ( http://www.extend-project.de) Nature of problem: This software provides a library of models for

  15. Analytical platform for metabolome analysis of microbial cells using methyl chloroformate derivatization followed by gas chromatography-mass spectrometry.

    Science.gov (United States)

    Smart, Kathleen F; Aggio, Raphael B M; Van Houtte, Jeremy R; Villas-Bôas, Silas G

    2010-09-01

    This protocol describes an analytical platform for the analysis of intra- and extracellular metabolites of microbial cells (yeast, filamentous fungi and bacteria) using gas chromatography-mass spectrometry (GC-MS). The protocol is subdivided into sampling, sample preparation, chemical derivatization of metabolites, GC-MS analysis and data processing and analysis. This protocol uses two robust quenching methods for microbial cultures, the first of which, cold glycerol-saline quenching, causes reduced leakage of intracellular metabolites, thus allowing a more reliable separation of intra- and extracellular metabolites with simultaneous stopping of cell metabolism. The second, fast filtration, is specifically designed for quenching filamentous micro-organisms. These sampling techniques are combined with an easy sample-preparation procedure and a fast chemical derivatization reaction using methyl chloroformate. This reaction takes place at room temperature, in aqueous medium, and is less prone to matrix effect compared with other derivatizations. This protocol takes an average of 10 d to complete and enables the simultaneous analysis of hundreds of metabolites from the central carbon metabolism (amino and nonamino organic acids, phosphorylated organic acids and fatty acid intermediates) using an in-house MS library and a data analysis pipeline consisting of two free software programs (Automated Mass Deconvolution and Identification System (AMDIS) and R).

  16. Loss of the Otx2-Binding Site in the Nanog Promoter Affects the Integrity of Embryonic Stem Cell Subtypes and Specification of Inner Cell Mass-Derived Epiblast

    Directory of Open Access Journals (Sweden)

    Dario Acampora

    2016-06-01

    Full Text Available Mouse embryonic stem cells (ESCs and the inner cell mass (ICM-derived epiblast exhibit naive pluripotency. ESC-derived epiblast stem cells (EpiSCs and the postimplantation epiblast exhibit primed pluripotency. Although core pluripotency factors are well-characterized, additional regulators, including Otx2, recently have been shown to function during the transition from naive to primed pluripotency. Here we uncover a role for Otx2 in the control of the naive pluripotent state. We analyzed Otx2-binding activity in ESCs and EpiSCs and identified Nanog, Oct4, and Sox2 as direct targets. To unravel the Otx2 transcriptional network, we targeted the strongest Otx2-binding site in the Nanog promoter, finding that this site modulates the size of specific ESC-subtype compartments in cultured cells and promotes Nanog expression in vivo, predisposing ICM differentiation to epiblast. Otx2-mediated Nanog regulation thus contributes to the integrity of the ESC state and cell lineage specification in preimplantation development.

  17. Tenosynovial giant cell tumor presenting as a parotid gland mass: Expanding the differential diagnosis of giant cell-rich lesions in salivary glands

    Directory of Open Access Journals (Sweden)

    Ling Guo

    2014-01-01

    Full Text Available Tenosynovial giant cell tumors (TGCT are rare benign soft tissue tumors affecting mostly young adults. The most common affected sites include the knee, ankle, elbow, shoulder, and fingers. The temporomandibular joint is occasionally affected. Herein, we report a case of a 31-year-old Caucasian male who presented clinically with a parotid gland mass. The initial clinical and radiological work-up failed to reveal any involvement of the adjacent temporomandibular joint. Fine-needle aspiration revealed a cellular tumor composed of mononuclear and multinucleated giant cells with fibrosis and hemosiderin deposition. This was subsequently found to be a TGCT arising from the temporomandibular joint. Giant cell-rich lesions are uncommon in salivary glands. Herein, we describe the cytomorphology and clinico-radiographic features of this tumor with emphasis on the differential diagnosis of giant cell-rich lesions presenting in salivary glands. Despite its rare occurrence, this entity should be considered when giant cells are prominent in specimens acquired from this location.

  18. The Measurement of High-Density Lipoprotein Mediated Cholesterol Efflux from Macrophage Cells by Liquid Chromatography Tandem Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Mo Wang

    2014-11-01

    Full Text Available Background: Studies have shown a negative association between macrophage cholesterol efflux and atherosclerotic cardiovascular diseases (CVD. However, the current methods for measuring cholesterol efflux require a radioactive tracer and involve a variety of cell treatments, making the measurement of macrophage cholesterol efflux impractical for use in clinical laboratories. In this study, we developed a non-radioactive and precise LC/MS/MS method for the measurement of high-density lipoprotein (HDL mediated cholesterol efflux from J774 macrophages. Methods: J774 cells were seeded on 12-well plates at a density of 1.5×105 cells/ml in H-DMEM medium, and when the cells were approximately 80% confluent, they were incubated with H-DMEM medium containing 2% FBS, 0.5 μg/ml ACAT inhibitor Sandoz 58-035, and 20 μg/ml [3,4-13C]cholesterol for 6 h. After washing and equilibrating the cells, HDL samples were added at a final concentration of 7% and incubated for 8 h. The cells were lysed, and [3,4-13C]cholesterol and cholesterol were measured by LC/MS/MS. Cholesterol efflux was expressed as the percent decrease of cell [3,4-13C]cholesterol mass during the incubation. Results: When incubated with [3,4-13C]cholesterol enriched J774 cells, HDL mediated higher cell cholesterol efflux than influx compared to serum and isolated LDL; therefore, HDL was used as the extracellular acceptor. The results from healthy volunteers showed that the rate of cholesterol efflux was negatively correlated with weight, BMI, blood pressure, and FERHDL and positively correlated with HDL-C, HDL2-C, and apoAI levels. Conclusions: A LC/MS/MS method for the measurement of HDL mediated cholesterol efflux from macrophage cells has been established. This method is non-radioactive, precise and reliable and is potentially useful for the assessment of HDL function and cardiovascular disease risks.

  19. PRODUCTION OF EMBBRYONIC STEM CELLS FROM INNER CELL MASS OF BLASTOCYST ISOLATED BY ENZYMATIC AND IMMUNOSURGERY METHODS

    Directory of Open Access Journals (Sweden)

    Thomas Mata Hine

    2008-03-01

    Full Text Available The objective of the research is determining the ICM isolation method to produce ESC. Blastocyst stage of DDy mice embryos were used in this study. Zona pellucida of blastocysts were removed by 0.25% pronase, the ICM isolation were done by enzimatic or immunosurgery method, and then they were cultured in DMEM-high glucose supplemented with mercaptoethanol, gentamycin, fetal bovine serum, and cumulus cells as feeder layer. The result of the research indicated that immunosurgery method yielding attachment rate and number ESC colony 93.85% and 43.08%, respectively, higher (P<0.05 than enzimatic method that weree 79.63% and 18.52%, respectively, but the viability of ICM cells were equal (P >0.05 that are 93.59% in enzymatic method and 98.56% in immunosurgery method. This research concluded that immunosurgery more effective method for isolation of ICM and ESC production than enzymatic method.

  20. A propidium monoazide–quantitative PCR method for the detection and quantification of viable Enterococcus faecalis in large-volume samples of marine waters

    KAUST Repository

    Salam, Khaled W.

    2014-08-23

    The development of rapid detection assays of cell viability is essential for monitoring the microbiological quality of water systems. Coupling propidium monoazide with quantitative PCR (PMA-qPCR) has been successfully applied in different studies for the detection and quantification of viable cells in small-volume samples (0.25-1.00 mL), but it has not been evaluated sufficiently in marine environments or in large-volume samples. In this study, we successfully integrated blue light-emitting diodes for photoactivating PMA and membrane filtration into the PMA-qPCR assay for the rapid detection and quantification of viable Enterococcus faecalis cells in 10-mL samples of marine waters. The assay was optimized in phosphate-buffered saline and seawater, reducing the qPCR signal of heat-killed E. faecalis cells by 4 log10 and 3 log10 units, respectively. Results suggest that high total dissolved solid concentration (32 g/L) in seawater can reduce PMA activity. Optimal PMA-qPCR standard curves with a 6-log dynamic range and detection limit of 102 cells/mL were generated for quantifying viable E. faecalis cells in marine waters. The developed assay was compared with the standard membrane filter (MF) method by quantifying viable E. faecalis cells in seawater samples exposed to solar radiation. The results of the developed PMA-qPCR assay did not match that of the standard MF method. This difference in the results reflects the different physiological states of E. faecalis cells in seawater. In conclusion, the developed assay is a rapid (∼5 h) method for the quantification of viable E. faecalis cells in marine recreational waters, which should be further improved and tested in different seawater settings. © 2014 Springer-Verlag Berlin Heidelberg.

  1. Cajal body number and nucleolar size correlate with the cell body mass in human sensory ganglia neurons.

    Science.gov (United States)

    Berciano, Maria T; Novell, Mariona; Villagra, Nuria T; Casafont, Iñigo; Bengoechea, Rocio; Val-Bernal, J Fernado; Lafarga, Miguel

    2007-06-01

    This paper studies the cell size-dependent organization of the nucleolus and Cajal bodies (CBs) in dissociated human dorsal root ganglia (DRG) neurons from autopsy tissue samples of patients without neurological disease. The quantitative analysis of nucleoli with an anti-fibrillarin antibody showed that all neurons have only one nucleolus. However, the nucleolar volume and the number of fibrillar centers per nucleolus significantly increase as a function of cell body size. Immunostaining for coilin demonstrated the presence of numerous CBs in DRG neurons (up to 20 in large size neurons). The number of CBs per neuron correlated positively with the cell body volume. Light and electron microscopy immunocytochemical analysis revealed the concentration of coilin, snRNPs, SMN and fibrillarin in CBs of DRG neurons. CBs were frequently associated with the nucleolus, active chromatin domains and PML bodies, but not with telomeres. Our results support the view that the nucleolar volume and number of both fibrillar centers and CBs depend on the cell body mass, a parameter closely related to transcriptional and synaptic activity in mammalian neurons. Moreover, the unusual large number of CBs could facilitate the transfer of RNA processing components from CBs to nucleolar and nucleoplasmic sites of RNA processing.

  2. Key factors regulating the mass delivery of macromolecules to model cell membranes

    DEFF Research Database (Denmark)

    Campbell, Richard A.; Watkins, Erik B.; Jagalski, Vivien

    2014-01-01

    diffusion and continuous delivery. Neutron reflectometry measurements were carried out on supported lipid bilayers of varying charge and on hydrophilic silica surfaces. Translocation of the macromolecule across the membrane and adsorption of the lamellar aggregates occur only when the membrane (1...... of the aggregates to activate endocytosis pathways on specific cell types is discussed in the context of targeted drug delivery applications....

  3. A multi-fluid model to simulate heat and mass transfer in a PEM fuel cell

    DEFF Research Database (Denmark)

    Berning, Torsten; Odgaard, Madeleine; Kær, Søren Knudsen

    2011-01-01

    and leaves at the anode side. Only a fundamental understanding of the various mechanisms involved can lead to proper water management and hence can prevent "flooding" of either anode or cathode side while keeping the membrane humidified, and consequently can optimize the fuel cell performance and durability...

  4. Interferometric measurements of dry mass content in nuclei and cytoplasm in the life cycle of antheridial filaments cells of Chara vulgaris L. in their successive developmental stages

    Directory of Open Access Journals (Sweden)

    Hanna Kuran

    2015-01-01

    Full Text Available Interferometric measurements of the nucleus and cytoplasm dry mass during interphase in the successive stages of development of antheridial filaments of Chara vulgaris demonstrated that the dry mass and surface area of cell nuclei double in size in each of the successive generations of the filaments, whereas neither the surface nor the dry mass of the cytoplasm increase in such proportion in the same period. In the successive stages of development of the antheridial filaments the dry mass and surface area of the nuclei and cytoplasm gradually diminish.

  5. Detection of Only Viable Bacterial Spores Using a Live/Dead Indicator in Mixed Populations

    Science.gov (United States)

    Behar, Alberto E.; Stam, Christina N.; Smiley, Ronald

    2013-01-01

    This method uses a photoaffinity label that recognizes DNA and can be used to distinguish populations of bacterial cells from bacterial spores without the use of heat shocking during conventional culture, and live from dead bacterial spores using molecular-based methods. Biological validation of commercial sterility using traditional and alternative technologies remains challenging. Recovery of viable spores is cumbersome, as the process requires substantial incubation time, and the extended time to results limits the ability to quickly evaluate the efficacy of existing technologies. Nucleic acid amplification approaches such as PCR (polymerase chain reaction) have shown promise for improving time to detection for a wide range of applications. Recent real-time PCR methods are particularly promising, as these methods can be made at least semi-quantitative by correspondence to a standard curve. Nonetheless, PCR-based methods are rarely used for process validation, largely because the DNA from dead bacterial cells is highly stable and hence, DNA-based amplification methods fail to discriminate between live and inactivated microorganisms. Currently, no published method has been shown to effectively distinguish between live and dead bacterial spores. This technology uses a DNA binding photoaffinity label that can be used to distinguish between live and dead bacterial spores with detection limits ranging from 109 to 102 spores/mL. An environmental sample suspected of containing a mixture of live and dead vegetative cells and bacterial endospores is treated with a photoaffinity label. This step will eliminate any vegetative cells (live or dead) and dead endospores present in the sample. To further determine the bacterial spore viability, DNA is extracted from the spores and total population is quantified by real-time PCR. The current NASA standard assay takes 72 hours for results. Part of this procedure requires a heat shock step at 80 degC for 15 minutes before the

  6. Islet-selectivity of G-protein coupled receptor ligands evaluated for PET imaging of pancreatic {beta}-cell mass

    Energy Technology Data Exchange (ETDEWEB)

    Cline, Gary W., E-mail: gary.cline@yale.edu [Yale University School of Medicine (United States); Zhao, Xiaojian [Yale University School of Medicine (United States); Jakowski, Amy B.; Soeller, Walter C.; Treadway, Judith L. [Pfizer Global Research and Development, Pfizer Inc., Groton CT (United States)

    2011-09-02

    Highlights: {yields} We screened G-protein coupled receptors for imaging pancreatic. {yields} Database mining and immunohistochemistry identified GPCRs enriched in {beta}-cells. {yields} In vitro and in vivo assays were used to determine exocrine vs endocrine specificity. {yields} GPCR candidates for imaging of {beta}-cell mass are Prokineticin-1R, mGluR5, and GLP-1R. -- Abstract: A critical unmet need exists for methods to quantitatively measure endogenous pancreatic {beta}-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity to islet {beta}-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5), neuropeptide Y-2 receptor (NPY-2R), and glucagon-like peptide 1 receptor (GLP-1R). In vitro specificity ratios gave the following receptor rank order: PK-1R > GLP-1R > NPY-2R > mGluR5. The biodistribution rank order of selectivity to the pancreas was found to be PK-1R > VMAT2 {approx} GLP-1R > mGluR5. Favorable islet selectivity and biodistribution

  7. Fetal adrenal demedullation lowers circulating norepinephrine and attenuates growth restriction but not reduction of endocrine cell mass in an ovine model of intrauterine growth restriction.

    Science.gov (United States)

    Davis, Melissa A; Macko, Antoni R; Steyn, Leah V; Anderson, Miranda J; Limesand, Sean W

    2015-01-09

    Placental insufficiency is associated with fetal hypoglycemia, hypoxemia, and elevated plasma norepinephrine (NE) that become increasingly pronounced throughout the third trimester and contribute to intrauterine growth restriction (IUGR). This study evaluated the effect of fetal adrenal demedullation (AD) on growth and pancreatic endocrine cell mass. Placental insufficiency-induced IUGR was created by exposing pregnant ewes to elevated ambient temperatures during mid-gestation. Treatment groups consisted of control and IUGR fetuses with either surgical sham or AD at 98 days gestational age (dGA; term = 147 dGA), a time-point that precedes IUGR. Samples were collected at 134 dGA. IUGR-sham fetuses were hypoxemic, hypoglycemic, and hypoinsulinemic, and values were similar in IUGR-AD fetuses. Plasma NE concentrations were ~5-fold greater in IUGR-sham compared to control-sham, control-AD, and IUGR-AD fetuses. IUGR-sham and IUGR-AD fetuses weighed less than controls. Compared to IUGR-sham fetuses, IUGR-AD fetuses weighed more and asymmetrical organ growth was absent. Pancreatic β-cell mass and α-cell mass were lower in both IUGR-sham and IUGR-AD fetuses compared to controls, however, pancreatic endocrine cell mass relative to fetal mass was lower in IUGR-AD fetuses. These findings indicate that NE, independently of hypoxemia, hypoglycemia and hypoinsulinemia, influence growth and asymmetry of growth but not pancreatic endocrine cell mass in IUGR fetuses.

  8. Plasma Cell Leukemia Presenting as a Chest Wall Mass: A Case Report

    Directory of Open Access Journals (Sweden)

    Ahmed Ali

    2016-06-01

    Full Text Available Plasma cell leukemia (PCL is an uncommon neoplasm of plasma cells, with an aggressive clinical course and poor outcome, even with current standard of care. It can occur either de novo (primary PCL or as a progression of multiple myeloma (MM. This disease has unique diagnostic criteria but certain genetic markers and clinical features may overlap with MM. Due to the low prevalence of PCL, guidelines on its management are extrapolated from the management of MM and based on small retrospective studies and cases reports/series. We present an interesting case of PCL in a middle-aged African-American male, who was diagnosed incidentally after chest wall imaging for an unrelated complaint. The diagnostic approach, management and outcomes of PCL are discussed.

  9. OCT4 expression in outgrowth colonies derived from porcine inner cell masses and epiblasts

    DEFF Research Database (Denmark)

    Rasmussen, M A; Wolf, X A; Schauser, K

    2011-01-01

    on the relationship between OCT4 expression and embryonic stem cell (ESC)-like morphology. A total of 104 zona pellucida-enclosed and 101 hatched blastocysts were subjected to four different methods of ICM and epiblast isolation, respectively: Manual isolation, immunosurgery, immunosurgery with manual cleaning......, or whole blastocyst culture. OCs were established on mouse embryonic fibroblast (MEF) cells and categorized according to morphology and OCT4 staining. Although all isolation methods resulted in ESC-like OCs, immunosurgery with manual cleaning yielded significantly higher rates of ICM/epiblast attachment...... and subsequent ESC-like morphology, whereas no significant difference was found between ICM and epiblasts with respect to these characteristics. All ESC-like OCs showed nuclear OCT4 staining and expression of OCT4, NANOG and SOX2 as evaluated by RT-PCR. Upon initial passages, the expression of pluripotency...

  10. Malignant TFE3-rearranged perivascular epithelioid cell neoplasm (PEComa) presenting as a subcutaneous mass.

    Science.gov (United States)

    Shon, W; Kim, J; Sukov, W; Reith, J

    2016-03-01

    Perivascular epithelioid cell neoplasms (PEComas) are a group of mesenchymal tumours with concurrent melanocytic and myogenic differentiation. Although many cases are sporadic, PEComas can be associated with tuberous sclerosis. A distinct subset of deep-seated PEComas has been shown to carry TFE3 fusions. To our knowledge, this is the first reported case of primary subcutaneous malignant PEComa with molecular confirmation of TFE3 gene rearrangement. © 2015 British Association of Dermatologists.

  11. Acyl chains of phospholipase D transphosphatidylation products in Arabidopsis cells: a study using multiple reaction monitoring mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Dominique Rainteau

    Full Text Available BACKGROUND: Phospholipases D (PLD are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA. PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor. METHODOLOGY/PRINCIPAL FINDINGS: Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA. As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut, which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18:2- and 16:0/18:3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates. CONCLUSIONS: MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18:2- and 16:0/18:3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.

  12. Acyl Chains of Phospholipase D Transphosphatidylation Products in Arabidopsis Cells: A Study Using Multiple Reaction Monitoring Mass Spectrometry

    Science.gov (United States)

    Rainteau, Dominique; Humbert, Lydie; Delage, Elise; Vergnolle, Chantal; Cantrel, Catherine; Maubert, Marie-Anne; Lanfranchi, Sandrine; Maldiney, Régis; Collin, Sylvie; Wolf, Claude; Zachowski, Alain; Ruelland, Eric

    2012-01-01

    Background Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor. Methodology/Principal findings Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates. Conclusions MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains. PMID:22848682

  13. Coscheduling in Clusters: Is It a Viable Alternative?

    Energy Technology Data Exchange (ETDEWEB)

    Choi, G S; Kim, J H; Ersoz, D; Yoo, A B; Das, C R

    2003-11-10

    than spin-based techniques like PB on a Linux platform. Third, the proposed HYBRID scheduling provides the best performance-energy behavior and can be implemented on any cluster with little effort. All these results suggest that blocking-based coscheduling techniques are viable candidates to be used instead of batching scheme for significant performance-energy benefits.

  14. Expression of adhesion molecules, chemokines and matrix metallo- proteinases (MMPs) in viable and degenerating stage of Taenia solium metacestode in swine neurocysticercosis.

    Science.gov (United States)

    Singh, Satyendra K; Singh, Aloukick K; Prasad, Kashi N; Singh, Amrita; Singh, Avinash; Rai, Ravi P; Tripathi, Mukesh; Gupta, Rakesh K; Husain, Nuzhat

    2015-11-30

    Neurocysticercosis (NCC) is a parasitic infection of central nervous system (CNS). Expression of adhesion molecules, chemokines and matrix metalloproteinases (MMPs) were investigated on brain tissues surrounding viable (n=15) and degenerating cysticerci (n=15) of Taenia solium in swine by real-time RT-PCR and ELISA. Gelatin gel zymography was performed for MMPs activity. ICAM-1 (intercellular adhesion molecule-1), E-selectin, MIP-1α (macrophage inflammatory protein-1α), Eotaxin-1 and RANTES (regulated on activation, normal T cell expressed and secreted) were associated with degenerating cysticerci (cysts). However, VCAM-1 (vascular cell adhesion molecule-1), MCP-1 (monocyte chemotactic protein-1), MMP-2 and MMP-9 were associated with both viable and degenerating cysts. In conclusion, viable and degenerating cysticerci have different immune molecule profiles and role of these molecules in disease pathogenesis needs to be investigated. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. The application of inductively coupled plasma dynamic reaction cell mass spectrometry for measurement of selenium isotopes, isotope ratios and chromatographic detection of selenoamino acids

    DEFF Research Database (Denmark)

    Sloth, Jens Jørgen; Larsen, Erik Huusfeldt

    2000-01-01

    ratios was close to the theoretical values for selenium concentrations at 1 and 10 ng ml(-1). The accuracy of the isotope ratios, however, was improved by correcting the count rate of all selenium isotopes equivalent to the formation of SeH at 9.6 +/- 0.5% one mass unit above the selenium isotopes....... A linear relationship (r mass from the Se-80 reference isotope. This indicated that the error was caused by mass bias. The slope of the curve at -3.0% error per mass unit can be used for correction of the measured......Inductively coupled plasma dynamic reaction cell mass spectrometry (ICP-DRC-MS) was characterised for the detection of the six naturally occurring selenium isotopes. The potentially interfering argon dimers at the selenium masses m/z 74, 76, 78 and 80 were reduced in intensity by approximately five...

  16. Detection and quantification of viable Bacillus cereus group species in milk by propidium monoazide quantitative real-time PCR.

    Science.gov (United States)

    Cattani, Fernanda; Barth, Valdir C; Nasário, Jéssica S R; Ferreira, Carlos A S; Oliveira, Sílvia D

    2016-04-01

    The Bacillus cereus group includes important spore-forming bacteria that present spoilage capability and may cause foodborne diseases. These microorganisms are traditionally evaluated in food using culturing methods, which can be laborious and time-consuming, and may also fail to detect bacteria in a viable but nonculturable state. The purpose of this study was to develop a quantitative real-time PCR (qPCR) combined with a propidium monoazide (PMA) treatment to analyze the contamination of UHT milk by B. cereus group species viable cells. Thirty micrograms per milliliter of PMA was shown to be the most effective concentration for reducing the PCR amplification of extracellular DNA and DNA from dead cells. The quantification limit of the PMA-qPCR assay was 7.5 × 10(2) cfu/mL of milk. One hundred thirty-five UHT milk samples were analyzed to evaluate the association of PMA to qPCR to selectively detect viable cells. The PMA-qPCR was able to detect B. cereus group species in 44 samples (32.6%), whereas qPCR without PMA detected 78 positive samples (57.8%). Therefore, the PMA probably inhibited the amplification of DNA from cells that were killed during UHT processing, which avoided an overestimation of bacterial cells when using qPCR and, thus, did not overvalue potential health risks. A culture-based method was also used to detect and quantify B. cereus sensu stricto in the same samples and showed positive results in 15 (11.1%) samples. The culture method and PMA-qPCR allowed the detection of B. cereus sensu stricto in quantities compatible with the infective dose required to cause foodborne disease in 3 samples, indicating that, depending on the storage conditions, even after UHT treatment, infective doses may be reached in ready-to-consume products. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  17. Optimizing Viable Leukocyte Sampling from the Female Genital Tract for Clinical Trials: An International Multi-Site Study

    Science.gov (United States)

    De Rosa, Stephen C.; Martinson, Jeffrey A.; Plants, Jill; Brady, Kirsten E.; Gumbi, Pamela P.; Adams, Devin J.; Vojtech, Lucia; Galloway, Christine G.; Fialkow, Michael; Lentz, Gretchen; Gao, Dayong; Shu, Zhiquan; Nyanga, Billy; Izulla, Preston; Kimani, Joshua; Kimwaki, Steve; Bere, Alfred; Moodie, Zoe; Landay, Alan L.; Passmore, Jo-Ann S.; Kaul, Rupert; Novak, Richard M.; McElrath, M. Juliana; Hladik, Florian

    2014-01-01

    Background Functional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear. Methods and Findings We enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (pbiopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4+ T cells in the female genital tract express the α4β7 integrin, an HIV envelope-binding mucosal homing receptor. Conclusions CVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention. PMID:24454917

  18. Eradication of high viable loads of Listeria monocytogenes contaminating food-contact surfaces

    Directory of Open Access Journals (Sweden)

    Silvia ede Candia

    2015-07-01

    Full Text Available This study demonstrates the efficacy of cold gaseous ozone treatments at low concentrations in the eradication of high Listeria monocytogenes viable cell loads from glass, polypropylene, stainless steel and expanded polystyrene food-contact surfaces. Using a step by step approach, involving the selection of the most resistant strain-surface combinations, 11 Listeria spp. strains resulted inactivated by a continuous ozone flow at 1.07 mg m-3 after 24 or 48 h of cold incubation, depending on both strain and surface evaluated. Increasing the inoculum level to 9 log CFU coupon-1, the best inactivation rate was obtained after 48h of treatment at 3.21 mg m-3 ozone concentration when cells were deposited onto stainless steel and expanded polystyrene coupons, resulted the most resistant food-contact surfaces in the previous assays.The addition of naturally microbiologically contaminated meat extract to a high load of L. monocytogenes LMG 23775 cells, the most resistant strain out of the 11 assayed Listeria spp. strains, led to its complete inactivation after four days of treatment.To the best of our knowledge, this is the first report describing the survival of L. monocytogenes and the effect of ozone treatment under cold storage conditions on expanded polystyrene, a commonly-used material in food packaging. These results could be useful for reducing pathogen cross-contamination phenomena during cold food storage.

  19. Analysis of the Listeria cell wall proteome by two-dimensional nanoliquid chromatography coupled to mass spectrometry.

    Science.gov (United States)

    Calvo, Enrique; Pucciarelli, M Graciela; Bierne, Hélène; Cossart, Pascale; Albar, Juan Pablo; García-Del Portillo, Francisco

    2005-02-01

    Genome analyses have revealed that the Gram-positive bacterial species Listeria monocytogenes and L. innocua contain a large number of genes encoding surface proteins predicted to be covalently bound to the cell wall (41 and 34, respectively). The function of most of these proteins is unknown and they have not even been identified biochemically. Here, we report the first characterization of the Listeria cell wall proteome using a nonelectrophoretic approach. The material analyzed consisted of a peptide mixture obtained from a cell wall extract insoluble in boiling 4% SDS. This extract, containing peptidoglycan (intrinsically resistant to proteases) and strongly associated proteins, was digested with trypsin in a solution with 0.01% SDS, used to favor protein digestion throughout the peptidoglycan. The resulting complex peptide mixture was fractionated and analyzed by two-dimensional nanoliquid chromatography coupled to ion-trap mass spectrometry. A total of 30 protein species were unequivocally identified in cell wall extracts of the genome strains L. monocytogenes EGD-e (19 proteins) and L. innocua CLIP11262 (11 proteins). Among them, 20 proteins bearing an LPXTG motif recognized for covalent anchoring to the peptidoglycan were identified. Other proteins detected included peptidoglycan-lytic enzymes, a penicillin-binding protein, and proteins bearing an NXZTN motif recently proposed to direct protein anchoring to the peptidoglycan. The marked sensitivity of the method makes it highly attractive in the post-genome era for defining the cell wall proteome in any bacterial species. This information will be useful to study novel protein-peptidoglycan associations and to rapidly identify new targets in the surface of important bacterial pathogens.

  20. Simultaneous determination of the flavonoids robinin and kaempferol in human breast cancer cells by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Tsiklauri, Lia; An, Guohua; Ruszaj, Donna M; Alaniya, Mary; Kemertelidze, Ether; Morris, Marilyn E

    2011-04-28

    An accurate, precise and sensitive method was developed and validated for the simultaneous quantification of the flavonoid glycoside robinin, and its algycone kaempferol in human breast cancer MCF-7 cells. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C(18) column using a mobile phase consisting of (A) water with 0.025% formic acid and 1mM ammonium formate and (B) acetonitrile with 0.025% formic acid. Rutin was used as the internal standard for robinin and fisetin as the internal standard for kaempferol. The assay had a limit of detection of 0.1ng/ml for both compounds when present in cell lysate. The calibration curves were linear from 1 to 250ng/ml (r>0.999) for each compound. The intra- and inter-day coefficients of variation were less than 10% and intra- and inter-day accuracies were within 11%. This assay was successfully applied in a robinin cellular uptake study to determine the intracellular concentrations of robinin in MCF-7 cells. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. High energy efficiency and high power density proton exchange membrane fuel cells: Electrode kinetics and mass transport

    Science.gov (United States)

    Srinivasan, Supramaniam; Velev, Omourtag A.; Parthasathy, Arvind; Manko, David J.; Appleby, A. John

    1991-01-01

    The development of proton exchange membrane (PEM) fuel cell power plants with high energy efficiencies and high power densities is gaining momentum because of the vital need of such high levels of performance for extraterrestrial (space, underwater) and terrestrial (power source for electric vehicles) applications. Since 1987, considerable progress has been made in achieving energy efficiencies of about 60 percent at a current density of 200 mA/sq cm and high power densities (greater than 1 W/sq cm) in PEM fuel cells with high (4 mg/sq cm) or low (0.4 mg/sq cm) platinum loadings in electrodes. The following areas are discussed: (1) methods to obtain these high levels of performance with low Pt loading electrodes - by proton conductor impregnation into electrodes, localization of Pt near front surface; (2) a novel microelectrode technique which yields electrode kinetic parameters for oxygen reduction and mass transport parameters; (3) demonstration of lack of water transport from anode to cathode; (4) modeling analysis of PEM fuel cell for comparison with experimental results and predicting further improvements in performance; and (5) recommendations of needed research and development for achieving the above goals.

  2. Colpoda secrete viable Listeria monocytogenes within faecal pellets.

    Science.gov (United States)

    Raghu Nadhanan, Rethish; Thomas, Connor J

    2014-02-01

    Transmission electron microscopy was used to demonstrate that co-cultures of the ciliate Colpoda RR (an environmental isolate) and Colpoda MLS-5 (a food processing environment isolate) with the pathogenic Listeria monocytogenes DRDC8 resulted in secretion of faecal pellets containing intact DRDC8 cells. A green fluorescent protein expressing variant of DRDC8 was used in co-cultures to confirm that the pellet-associated bacterial cells were L. monocytogenes. Viability was confirmed by plate counts, and assay of microbial respiratory activity-proved DRDC8 cells present within faecal pellets was metabolically active. Following treatment of faecal pellets secreted by Colpoda RR and MLS-5 with gentamycin and sodium hypochlorite (NaOCl), no loss of viability of the pellet-located DRDC8 cells was observed, indicating that faecal pellet encapsulated DRDC8 cells are resistant to biocidal agents. This work suggests that Colpoda-derived faecal pellets may provide a mechanism for transmission of L. monocytogenes and other pathogenic bacteria. Furthermore, bacteria encapsulated by faecal pellets may be resistant to disinfectants and cleaning agents used in food manufacturing and preparation facilities. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  3. Effect of myocardial revascularisation on left ventricular systolic function in patients with and without viable myocardium: should non-viable segments be revascularised?

    Science.gov (United States)

    Stipac, Alja Vlahovic; Stankovic, Ivan; Vidakovic, Radosav; Putnikovic, Biljana; Ilic, Ivan; Milicic, Biljana; Neskovic, Aleksandar N

    2013-12-01

    To assess the effect of surgical revascularisation on left ventricular (LV) systolic function in patients with viable and non-viable dysfunctional LV segments determined by low dose dobutamine stress echocardiography (DSE). Prospective observational cohort study. Single tertiary care centre. Consecutive patients referred to surgical revascularisation (n=115). DSE and surgical revascularisation. Functional recovery defined as increase in ejection fraction ≥ 5% 1 year after revascularisation in patients with and without viable myocardium (viability defined as improvement of contractility in ≥ 4 LV segments on DSE). The mean age, ejection fraction and wall motion score index (WMSi) of patients were 59 ± 9 years, 44 ± 9% and 1.82 ± 0.31, respectively. There was no difference between DSE positive and DSE negative patients for any of those parameters at baseline study (p>0.05 for all). After 12 months, the ejection fraction increased 11 ± 1% in patients with viable myocardium vs 7 ± 1% in patients without viable myocardium (p=0.002). Moreover, in patients with viable myocardium, the greatest increase of ejection fraction occurred 1 month after surgery (9 ± 1%), whereas in those patients with negative DSE the ejection fraction increased more gradually (2±1% after 1 month, p=0.002 between groups for 1 month vs preoperative value), but still improved after 12 months follow-up (pmyocardial revascularisation. Functional recovery continuously occurs throughout the first year after surgical treatment.

  4. Mass and Heat Transfer in Ion-Exchange Membranes Applicable to Solid Polymer Fuel Cells

    Energy Technology Data Exchange (ETDEWEB)

    Otteroey, M.

    1996-04-01

    In this doctoral thesis, an improved emf method for determination of transference numbers of two counter ions in ion-exchange membranes is presented. Transference numbers were obtained as a continuous function of the composition. The method avoids problems with diffusion by using a stack of membranes. Water transference coefficients in ion-exchange membranes is discussed and reversible and irreversible water transfer is studied by emf methods. Efforts were made to get data relevant to the solid polymer fuel cell. The results support the findings of other researchers that the reversible water transfer is lower than earlier predicted. A chapter on the conductivity of ion-exchange membranes establishes a method to separate the very thin liquid layers surrounding the membranes in a stack. Using the method it was found that the conductivity is obtained with high accuracy and that the liquid layer in a membrane stack can contribute significantly to the total measured resistance. A four point impedance method was tested to measure the conductivity of membranes under fuel cell conditions. Finally, there is a discussion of reversible heat effects and heat transfer in ion-exchange membranes. 155 refs., 45 figs., 13 tabs.

  5. Study of cell antigens and intracellular DNA by identification of element-containing labels and metallointercalators using inductively coupled plasma mass spectrometry.

    Science.gov (United States)

    Ornatsky, O I; Lou, X; Nitz, M; Schäfer, S; Sheldrick, W S; Baranov, V I; Bandura, D R; Tanner, S D

    2008-04-01

    The enumeration of absolute cell numbers and cell proliferation in clinical samples is important for diagnostic and research purposes. Detection of cellular DNA with fluorescent dyes is the most commonly used approach for cell enumeration in cytometry. Inductively coupled plasma mass spectrometry (ICPMS) has been recently introduced to the field of protein and cell surface antigen identification via ICPMS-linked immunoassays using element-labeled affinity reagents such as gold and lanthanide-conjugated antibodies. In the present work, we describe novel methods for using metallointercalators that irreversibly bind DNA and low concentrations of rare earth metals added to cell growth media for rapid and sensitive measurement of cell numbers by mass spectrometry. We show that Ir- and Rh-containing metallointercalators are useful reagents for labeling cells and normalizing signals when studying antigen expression on different types and numbers of cells. Results are presented for solution analysis performed by conventional ICPMS and compared to measurements obtained on the novel flow cytometer mass spectrometer (FC-MS) instrument, designed to analyze multiple antigens and DNA simultaneously in single cells.

  6. A technique for determining viable military logistics support alternatives

    Science.gov (United States)

    Hester, Jesse Stuart

    A look at today's US military will see them operating much beyond the scope of protecting and defending the United States. These operations now consist of, but are not limited to humanitarian aid, disaster relief, peace keeping, and conflict resolution. This broad spectrum of operational environments has necessitated a transformation of the individual military services to a hybrid force that is attempting to leverage the inherent and emerging capabilities and strengths of all those under the umbrella of the Department of Defense (DOD), this concept has been coined Joint Operations. Supporting Joint Operations requires a new approach to determining a viable military logistics support system. The logistics architecture for these operations has to accommodate scale, time, varied mission objectives, and imperfect information. Compounding the problem is the human in the loop (HITL) decision maker (DM) who is a necessary component for quickly assessing and planning logistics support activities. Past outcomes are not necessarily good indicators of future results, but they can provide a reasonable starting point for planning and prediction of specific needs for future requirements. Adequately forecasting the necessary logistical support structure and commodities needed for any resource intensive environment has progressed well beyond stable demand assumptions to one in which dynamic and nonlinear environments can be captured with some degree of fidelity and accuracy. While these advances are important, a holistic approach that allows exploration of the operational environment or design space does not exist to guide the military logistician in a methodical way to support military forecasting activities. To bridge this capability gap, a method called Adaptive Technique for Logistics Architecture Solutions (ATLAS) has been developed. This method provides a process that facilitates the use of techniques and tools that filter and provide relevant information to the DM. By doing

  7. Numerical evaluation of oxide growth in metallic support microstructures of Solid Oxide Fuel Cells and its influence on mass transport

    DEFF Research Database (Denmark)

    Reiss, Georg; Frandsen, Henrik Lund; Persson, Åsa Helen

    2015-01-01

    is evaluated by determining an effective diffusion coefficient and the equivalent electrical area specific resistance (ASR) due to diffusion over time. It is thus possible to assess the applicability (in terms of corrosion behaviour) of potential metallic supports without costly long-term experiments......Metal-supported Solid Oxide Fuel Cells (SOFCs) are developed as a durable and cost-effective alternative to the state-of-the-art cermet SOFCs. This novel technology offers new opportunities but also new challenges. One of them is corrosion of the metallic support, which will decrease the long......-term performance of the SOFCs. In order to understand the implications of the corrosion on the mass-transport through the metallic support, a corrosion model is developed that is capable of determining the change of the porous microstructure due to oxide scale growth. The model is based on high...

  8. Mass and Momentum Transport in Microcavities for Diffusion-Dominant Cell Culture Applications

    Science.gov (United States)

    Yew, Alvin G.; Pinero, Daniel; Hsieh, Adam H.; Atencia, Javier

    2012-01-01

    For the informed design of microfluidic devices, it is important to understand transport phenomena at the microscale. This letter outlines an analytically-driven approach to the design of rectangular microcavities extending perpendicular to a perfusion microchannel for microfluidic cell culture devices. We present equations to estimate the spatial transition from advection- to diffusion-dominant transport inside cavities as a function of the geometry and flow conditions. We also estimate the time required for molecules, such as nutrients or drugs to travel from the microchannel to a given depth into the cavity. These analytical predictions can facilitate the rational design of microfluidic devices to optimize and maintain long-term, physiologically-based culture conditions with low fluid shear stress.

  9. Lipogenic metabolism: a viable target for prostate cancer treatment?

    Directory of Open Access Journals (Sweden)

    Mengmeng Liang

    2014-10-01

    Full Text Available Cancer cells often depend on altered metabolism compared with their normal counterparts. [1],[2],[3],[4] As observed in 1924 by Otto Warburg, cancer cells show preferential glucose consumption by way of aerobic glycolysis while normal cells generally assume mitochondrial oxidative phosphorylation. [4] Another metabolic hallmark of carcinogenesis is altered lipid metabolism, whereby cancer cells may adopt enhanced de novo lipid production (lipogenesis. [1],[2],[3] Enhanced lipid metabolism is also observed in individuals with metabolic syndromes potentially a consequence of increasing popularity of the Standard American Diet, composed of high levels of saturated fats and carbohydrates. [5] A growing body of epidemiological data indicates a positive correlation between the occurrence of metabolic syndromes, such as cardiovascular disease, obesity, type-2 diabetes and associated hyperinsulemia, with the aggressiveness of cancer. [6],[7],[8],[9] Remarkably, it is estimated that for every 1% reduction in saturated fats, replaced by polyunsaturated, there would be a 2%-3% reduction in cardiovascular disease. [10] Thus, it is conceivable that an equally remarkable attenuation in cancer progression might be achieved with such a reduction in lipid accumulation.

  10. Increased Power in Sediment Microbial Fuel Cell: Facilitated Mass Transfer via a Water-Layer Anode Embedded in Sediment.

    Science.gov (United States)

    Lee, Yoo Seok; An, Junyeong; Kim, Bongkyu; Park, HyunJun; Kim, Jisu; Chang, In Seop

    2015-01-01

    We report a methodology for enhancing the mass transfer at the anode electrode of sediment microbial fuel cells (SMFCs), by employing a fabric baffle to create a separate water-layer for installing the anode electrode in sediment. The maximum power in an SMFC with the anode installed in the separate water-layer (SMFC-wFB) was improved by factor of 6.6 compared to an SMFC having the anode embedded in the sediment (SMFC-woFB). The maximum current density in the SMFC-wFB was also 3.9 times higher (220.46 mA/m2) than for the SMFC-woFB. We found that the increased performance in the SMFC-wFB was due to the improved mass transfer rate of organic matter obtained by employing the water-layer during anode installation in the sediment layer. Acetate injection tests revealed that the SMFC-wFB could be applied to natural water bodies in which there is frequent organic contamination, based on the acetate flux from the cathode to the anode.

  11. Determination of polymer electrolyte membrane (PEM) degradation products in fuel cell water using electrospray ionization tandem mass spectrometry.

    Science.gov (United States)

    Zedda, Marco; Tuerk, Jochen; Peil, Stefan; Schmidt, Torsten C

    2010-12-30

    Within the scope of research of membrane degradation phenomena during fuel cell operation a reliable analytical procedure for the extraction, detection and quantification of possible membrane oxidation products has been developed. These oxidation products originate from the attack of hydroxyl or peroxyl radicals on the membrane polymer. Such radicals are formed in situ (during fuel cell operation) or ex situ (Fenton test as oxidative stress simulation). The analysis of membrane oxidation products was carried out by electrospray ionization tandem mass spectrometry. Five potential membrane oxidation products (4-hydroxybenzoic acid (4-HBA), 4-hydroxybenzaldehyde (4-HBAD), 4,4-biphenol (4,4-BP), 4-hydroxybenzenesulfonate (4-HBS), and 4,4-sulfonylbiphenol (4,4-SBP)) were selected based on the molecular structure of the sulfonated polyarylether membrane used. In conjunction with the development of a multiple reaction monitoring (MRM) method, the ionization and fragmentation of the selected compounds were investigated. For 4,4-BP a molecular ion (M(+•) ) was observed in the positive ionization mode and used for MRM method development. Reproducible extraction of the model compounds was achieved using a mixed-mode sorbent material with both weak anion-exchange and reversed-phase retention properties. By using the developed analytical procedure, the identities of two membrane degradation products (4-HBA and 4-HBAD) were determined in situ and ex situ. In addition to the investigation of membrane degradation phenomena, the combination of extraction on a mixed-mode sorbent material and tandem mass spectrometric detection is attractive for the analysis of aromatic sulfonic acids, phenolic acids and phenols. Copyright © 2010 John Wiley & Sons, Ltd.

  12. Eight-Shaped Hatching Increases the Risk of Inner Cell Mass Splitting in Extended Mouse Embryo Culture.

    Directory of Open Access Journals (Sweden)

    Zheng Yan

    Full Text Available Increased risk of monozygotic twinning (MZT has been shown to be associated with assisted reproduction techniques, particularly blastocyst culture. Interestingly, inner cell mass (ICM splitting in human '8'-shaped hatching blastocysts that resulted in MZT was reported. However, the underlying cause of MZT is not known. In this study, we investigated in a mouse model whether in vitro culture leads to ICM splitting and its association with hatching types. Blastocyst hatching was observed in: (i in vivo developed blastocysts and (ii-iii in vitro cultured blastocysts following in vivo or in vitro fertilization. We found that '8'-shaped hatching occurred with significantly higher frequency in the two groups of in vitro cultured blastocysts than in the group of in vivo developed blastocysts (24.4% and 20.4% versus 0.8%, respectively; n = 805, P < 0.01. Moreover, Oct4 immunofluorescence staining was performed to identify the ICM in the hatching and hatched blastocysts. Scattered and split distribution of ICM cells was observed around the small zona opening of '8'-shaped hatching blastocysts. This occurred at a high frequency in the in vitro cultured groups. Furthermore, we found more double OCT4-positive masses, suggestive of increased ICM splitting in '8'-shaped hatching and hatched blastocysts than in 'U'-shaped hatching and hatched blastocysts (12.5% versus 1.9%, respectively; n = 838, P < 0.01. Therefore, our results demonstrate that extended in vitro culture can cause high frequencies of '8'-shaped hatching, and '8'-shaped hatching that may disturb ICM herniation leading to increased risk of ICM splitting in mouse blastocysts. These results may provide insights into the increased risk of human MZT after in vitro fertilization and blastocyst transfer.

  13. Regeneration of viable oil palm plants from protoplasts by optimizing media components, growth regulators and cultivation procedures.

    Science.gov (United States)

    Masani, Mat Yunus Abdul; Noll, Gundula; Parveez, Ghulam Kadir Ahmad; Sambanthamurthi, Ravigadevi; Prüfer, Dirk

    2013-09-01

    Oil palm protoplasts are suitable as a starting material for the production of oil palm plants with new traits using approaches such as somatic hybridization, but attempts to regenerate viable plants from protoplasts have failed thus far. Here we demonstrate, for the first time, the regeneration of viable plants from protoplasts isolated from cell suspension cultures. We achieved a protoplast yield of 1.14×10(6) per gram fresh weight with a viability of 82% by incubating the callus in a digestion solution comprising 2% cellulase, 1% pectinase, 0.5% cellulase onuzuka R10, 0.1% pectolyase Y23, 3% KCl, 0.5% CaCl2 and 3.6% mannitol. The regeneration of protoplasts into viable plants required media optimization, the inclusion of plant growth regulators and the correct culture technique. Microcalli derived from protoplasts were obtained by establishing agarose bead cultures using Y3A medium supplemented with 10μM naphthalene acetic acid, 2μM 2,4-dichlorophenoxyacetic acid, 2μM indole-3-butyric acid, 2μM gibberellic acid and 2μM 2-γ-dimethylallylaminopurine. Small plantlets were regenerated from microcalli by somatic embryogenesis after successive subculturing steps in medium with limiting amounts of growth regulators supplemented with 200mg/l ascorbic acid. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. Articular Cartilage Repair Using Marrow Stimulation Augmented with a Viable Chondral Allograft: 9-Month Postoperative Histological Evaluation

    Directory of Open Access Journals (Sweden)

    James K. Hoffman

    2015-01-01

    Full Text Available Marrow stimulation is frequently employed to treat focal chondral defects of the knee. However, marrow stimulation typically results in fibrocartilage repair tissue rather than healthy hyaline cartilage, which, over time, predisposes the repair to failure. Recently, a cryopreserved viable chondral allograft was developed to augment marrow stimulation. The chondral allograft is comprised of native viable chondrocytes, chondrogenic growth factors, and extracellular matrix proteins within the superficial, transitional, and radial zones of hyaline cartilage. Therefore, host mesenchymal stem cells that infiltrate the graft from the underlying bone marrow following marrow stimulation are provided with the optimal microenvironment to undergo chondrogenesis. The present report describes treatment of a trochlear defect with marrow stimulation augmented with this novel chondral allograft, along with nine month postoperative histological results. At nine months, the patient demonstrated complete resolution of pain and improvement in function, and the repair tissue consisted of 85% hyaline cartilage. For comparison, a biopsy obtained from a patient 8.2 months after treatment with marrow stimulation alone contained only 5% hyaline cartilage. These outcomes suggest that augmenting marrow stimulation with the viable chondral allograft can eliminate pain and improve outcomes, compared with marrow stimulation alone.

  15. Real-time detection of viable microorganisms by intracellular phototautomerism

    Directory of Open Access Journals (Sweden)

    Schuren Frank

    2010-06-01

    Full Text Available Abstract Background To date, the detection of live microorganisms present in the environment or involved in infections is carried out by enumeration of colony forming units on agar plates, which is time consuming, laborious and limited to readily cultivable microorganisms. Although cultivation-independent methods are available, they involve multiple incubation steps and do mostly not discriminate between dead or live microorganisms. We present a novel generic method that is able to specifically monitor living microorganisms in a real-time manner. Results The developed method includes exposure of cells to a weak acid probe at low pH. The neutral probe rapidly permeates the membrane and enters the cytosol. In dead cells no signal is obtained, as the cytosolic pH reflects that of the acidic extracellular environment. In live cells with a neutral internal pH, the probe dissociates into a fluorescent phototautomeric anion. After reaching peak fluorescence, the population of live cells decays. This decay can be followed real-time as cell death coincides with intracellular acidification and return of the probe to its uncharged non-fluorescent state. The rise and decay of the fluorescence signal depends on the probe structure and appears discriminative for bacteria, fungi, and spores. We identified 13 unique probes, which can be applied in the real-time viability method described here. Under the experimental conditions used in a microplate reader, the reported method shows a detection limit of 106 bacteria ml-1, while the frequently used LIVE/DEAD BacLight™ Syto9 and propidium iodide stains show detection down to 106 and 107 bacteria ml-1, respectively. Conclusions We present a novel fluorescence-based method for viability assessment, which is applicable to all bacteria and eukaryotic cell types tested so far. The RTV method will have a significant impact in many areas of applied microbiology including research on biocidal activity, improvement of

  16. Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry.

    Science.gov (United States)

    Doneanu, Catalin E; Xenopoulos, Alex; Fadgen, Keith; Murphy, Jim; Skilton, St John; Prentice, Holly; Stapels, Martha; Chen, Weibin

    2012-01-01

    Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a "discovery" assay, the latter as a "monitoring" assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique ("Hi3" method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally

  17. Determination of membrane degradation products in the product water of polymer electrolyte membrane fuel cells using liquid chromatography mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Zedda, Marco

    2011-05-12

    The predominant long term failure of polymer electrolyte membranes (PEM) is caused by hydroxyl radicals generated during fuel cell operation. These radicals attack the polymer, leading to chain scission, unzipping and consequently to membrane decomposition products. The present work has investigated decomposition products of novel sulfonated aromatic hydrocarbon membranes on the basis of a product water analysis. Degradation products from the investigated membrane type and the possibility to detect these compounds in the product water for diagnostic purposes have not been discovered yet. This thesis demonstrates the potential of solid phase extraction and liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) for the extraction, separation, characterization, identification and quantification of membrane degradation products in the product water of fuel cells. For this purpose, several polar aromatic hydrocarbons with different functional groups were selected as model compounds for the development of reliable extraction, separation and detection methods. The results of this thesis have shown that mixed mode sorbent materials with both weak anion exchange and reversed phase retention properties are well suited for reproducible extraction of both molecules and ions from the product water. The chromatographic separation of various polar aromatic hydrocarbons was achieved by means of phase optimized liquid chromatography using a solvent gradient and on a C18 stationary phase. Sensitive and selective detection of model compounds could be successfully demonstrated by the analysis of the product water using tandem mass spectrometry. The application of a hybrid mass spectrometer (Q Trap) for the characterization of unknown polar aromatic hydrocarbons has led to the identification and confirmation of 4-hydroxybenzoic acid in the product water. In addition, 4-HBA could be verified as a degradation product resulting from PEM decomposition by hydroxyl radicals using an

  18. The simple fabrication of nanorods mass production for the dye-sensitized solar cell

    Directory of Open Access Journals (Sweden)

    Dharmanto

    2017-01-01

    Full Text Available ZnO nanorods were successfully synthesized by the plasma torch with the zinc powder as the source of ZnO. Several testing was conducted to examine the results of ZnO nanoparticles among others are X-ray fluorescence (XRF, Scanning Electron Microscopy (SEM, and X-ray diffraction (XRD. The results of ZnO nanorods diameter vary from 87 nm to 263 nm which can be seen from SEM images and it is length varies from 300 to 1000 nm. It was found from XRD data that a sharp peak occured at 36.253°. It is indicated a good crystal growth and agreed well with the standard data (JCPDF-ICDD card no.: 36-1451. Effects of electrical current variations of plasma torch of 20, 25, and 30 Amperes on the size of ZnO nanorods are indicated from aspect ratio (72.85, 87.42, and 103 nm. The effects of electrical current of plasma torch on the purity of ZnO were about 95.61%, 98.46%, and 96.49% respectively. The performance of the solar cell at the time with the values of Voc, Jsc, FF, and efficiency are 0.466 V, 1.524 mA/cm 2,, 51.95%, and 0.37% respectively. ZnO nanorods were successfully synthesized by the plasma torch with the zinc powder as the source of ZnO.

  19. Birth of viable puppies derived from breeding cloned female dogs with a cloned male.

    Science.gov (United States)

    Park, J E; Hong, S G; Kang, J T; Oh, H J; Kim, M K; Kim, M J; Kim, H J; Kim, D Y; Jang, G; Lee, B C

    2009-09-15

    Since the establishment of production of viable cloned dogs by somatic cell nucleus transfer, great concern has been given to the reproductive abilities of these animals (Canis familiaris). Therefore, we investigated reproductive activity of cloned dogs by (1) performing sperm analysis using computer-assisted sperm analysis and early embryonic development, (2) assessing reproductive cycling by measuring serum progesterone (P4) levels and performing vaginal cytology, and (3) breeding cloned dogs using artificial insemination. Results showed that most parameters of sperm motility in a cloned male dog were within the reference range, and in vivo-matured oocytes from a noncloned female were successfully fertilized by spermatozoa from a cloned male dog and develop normally to the 8-cell stage. Three cloned female dogs displayed normal patterns of P4 levels and morphologic changes of the vaginal epithelium. Two cloned female dogs became pregnant using semen from a cloned male dog and successfully delivered 10 puppies by natural labor. In conclusion, these data demonstrated that both cloned male and female dogs are fertile, and their puppies are currently alive and healthy with normal growth patterns.

  20. Quantification of excretory renal function and urinary protein excretion by determination of body cell mass using bioimpedance analysis.

    Science.gov (United States)

    Flury, Stefan; Trachsler, Johannes; Schwarz, Albin; Ambühl, Patrice M

    2015-10-27

    Creatinine clearance (CrCl) based on 24 h urine collection is an established method to determine glomerular filtration rate (GFR). However, its measurement is cumbersome and the results are frequently inaccurate. The aim of this study was to develop an alternative method to predict CrCl and urinary protein excretion based on plasma creatinine and the quantification of muscle mass through bioimpedance analysis (BIA). In 91 individuals with normal and impaired renal function CrCl was measured from 24 h urine excretion and plasma creatinine concentration. A model to predict 24 h-creatininuria was developed from various measurements assessing muscle mass such as body cell mass (BCM) and fat free mass (FFM) obtained by BIA, skinfold caliper and other techniques (training group, N = 60). Multivariate regression analysis was performed to predict 24 h-creatininuria and to calculate CrCl. A validation group (N = 31) served to compare predicted and measured CrCl. Overall (accuracy, bias, precision, correlation) the new BIA based prediction model performed substantially better compared with measured CrCl (P15 = 87 %, bias = 0, IQR of differences = 7.9 mL/min/1.73 m(2), R = 0.972) versus established estimation formulas such as the 4vMDRD (P15 =  6 %, bias = -8.3 mL/min/1.73 m(2), IQR = 13.7 mL/min/1.73 m(2), R = 0.935), CKD-EPI (P15 = 29 %, bias = -7.0 mL/min/1.73 m(2), IQR = 12.1 mL/min/1.73 m(2), R = 0.932, Cockcroft-Gault equations (P15 = 55 %, bias = -4.4 mL/min/1.73 m(2), IQR = 9.0 mL/min/1.73 m(2), R = 0.920). The superiority of the new method over established prediction formulas was most obvious in a subgroup of individuals with BMI > 30 kg/m(2) and in a subgroup with CrCl > 60 mL/min/1.73 m(2). Moreover, 24 h urinary protein excretion could be estimated accurately by normalization with 24 h-creatininuria derived from BIA based BCM. Prediction of CrCl based on estimated urinary creatinine excretion determined from measurement of BCM by BIA technique is both

  1. The Role of Mitochondria from Mature Oocyte to Viable Blastocyst

    Directory of Open Access Journals (Sweden)

    Scott Chappel

    2013-01-01

    Full Text Available The oocyte requires a vast supply of energy after fertilization to support critical events such as spindle formation, chromatid separation, and cell division. Until blastocyst implantation, the developing zygote is dependent on the existing pool of mitochondria. That pool size within each cell decreases with each cell division. Mitochondria obtained from oocytes of women of advanced reproductive age harbor DNA deletions and nucleotide variations that impair function. The combination of lower number and increased frequency of mutations and deletions may result in inadequate mitochondrial activity necessary for continued embryo development and cause pregnancy failure. Previous reports suggested that mitochondrial activity within oocytes may be supplemented by donor cytoplasmic transfer at the time of intracytoplasmic sperm injection (ICSI. Those reports showed success; however, safety concerns arose due to the potential of two distinct populations of mitochondrial genomes in the offspring. Mitochondrial augmentation of oocytes is now reconsidered in light of our current understanding of mitochondrial function and the publication of a number of animal studies. With a better understanding of the role of this organelle in oocytes immediately after fertilization, blastocyst and offspring, mitochondrial augmentation may be reconsidered as a method to improve oocyte quality.

  2. Cybernetically sound organizational structures II: Relating de Sitter's design theory to Beer's viable system model

    NARCIS (Netherlands)

    Achterbergh, J.M.I.M.; Vriens, D.J.

    2011-01-01

    - Purpose – The purpose of this paper is to show how the viable system model (VSM) and de Sitter's design theory can complement each other in the context of the diagnosis and design of viable organizations. - Design/methodology/approach – Key concepts from Beer's model and de Sitter's design

  3. Stable quasi-solid-state dye-sensitized solar cell using a diamide derivative as low molecular mass organogelator

    Science.gov (United States)

    Tao, Li; Huo, Zhipeng; Dai, Songyuan; Zhu, Jun; Zhang, Changneng; Huang, Yang; Zhang, Bing; Yao, Jianxi

    2014-09-01

    High stability is a significant target for practical applications of dye-sensitized solar cells. 2-(1-oxododecyl)hydrazide, a diamide derivative, is synthesized and applied in quasi-solid-state dye-sensitized solar cells (QS-DSSCs) as a low molecular mass organogelator (LMOG). It is noteworthy that the transition temperature from gel state to liquid state (Tgel) of this gel electrolyte is 125.2 °C, which ensures the gel state of the electrolyte at the DSSC operating temperature. The influences of the gel electrolyte on the kinetic processes of electron transport and recombination are investigated. The diffusion of redox species in the gel electrolyte is hindered by the crosslinked network, and the decreased electron recombination lifetime indicates an increased electron recombination in QS-DSSC. Significantly, the QS-DSSC exhibits excellent thermal and light-soaking stabilities during accelerated aging tests for 1000 h. Especially, there is almost no change in the short-circuit current density (Jsc) in the QS-DSSC, while the Jsc of the liquid electrolyte based DSSC decreases to 79-90% of their initial values. These results are very important for the application and commercialization of DSSCs.

  4. Design and characterisation of a novel in vitro skin diffusion cell system for assessing mass casualty decontamination systems.

    Science.gov (United States)

    Matar, H; Larner, J; Kansagra, S; Atkinson, K L; Skamarauskas, J T; Amlot, R; Chilcott, R P

    2014-06-01

    The efficient removal of contaminants from the outer surfaces of the body can provide an effective means of reducing adverse health effects associated with incidents involving the accidental or deliberate release of hazardous materials. Showering with water is frequently used by first responders as a rapid method of mass casualty decontamination (MCD). However, there is a paucity of data on the generic effectiveness and safety of aqueous decontamination systems. To address these issues, we have developed a new in vitro skin diffusion cell system to model the conditions of a common MCD procedure ("ladder pipe system"). The new diffusion cell design incorporates a showering nozzle, an air sampling port for measurement of vapour loss and/aerosolisation, adjustable (horizontal to vertical) skin orientation and a circulating manifold system (to maintain a specified flow rate, temperature and pressure of shower water). The dermal absorption characteristics of several simulants (Invisible Red S, curcumin and methyl salicylate) measured with the new in vitro model were in good agreement with previous in vitro and in vivo studies. Moreover, these initial studies have indicated that whilst flow rate and water temperature are important factors for MCD, the presence of clothing during showering may (under certain circumstances) cause transfer and spreading of contaminants to the skin surface. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Mass Cytometry and Topological Data Analysis Reveal Immune Parameters Associated with Complications after Allogeneic Stem Cell Transplantation.

    Science.gov (United States)

    Lakshmikanth, Tadepally; Olin, Axel; Chen, Yang; Mikes, Jaromir; Fredlund, Erik; Remberger, Mats; Omazic, Brigitta; Brodin, Petter

    2017-08-29

    Human immune systems are variable, and immune responses are often unpredictable. Systems-level analyses offer increased power to sort patients on the basis of coordinated changes across immune cells and proteins. Allogeneic stem cell transplantation is a well-established form of immunotherapy whereby a donor immune system induces a graft-versus-leukemia response. This fails when the donor immune system regenerates improperly, leaving the patient susceptible to infections and leukemia relapse. We present a systems-level analysis by mass cytometry and serum profiling in 26 patients sampled 1, 2, 3, 6, and 12 months after transplantation. Using a combination of machine learning and topological data analyses, we show that global immune signatures associated with clinical outcome can be revealed, even when patients are few and heterogeneous. This high-resolution systems immune monitoring approach holds the potential for improving the development and evaluation of immunotherapies in the future. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Depth Profile Analysis of Amorphous Silicon Thin Film Solar Cells by Pulsed Radiofrequency Glow Discharge Time of Flight Mass Spectrometry

    Science.gov (United States)

    Alvarez-Toral, Aitor; Sanchez, Pascal; Menéndez, Armando; Pereiro, Rosario; Sanz-Medel, Alfredo; Fernández, Beatriz

    2015-02-01

    Among the different solar cell technologies, amorphous silicon (a-Si:H) thin film solar cells (TFSCs) are today very promising and, so, TFSCs analytical characterization for quality control issues is increasingly demanding. In this line, depth profile analysis of a-Si:H TFSCs on steel substrate has been investigated by using pulsed radiofrequency glow discharge-time of flight mass spectrometry (rf-PGD-TOFMS). First, to discriminate potential polyatomic interferences for several analytes (e.g., 28Si+, 31P+, and 16O+) appropriate time positions along the GD pulse profile were selected. A multi-matrix calibration approach, using homogeneous certified reference materials without hydrogen as well as coated laboratory-made standards containing hydrogen, was employed for the methodological calibration. Different calibration strategies (in terms of time interval selection on the pulse profile within the afterglow region) have been compared, searching for optimal calibration graphs correlation. Results showed that reliable and fast quantitative depth profile analysis of a-Si:H TFSCs by rf-PGD-TOFMS can be achieved.

  7. Hymenolepis nana: immunity against oncosphere challenge in mice previously given viable or non-viable oncospheres of H. nana, H. diminuta, H. microstoma and Taenia taeniaeformis.

    Science.gov (United States)

    Ito, A; Onitake, K; Sasaki, J; Takami, T

    1991-04-01

    When mice, previously given oral inoculation with viable oncospheres of the heterologous cestode species (Hymenolepis diminuta, H. microstoma, Taenia taeniaeformis) and the homologous one (H. nana), were challenged with oncospheres of H. nana 4 days after the primary inoculation, they showed strong and complete resistance to H. nana challenge, respectively. However, the resistance was not evoked in mice given either infective eggs of Toxocara canis or non-viable oncospheres of all cestode species examined. Congenitally athymic nude mice given viable oncospheres did not show any resistance to H. nana either. Eosinophil infiltration around cysticercoids of H. nana in the intestinal villi appeared to be more prominent in mice previously given viable oncospheres of H. diminuta than in mice given non-viable oncospheres or PBS only. Some of the eosinophils in the villus harboring cysticercoid(s) of H. nana invaded the epithelia in the former, whereas all eosinophils remained in the lamina propria in the latter. There was almost no eosinophil infiltration in nude mice. Microscopic observations revealed that oncospheres of H. diminuta, which require beetles as the intermediate host like H. microstoma, could invade the mouse intestinal tissue. Therefore, it is strongly suggested that the strong cross resistance to H. nana in mice, induced by oncospheres of all heterologous cestode species, is thymus-dependent and due to oncospheral invasion into the intestinal tissue of mice.

  8. Plant defense responses in opium poppy cell cultures revealed by liquid chromatography-tandem mass spectrometry proteomics.

    Science.gov (United States)

    Zulak, Katherine G; Khan, Morgan F; Alcantara, Joenel; Schriemer, David C; Facchini, Peter J

    2009-01-01

    Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids, including the narcotic analgesic morphine and the antimicrobial agent sanguinarine. In contrast to the plant, cell cultures of opium poppy do not accumulate alkaloids constitutively but produce sanguinarine in response to treatment with certain fungal-derived elicitors. The induction of sanguinarine biosynthesis provides a model platform to characterize the regulation of benzylisoquinoline alkaloid pathways and other defense responses. Proteome analysis of elicitor-treated opium poppy cell cultures by two-dimensional denaturing-polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry facilitated the identification of 219 of 340 protein spots based on peptide fragment fingerprint searches of a combination of databases. Of the 219 hits, 129 were identified through pre-existing plant proteome databases, 63 were identified by matching predicted translation products in opium poppy-expressed sequence tag databases, and the remainder shared evidence from both databases. Metabolic enzymes represented the largest category of proteins and included S-adenosylmethionine synthetase, several glycolytic, and a nearly complete set of tricarboxylic acid cycle enzymes, one alkaloid, and several other secondary metabolic enzymes. The abundance of chaperones, heat shock proteins, protein degradation factors, and pathogenesis-related proteins provided a comprehensive proteomics view on the coordination of plant defense responses. Qualitative comparison of protein abundance in control and elicitor-treated cell cultures allowed the separation of induced and constitutive or suppressed proteins. DNA microarrays were used to corroborate increases in protein abundance with a corresponding induction in cognate transcript levels.

  9. A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

    Science.gov (United States)

    Pooley, Hannah B.; de Silva, Kumudika; Purdie, Auriol C.; Begg, Douglas J.; Whittington, Richard J.

    2016-01-01

    ABSTRACT Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method

  10. Mass spectrometry analysis of adipose-derived stem cells reveals a significant effect of hypoxia on pathways regulating extracellular matrix.

    Science.gov (United States)

    Riis, Simone; Stensballe, Allan; Emmersen, Jeppe; Pennisi, Cristian Pablo; Birkelund, Svend; Zachar, Vladimir; Fink, Trine

    2016-04-14

    Adipose-derived stem cells (ASCs) are being increasingly recognized for their potential to promote tissue regeneration and wound healing. These effects appear to be partly mediated by paracrine signaling pathways, and are enhanced during hypoxia. Mass spectrometry (MS) is a valuable tool for proteomic profiling of cultured ASCs, which may help to reveal the identity of the factors secreted by the cells under different conditions. However, serum starvation which is essentially required to obtain samples compatible with secretome analysis by MS can have a significant influence on ASCs. Here, we present a novel and optimized culturing approach based on the use of a clinically relevant serum-free formulation, which was used to assess the effects of hypoxia on the ASC proteomic profile. Human ASCs from three human donors were expanded in StemPro® MSC SFM XenoFree medium. Cells were cultured for 24 h in serum- and albumin-free supplements in either normoxic (20 %) or hypoxic (1 %) atmospheres, after which the cells and conditioned medium were collected, subfractionated, and analyzed using MS. Prior to analysis, the secreted proteins were further subdivided into a secretome (>30 kDa) and a peptidome (3-30 kDa) fraction. MS analysis revealed the presence of 342, 98, and 3228 proteins in the normoxic ASC secretome, peptidome, and proteome, respectively. A relatively small fraction of the proteome (9.6 %) was significantly affected by hypoxia, and the most regulated proteins were those involved in extracellular matrix (ECM) synthesis and cell metabolism. No proteins were found to be significantly modulated by hypoxic treatment across all cultures for the secretome and peptidome samples. This study highlights ECM remodeling as a significant mechanism contributing to the ASC regenerative effect after hypoxic preconditioning, and further underscores considerable inter-individual differences in ASC response to hypoxia. The novel culture paradigm provides a basis for future

  11. Vibrio cholerae classical biotype is converted to the viable non-culturable state when cultured with the El Tor biotype.

    Science.gov (United States)

    Pradhan, Subhra; Mallick, Sanjaya K; Chowdhury, Rukhsana

    2013-01-01

    A unique event in bacterial epidemiology was the emergence of the El Tor biotype of Vibrio cholerae O1 and the subsequent rapid displacement of the existing classical biotype as the predominant cause of epidemic cholera. We demonstrate that when the El Tor and classical biotypes were cocultured in standard laboratory medium a precipitous decline in colony forming units (CFU) of the classical biotype occurred in a contact dependent manner. Several lines of evidence including DNA release, microscopy and flow cytometric analysis indicated that the drastic reduction in CFU of the classical biotype in cocultures was not accompanied by lysis, although when the classical biotype was grown individually in monocultures, lysis of the cells occurred concomitant with decrease in CFU starting from late stationary phase. Furthermore, uptake of a membrane potential sensitive dye and protection of genomic DNA from extracellular DNase strongly suggested that the classical biotype cells in cocultures retained viability in spite of loss of culturability. These results suggest that coculturing the classical biotype with the El Tor biotype protects the former from lysis allowing the cells to remain viable in spite of the loss of culturability. The stationary phase sigma factor RpoS may have a role in the loss of culturability of the classical biotype in cocultures. Although competitive exclusion of closely related strains has been reported for several bacterial species, conversion of the target bacterial population to the viable non-culturable state has not been demonstrated previously and may have important implications in the evolution of bacterial strains.

  12. The effect of formulation on the penetration of coated and uncoated zinc oxide nanoparticles into the viable epidermis of human skin in vivo.

    Science.gov (United States)

    Leite-Silva, Vânia R; Le Lamer, Marina; Sanchez, Washington Y; Liu, David C; Sanchez, Washington H; Morrow, Isabel; Martin, Darren; Silva, Heron D T; Prow, Tarl W; Grice, Jeffrey E; Roberts, Michael S

    2013-06-01

    The use of nanoparticulate zinc oxide (ZnO-NP) in sunscreens and other cosmetic products has raised public health concerns. The two key issues are the extent of exposure to ZnO-NP and the likely hazard after the application of ZnO-NP in sunscreen and cosmetic products to humans in vivo. Our aims were to assess exposure by the extent of ZnO-NP penetration into the viable epidermis and hazard by changes in the viable epidermal redox state for a number of topical products. Of particular interest is the role of the particle coating, formulation used, and the presence of any enhancers. Multiphoton tomography with fluorescence lifetime imaging microscopy (MPT-FLIM) was used to simultaneously observe ZnO-NP penetration and potential metabolic changes within the viable epidermis of human volunteers after topical application of various ZnO-NP products. Coated and uncoated ZnO-NP remained in the superficial layers of the SC and in the skin furrows. We observed limited penetration of coated ZnO-NP dispersed in a water-in-oil emulsion formulation, which was predominantly localized adjacent to the skin furrow. However, the presence of ZnO-NP in the viable epidermis did not alter the metabolic state or morphology of the cells. In summary, our data suggest that some limited penetration of coated and uncoated ZnO-NP may occur into viable stratum granulosum epidermis adjacent to furrows, but that the extent is not sufficient to affect the redox state of those viable cells. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  13. Growth Phase, Oxygen, Temperature and Starvation Affect the Development of Viable but Non-Culturable State of Vibrio cholerae

    Directory of Open Access Journals (Sweden)

    Bin eWu

    2016-03-01

    Full Text Available AbstractVibrio cholerae can enter into a viable but non-culturable (VBNC state in order to survive in unfavourable environments. In this study, we studied the roles of five physicochemical and microbiological factors or states, namely, different strains, growth phases, oxygen, temperature, and starvation, on the development of VBNC of V. cholerae in artificial sea water (ASW. Different strains of the organism, the growth phase, and oxygen levels affected the progress of VBNC development. It was found that the VBNC state was induced faster in V. cholerae serogroup O1 classical biotype strain O395 than in O1 El Tor biotype strains C6706 and N16961. When cells in different growth phases were used for VBNC induction, stationary-phase cells lost their culturability more quickly than exponential-phase cells, while induction of a totally non-culturable state took longer to achieve for stationary-phase cells in all three strains, suggesting that heterogeneity of cells should be considered. Aeration strongly accelerated the loss of culturability. During the development of the VBNC state, the culturable cell count under aeration conditions was almost 106-fold lower than under oxygen-limited conditions for all three strains. The other two factors, temperature and nutrients-rich environment, may prevent the induction of VBNC cells. At 22°C or 37°C in ASW, most of the cells rapidly died and the culturable cell count reduced from about 108 CFU/mL to 106–105 CFU/mL. The total cell counts showed that cells that lost viability were decomposed, and the viable cell counts were the same as culturable cell counts, indicating that the cells did not reach the VBNC state. VBNC state development was blocked when ASW was supplied with Luria-Bertani broth (LB, but it was not affected in ASW with M9, suggesting that specific nutrients in LB may prevent the development of VBNC state. These results revealed that the five factors evaluated in this study had different

  14. Detection and Quantification of Viable and Nonviable Trypanosoma cruzi Parasites by a Propidium Monoazide Real-Time Polymerase Chain Reaction Assay

    Science.gov (United States)

    Cancino-Faure, Beatriz; Fisa, Roser; Alcover, M. Magdalena; Jimenez-Marco, Teresa; Riera, Cristina

    2016-01-01

    Molecular techniques based on real-time polymerase chain reaction (qPCR) allow the detection and quantification of DNA but are unable to distinguish between signals from dead or live cells. Because of the lack of simple techniques to differentiate between viable and nonviable cells, the aim of this study was to optimize and evaluate a straightforward test based on propidium monoazide (PMA) dye action combined with a qPCR assay (PMA-qPCR) for the selective quantification of viable/nonviable epimastigotes of Trypanosoma cruzi. PMA has the ability to penetrate the plasma membrane of dead cells and covalently cross-link to the DNA during exposure to bright visible light, thereby inhibiting PCR amplification. Different concentrations of PMA (50–200 μM) and epimastigotes of the Maracay strain of T. cruzi (1 × 105–10 parasites/mL) were assayed; viable and nonviable parasites were tested and quantified by qPCR with a TaqMan probe specific for T. cruzi. In the PMA-qPCR assay optimized at 100 μM PMA, a significant qPCR signal reduction was observed in the nonviable versus viable epimastigotes treated with PMA, with a mean signal reduction of 2.5 logarithm units and a percentage of signal reduction > 98%, in all concentrations of parasites assayed. This signal reduction was also observed when PMA-qPCR was applied to a mixture of live/dead parasites, which allowed the detection of live cells, except when the concentration of live parasites was low (10 parasites/mL). The PMA-qPCR developed allows differentiation between viable and nonviable epimastigotes of T. cruzi and could thus be a potential method of parasite viability assessment and quantification. PMID:27139452

  15. Numerical Study of the Dynamic Response of Heat and Mass Transfer to Operation Mode Switching of a Unitized Regenerative Fuel Cell

    Directory of Open Access Journals (Sweden)

    Hong Xiao

    2016-12-01

    Full Text Available Knowledge concerning the complicated changes of mass and heat transfer is desired to improve the performance and durability of unitized regenerative fuel cells (URFCs. In this study, a transient, non-isothermal, single-phase, and multi-physics mathematical model for a URFC based on the proton exchange membrane is generated to investigate transient responses in the process of operation mode switching from fuel cell (FC to electrolysis cell (EC. Various heat generation mechanisms, including Joule heat, reaction heat, and the heat attributed to activation polarizations, have been considered in the transient model coupled with electrochemical reaction and mass transfer in porous electrodes. The polarization curves of the steady-state models are validated by experimental data in the literatures. Numerical results reveal that current density, gas mass fractions, and temperature suddenly change with the sudden change of operating voltage in the mode switching process. The response time of temperature is longer than that of current density and gas mass fractions. In both FC and EC modes, the cell temperature and gradient of gas mass fraction in the oxygen side are larger than that in the hydrogen side. The temperature difference of the entire cell is less than 1.5 K. The highest temperature appears at oxygen-side catalyst layer under the FC mode and at membrane under a more stable EC mode. The cell is exothermic all the time. These dynamic responses and phenomena have important implications for heat analysis and provide proven guidelines for the improvement of URFCs mode switching.

  16. Analysis of cancer cell lipids using matrix-assisted laser desorption/ionization 15-T Fourier transform ion cyclotron resonance mass spectrometry.

    Science.gov (United States)

    Yang, Hyo-Jik; Park, Kyu Hwan; Lim, Dong Wan; Kim, Hyun Sik; Kim, Jeongkwon

    2012-03-30

    A combination of methodologies using the extremely high mass accuracy and resolution of 15-T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) was introduced for the identification of intact cancer cell phospholipids. Lipids from a malignant glioma cell line were initially analyzed at a resolution of >200,000 and identified by setting the mass tolerance to ±1 mDa using matrix-assisted laser desorption/ionization (MALDI) 15-T FT-ICR MS in positive ion mode. In most cases, a database search of potential lipid candidates using the exact masses of the lipids yielded only one possible chemical composition. Extremely high mass accuracy (800,000), yielded well-resolved isotopic fine structures allowing for the identification of lipids by MALDI 15-T FT-ICR MS without using tandem mass spectrometric (MS/MS) analysis. Using this method, a total of 38 unique lipids were successfully identified. Copyright © 2012 John Wiley & Sons, Ltd.

  17. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS: A New Tool for the Analysis of Toxicological Effects on Single Cell Level

    Directory of Open Access Journals (Sweden)

    Harald Jungnickel

    2016-02-01

    Full Text Available Single cell imaging mass spectrometry opens up a complete new perspective for strategies in toxicological risk assessment and drug discovery. In particular, time-of-flight secondary ion mass spectrometry (ToF-SIMS with its high spatial and depth resolution is becoming part of the imaging mass spectrometry toolbox used for single cell analysis. Recent instrumentation advancements in combination with newly developed cluster ion guns allow 3-dimensional reconstruction of single cells together with a spatially resolved compound location and quantification on nanoscale depth level. The exact location and quantification of a single compound or even of a set of compounds is no longer restricted to the two dimensional space within single cells, but is available for voxels, a cube-sized 3-dimensional space, rather than pixels. The information gathered from one voxel is further analysed using multivariate statistical methodology like maximum autocorrelation factors to co-locate the compounds of interest within intracellular organelles like nucleus, mitochondria or golgi apparatus. Furthermore, the cell membrane may be resolved, including adhering compounds and potential changes of the lipid patterns. The generated information can be used further for a first evaluation of intracellular target specifity of new drug candidates or for the toxicological risk assessment of environmental chemicals and their intracellular metabolites. Additionally, single cell lipidomics and metabolomics enable for the first time an in-depth understanding of the activation or inhibition of cellular biosynthesis and signalling pathways.

  18. Survival of Erwinia amylovora in mature apple fruit calyces through the viable but nonculturable (VBNC) state.

    Science.gov (United States)

    Ordax, M; Biosca, E G; Wimalajeewa, S C; López, M M; Marco-Noales, E

    2009-07-01

    Survival of Erwinia amylovora, causal agent of fire blight in pome fruits and other rosaceous plants, was monitored inside mature apples calyces under some storage conditions utilized in fruit. Apple fruit calyces inoculated with two E. amylovora strains and their respective GFP-marked strains were maintained at 26 degrees and 5 degrees C, and the effect of copper treatment was assayed at 0.01 and 0.1 mmol l(-1) CuSO4. In nontreated apples at 26 degrees C, part of the population of E. amylovora survived in the 'viable but nonculturable' (VBNC) state, whereas at 5 degrees C the majority of the population retained culturability. In copper-treated apples, the whole population adopted the VBNC state irrespective of temperature. Regardless of temperature, copper and inoculum dose, VBNC cells recovered culturability and pathogenicity in King's B broth or by host plant passage. Erwinia amylovora survived for at least 35 days in mature apple calyces. Besides, the ability of the pathogen in the VBNC state to regain culturability and pathogenicity suggests that the apple fruit could be a potential carrier of E. amylovora contributing to the spreading of fire blight disease. The risk of E. amylovora dissemination through mature fruit transport, although low, has been demonstrated, and should be considered in pest risk assessments.

  19. Intrasplenic masses of ``preserved`` functioning splenic tissue in sickle cell disease: correlation of imaging findings (CT, ultrasound, MRI, and nuclear scintigraphy)

    Energy Technology Data Exchange (ETDEWEB)

    Levin, T.L. [Department of Radiology, Columbia-Presbyterian Medical Center, Babies and Children`s Hospital of New York, 3959 Broadway, BHN 3-318, New York, NY 10032 (United States); Berdon, W.E. [Department of Radiology, Columbia-Presbyterian Medical Center, Babies and Children`s Hospital of New York, 3959 Broadway, BHN 3-318, New York, NY 10032 (United States); Haller, J.O. [Department of Radiology, SUNY Downstate Medical Center, Brooklyn, New York (United States); Ruzal-Shapiro, C. [Department of Radiology, Columbia-Presbyterian Medical Center, Babies and Children`s Hospital of New York, 3959 Broadway, BHN 3-318, New York, NY 10032 (United States); Hurlet-Jenson, A. [Department of Pediatrics, Columbia-Presbyterian Medical Center, Babies and Children`s Hospital of New York, New York (United States)

    1996-09-01

    Purpose. We studied six patients with sickle cell disease (SSD), five homozygous for sickle cell anemia and one with sickle beta-thalassemia, in whom rounded intrasplenic masses proved to be preserved functioning splenic tissue. Materials and methods. Available images including computed tomography, ultrasonography, bone scans (Tc-99m MDP), liver spleen scans (Tc-99m sulfur colloid), and MRI were evaluated. Results. The masses were low density on CT (in an otherwise calcified spleen), hypoechoic relative to the echogenic spleen on US, and had the imaging characteristics of normal spleen on MRI. They failed to accumulate Tc-99m MDP but did demonstrate uptake of Tc-99m sulfur colloid. Conclusion. In a patient with SSD and intrasplenic masses, proper correlation of multiple imaging modalities will establish the diagnosis of functioning splenic tissue and avoid mistaken diagnosis of splenic abscess or infarction. (orig.). With 2 figs., 1 tab.

  20. Body Mass Index and von Hippel-Lindau Gene Mutations in Clear-cell Renal Cancer: Results of the Netherlands Cohort Study on Diet and Cancer

    NARCIS (Netherlands)

    Smits, K.M.; Schouten, L.J.; Hudak, E.; Verhage, B.; Dijk, B.A.C. van; Hulsbergen - Kaa, C.A. van de; Goldbohm, R.A.; Oosterwijk, E.; Brandt, P.A. van den

    2010-01-01

    Purpose: Body mass index (BMI) is an important risk factor for clear-cell renal cancer (cc-RCC). A common molecular alteration in cc-RCC is loss-of-function of the von Hippel-Lindau (VHL) gene. We evaluated the association between BMI and VHL mutations in cc-RCC by using data from the Netherlands

  1. Semi-Quantitative Mass Spectrometry in AML Cells Identifies New Non-Genomic Targets of the EZH2 Methyltransferase.

    Science.gov (United States)

    Sbirkov, Yordan; Kwok, Colin; Bhamra, Amandeep; Thompson, Andrew J; Gil, Veronica; Zelent, Arthur; Petrie, Kevin

    2017-07-05

    Alterations to the gene encoding the EZH2 (KMT6A) methyltransferase, including both gain-of-function and loss-of-function, have been linked to a variety of haematological malignancies and solid tumours, suggesting a complex, context-dependent role of this methyltransferase. The successful implementation of molecularly targeted therapies against EZH2 requires a greater understanding of the potential mechanisms by which EZH2 contributes to cancer. One aspect of this effort is the mapping of EZH2 partner proteins and cellular targets. To this end we performed affinity-purification mass spectrometry in the FAB-M2 HL-60 acute myeloid leukaemia (AML) cell line before and after all-trans retinoic acid-induced differentiation. These studies identified new EZH2 interaction partners and potential non-histone substrates for EZH2-mediated methylation. Our results suggest that EZH2 is involved in the regulation of translation through interactions with a number of RNA binding proteins and by methylating key components of protein synthesis such as eEF1A1. Given that deregulated mRNA translation is a frequent feature of cancer and that eEF1A1 is highly expressed in many human tumours, these findings present new possibilities for the therapeutic targeting of EZH2 in AML.

  2. Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction.

    Science.gov (United States)

    Abelin, Jennifer G; Keskin, Derin B; Sarkizova, Siranush; Hartigan, Christina R; Zhang, Wandi; Sidney, John; Stevens, Jonathan; Lane, William; Zhang, Guang Lan; Eisenhaure, Thomas M; Clauser, Karl R; Hacohen, Nir; Rooney, Michael S; Carr, Steven A; Wu, Catherine J

    2017-02-21

    Identification of human leukocyte antigen (HLA)-bound peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is poised to provide a deep understanding of rules underlying antigen presentation. However, a key obstacle is the ambiguity that arises from the co-expression of multiple HLA alleles. Here, we have implemented a scalable mono-allelic strategy for profiling the HLA peptidome. By using cell lines expressing a single HLA allele, optimizing immunopurifications, and developing an application-specific spectral search algorithm, we identified thousands of peptides bound to 16 different HLA class I alleles. These data enabled the discovery of subdominant binding motifs and an integrative analysis quantifying the contribution of factors critical to epitope presentation, such as protein cleavage and gene expression. We trained neural-network prediction algorithms with our large dataset (>24,000 peptides) and outperformed algorithms trained on datasets of peptides with measured affinities. We thus demonstrate a strategy for systematically learning the rules of endogenous antigen presentation. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Transfection of the inner cell mass and lack of a unique DNA sequence affecting the uptake of exogenous DNA by sperm as shown by dideoxy sequencing analogues.

    Science.gov (United States)

    Cabrera, M; Chan, P J; Kalugdan, T H; King, A

    1997-02-01

    The purpose of this study was to determine whether exogenous DNA internalized into blastocysts after transference from DNA-carrier sperm are localized at the inner cell mass or trophoblast cells and to identify differences in uptake of exogenous DNA fragments by sperm due to unique DNA sequences. Mouse blastocysts at the hatching stage were exposed to migrating human sperm cells carrying exogenous DNA fragments synthesized from the E6-E7 conserved gene regions of human papillomavirus (HPV) types 16 and 18. After an interaction period of 2 hr, the transfected blastocysts were washed several times to remove extraneous sperm and the blastocysts were dissected into groups of cells derived from the inner cell mass and trophoblasts. The cells were analyzed by polymerase chain reaction (PCR) for the presence of HPV DNA fragments. In the second part of the experiment, thawed donor (N = 10) sperm cells were pooled, washed, and divided into two fractions. The first (control) fraction was added with formalin and further divided and added with a 35S-radiolabeled G, A, T, or C sequencing mixture. The second fraction was similarly treated but the formalin step was omitted from the treatment. After an hour of incubation at 37 degrees C, the sperm specimens were washed several times by centrifugation and DNA extracted by the GeneReleaser method. The extracted DNA were processed on sequence gels, and the autoradiographs analyzed. Mouse blastocysts transfected by carrier sperm with DNA from HPV types 16 and 18 showed localization of the HPV DNA to both the inner cell mass and trophoblast cells. Negative controls consisting of untreated human sperm and untreated mouse blastocysts did not reveal any evidence of HPV DNA. The positive sperm control generated expected DNA fragments from HPV types 16 and 18. In the second experiment, the intensities of the DNA fragments in the G, A, T, and C columns from low to high molecular weights were not different from the positive control bands

  4. Ciliary neurotrophic factor (CNTF) protects non-obese Swiss mice against type 2 diabetes by increasing beta cell mass and reducing insulin clearance.

    Science.gov (United States)

    Rezende, L F; Santos, G J; Santos-Silva, J C; Carneiro, E M; Boschero, A C

    2012-05-01

    Ciliary neurotrophic factor (CNTF) improves metabolic variables of obese animals with characteristics of type 2 diabetes, mainly by reducing insulin resistance. We evaluated whether CNTF was able to improve other metabolic variables in mouse models of type 2 diabetes, such as beta cell mass and insulin clearance, and whether CNTF has any effect on non-obese mice with characteristics of type 2 diabetes. Neonatal mice were treated with 0.1 mg/kg CNTF or citrate buffer via intraperitoneal injections, before injection of 250 mg/kg alloxan. HEPG2 cells were cultured for 3 days in the presence of citrate buffer, 1 nmol/l CNTF or 50 mmol/l alloxan or a combination of CNTF and alloxan. Twenty-one days after treatment, we determined body weight, epididymal fat weight, blood glucose, plasma insulin, NEFA, glucose tolerance, insulin resistance, insulin clearance and beta cell mass. Finally, we assessed insulin receptor and protein kinase B phosphorylation in peripheral organs, as well as insulin-degrading enzyme (IDE) protein production and alternative splicing in the liver and HEPG2 cells. CNTF improved insulin sensitivity and beta cell mass, while reducing glucose-stimulated insulin secretion and insulin clearance in Swiss mice, improving glucose handling in a non-obese type 2 diabetes model. This effect was associated with lower IDE production and activity in liver cells. All these effects were observed even at 21 days after CNTF treatment. CNTF protection against type 2 diabetes is partially independent of the anti-obesity actions of CNTF, requiring a reduction in insulin clearance and increased beta cell mass, besides increased insulin sensitivity. Furthermore, knowledge of the long-term effects of CNTF expands its pharmacological relevance.

  5. Long-term ketogenic diet causes glucose intolerance and reduced β- and α-cell mass but no weight loss in mice.

    Science.gov (United States)

    Ellenbroek, Johanne H; van Dijck, Laura; Töns, Hendrica A; Rabelink, Ton J; Carlotti, Françoise; Ballieux, Bart E P B; de Koning, Eelco J P

    2014-03-01

    High-fat, low-carbohydrate ketogenic diets (KD) are used for weight loss and for treatment of refractory epilepsy. Recently, short-time studies in rodents have shown that, besides their beneficial effect on body weight, KD lead to glucose intolerance and insulin resistance. However, the long-term effects on pancreatic endocrine cells are unknown. In this study we investigate the effects of long-term KD on glucose tolerance and β- and α-cell mass in mice. Despite an initial weight loss, KD did not result in weight loss after 22 wk. Plasma markers associated with dyslipidemia and inflammation (cholesterol, triglycerides, leptin, monocyte chemotactic protein-1, IL-1β, and IL-6) were increased, and KD-fed mice showed signs of hepatic steatosis after 22 wk of diet. Long-term KD resulted in glucose intolerance that was associated with insufficient insulin secretion from β-cells. After 22 wk, insulin-stimulated glucose uptake was reduced. A reduction in β-cell mass was observed in KD-fed mice together with an increased number of smaller islets. Also α-cell mass was markedly decreased, resulting in a lower α- to β-cell ratio. Our data show that long-term KD causes dyslipidemia, a proinflammatory state, signs of hepatic steatosis, glucose intolerance, and a reduction in β- and α-cell mass, but no weight loss. This indicates that long-term high-fat, low-carbohydrate KD lead to features that are also associated with the metabolic syndrome and an increased risk for type 2 diabetes in humans.

  6. Detection of viable Helicobacter pylori inside free-living amoebae in wastewater and drinking water samples from Eastern Spain.

    Science.gov (United States)

    Moreno-Mesonero, Laura; Moreno, Yolanda; Alonso, José Luis; Ferrús, M Antonia

    2017-10-01

    Helicobacter pylori is one of the most concerning emerging waterborne pathogens. It has been suggested that it could survive in water inside free-living amoebae (FLA), but nobody has studied this relationship in the environment yet. Thus, we aimed to detect viable H. pylori cells from inside FLA in water samples. Sixty-nine wastewater and 31 drinking water samples were collected. FLA were purified and identified by PCR and sequencing. For exclusively detecting H. pylori inside FLA, samples were exposed to sodium hypochlorite and assayed by specific PMA-qPCR, DVC-FISH and culture. FLA were detected in 38.7% of drinking water and 79.7% of wastewater samples, even after disinfection. In wastewater, Acanthamoeba spp. and members of the family Vahlkampfiidae were identified. In drinking water, Acanthamoeba spp. and Echinamoeba and/or Vermamoeba were present. In 39 (58.2%) FLA-positive samples, H. pylori was detected by PMA-qPCR. After DVC-FISH, 21 (31.3%) samples harboured viable H. pylori internalized cells. H. pylori was cultured from 10 wastewater samples. To our knowledge, this is the first report that demonstrates that H. pylori can survive inside FLA in drinking water and wastewater, strongly supporting the hypothesis that FLA could play an important role in the transmission of H. pylori to humans. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  7. Escherichia coli detection using mTEC agar and fluorescent antibody direct viable counting on coastal recreational water samples.

    Science.gov (United States)

    Zimmerman, A M; Rebarchik, D M; Flowers, A R; Williams, J L; Grimes, D J

    2009-10-01

    Escherichia coli is the faecal indicator species recommended by the US Environmental Protection Agency (USEPA) for monitoring fresh recreational water. Viable but nonculturable (VBNC) E. coli are living cells that are dormant and not culturable using standard microbiological cultivation methods. This study reports a comparison between the mTEC culture method recommended by USEPA for E. coli enumeration and a fluorescent antibody-direct viable count (FA-DVC) method to visualize living E. coli cells with a microscope. Escherichia coli, faecal coliforms and Enterococcus were detected using standard methods recommended by the USEPA. VBNC E. coli was visualized with FA-DVC. Results were analysed with standard statistical methods (Pearson correlation; paired-sample t-test). Significantly higher numbers of E. coli were detected using the FA-DVC method than using the mTEC method. Escherichia coli results were also compared with faecal coliform (mFC broth) and Enterococcus (mEI agar) counts in the same samples. The results of this comparative study demonstrate that E. coli can be present in higher numbers than what are detected with standard culture methods. This study re-emphasizes the need for a rapid, accurate and precise method for detecting health risks to humans who use recreational waters.

  8. Bariatric surgery: a viable treatment option for patients with severe mental illness.

    Science.gov (United States)

    Shelby, Sarah R; Labott, Susan; Stout, Rebecca A

    2015-01-01

    Although bariatric surgery has become a recognized treatment for obesity, its utility among patients with severe psychiatric disorders has not been extensively studied. A few studies have reported similar weight loss outcomes in these patients, but psychiatric status after bariatric surgery has been studied only minimally, and it is unknown if exacerbation of the mental illness affects weight loss. The aim of this study was to shed greater light on the issue of serious mental illness and bariatric surgery. Specifically, do patients with a diagnosis of schizophrenia, bipolar I, and bipolar II have poorer weight loss outcomes postbariatric surgery than the general bariatric surgery population? Also, do patients with these diagnoses experience an exacerbation of psychiatric symptoms after bariatric surgery, and if so, is the exacerbation of these disorders linked to poorer weight loss results? Midwest university medical center. A medical record review of approximately 1500 bariatric patients in a Midwest university medical center was conducted to identify those patients with diagnoses of schizophrenia, bipolar I, and bipolar II. Information was gathered on bariatric surgery outcomes and changes in psychiatric status postsurgery. Eighteen patients were identified as undergoing bariatric surgery and having a diagnosis of schizophrenia, bipolar I, or bipolar II. Weight loss in this group was significant and comparable to expected outcomes of absolute weight lost, changes in body mass index, and percentage excess weight loss for patients in the typical bariatric population. Postsurgery psychiatric status was known on 10 patients. All 10 patients experienced some exacerbation of psychiatric problems yet weight loss outcomes were still as expected. Bariatric surgery is a viable obesity treatment option for patients with schizophrenia, bipolar I, and bipolar II disorders. Symptom exacerbations occurred postsurgery, although it is not clear if these were due to the surgery or

  9. Isolation of viable human hepatic progenitors from adult livers is possible even after 48 hours of cold ischemia.

    Science.gov (United States)

    Aupet, Sophie; Simoné, Gael; Heyd, Bruno; Bachellier, Philippe; Vidal, Isabelle; Richert, Lysiane; Martin, Hélène

    2013-07-01

    Liver transplantation, utilized routinely for end-stage liver disease, has been constrained by the paucity of organ donors, and is being complemented by alternative strategies such as liver cell transplantation. One of the most promising forms of liver cell transplantation is hepatic stem cell therapies, as the number of human hepatic stem cells (hHpSCs) and other early hepatic progenitor cells (HPCs) are sufficient to provide treatment for multiple patients from a single liver source. In the present study, human adult livers were exposed to cold ischemia and then processed after numbers, albeit somewhat lower, were obtained from those exposed to 48 h of cold ischemia. The yields are similar to those reported from livers with minimal exposure to ischemia. When cultured on plastic dishes and in Kubota's Medium, a serum-free medium designed for early lineage stage HPCs, colonies of rapidly expanding cells formed. They were confirmed to be probable hHpSCs by their ability to survive and expand on plastic and in Kubota's Medium for months, by co-expression of EpCAM and neural cell adhesion molecule, minimal if any albumin expression, with EpCAM found throughout the cells, and no expression of alpha-fetoprotein. The yields of viable EpCAM(+) cells were surprisingly large, and the numbers from a single donor liver are sufficient to treat approximately 50-100 patients given the numbers of EpCAM(+) cells currently used in hepatic stem cell therapies. Thus, cold ischemic livers for up to 48 h are a new source of cells that might be used for liver cell therapies.

  10. A high expression EGFR/cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening EGFR antagonists from Rhizoma Polygoni Cuspidati

    Directory of Open Access Journals (Sweden)

    Meng Sun

    2011-08-01

    Full Text Available The epidermal growth factor receptors (EGFRs in some tumor cells are significant targets for drug discovery. In this work, we have developed an EGFR cell membrane chromatography and online high performance liquid chromatography/mass spectrometry system for screening active component from Rhizoma Polygoni Cuspidati. As a result, resveratrol from Rhizoma Polygoni Cuspidati was found to be the active component acting on EGFR like gefitinib. There was a good relationship between their inhibiting effects on EGFR secretion and HEK293 EGFR cell growth in vitro. The EGFR/CMC-online-HPLC/MS system demonstrated fast and effective characteristics for screening leading compounds from traditional Chinese medicine.

  11. Clinically Viable Gene Expression Assays with Potential for Predicting Benefit from MEK Inhibitors.

    Science.gov (United States)

    Brant, Roz; Sharpe, Alan; Liptrot, Tom; Dry, Jonathan R; Harrington, Elizabeth A; Barrett, J Carl; Whalley, Nicky; Womack, Christopher; Smith, Paul; Hodgson, Darren R

    2017-03-15

    Purpose: To develop a clinically viable gene expression assay to measure RAS/RAF/MEK/ERK (RAS-ERK) pathway output suitable for hypothesis testing in non-small cell lung cancer (NSCLC) clinical studies. Experimental Design: A published MEK functional activation signature (MEK signature) that measures RAS-ERK functional output was optimized for NSCLC in silico NanoString assays were developed for the NSCLC optimized MEK signature and the 147-gene RAS signature. First, platform transfer from Affymetrix to NanoString, and signature modulation following treatment with KRAS siRNA and MEK inhibitor, were investigated in cell lines. Second, the association of the signatures with KRAS mutation status, dynamic range, technical reproducibility, and spatial and temporal variation was investigated in NSCLC formalin-fixed paraffin-embedded tissue (FFPET) samples. Results: We observed a strong cross-platform correlation and modulation of signatures in vitro Technical and biological replicates showed consistent signature scores that were robust to variation in input total RNA; conservation of scores between primary and metastatic tumor was statistically significant. There were statistically significant associations between high MEK ( P = 0.028) and RAS ( P = 0.003) signature scores and KRAS mutation in 50 NSCLC samples. The signatures identify overlapping but distinct candidate patient populations from each other and from KRAS mutation testing. Conclusions: We developed a technically and biologically robust NanoString gene expression assay of MEK pathway output, compatible with the quantities of FFPET routinely available. The gene signatures identified a different patient population for MEK inhibitor treatment compared with KRAS mutation testing. The predictive power of the MEK signature should be studied further in clinical trials. Clin Cancer Res; 23(6); 1471-80. ©2016 AACR See related commentary by Xue and Lito, p. 1365 . ©2016 American Association for Cancer Research.

  12. Immediate natural tooth pontic: A viable yet temporary prosthetic solution: A patient reported outcome

    Directory of Open Access Journals (Sweden)

    Sudhir Bhandari

    2012-01-01

    Conclusion: The concept of immediate pontic placement is surely a viable treatment option and promises an excellent transient esthetic solution for a lost tooth as well as enables good preparation of the extraction site for future prosthetic replacement.

  13. Improved identification of viable myocardium using second harmonic imaging during dobutamine stress echocardiography

    NARCIS (Netherlands)

    F. Sozzi (Fabiola); D. Poldermans (Don); J.J. Bax (Jeroen); A. Elhendy (Abdou); E.C. Vourvouri (Eleni); R. Valkema (Roelf); J. de Sutter; A.F.L. Schinkel (Arend); A. Borghetti; J.R.T.C. Roelandt (Jos)

    2001-01-01

    textabstractOBJECTIVE: To determine whether, compared with fundamental imaging, second harmonic imaging can improve the accuracy of dobutamine stress echocardiography for identifying viable myocardium, using nuclear imaging as a reference. PATIENTS: 30 patients with chronic left

  14. Marine environmental pollution stress detection through direct viable counts of bacteria

    Digital Repository Service at National Institute of Oceanography (India)

    Ramaiah, N.; Kenkre, V.D.; Verlecar, X.N.

    Direct viable counts (DVC) of bacteria were quantified from polluted and relatively less/non-polluted coastal locations during different seasons to assess whether they can be routinely monitored for an understanding of environmental stress(es...

  15. Cigarette smoke-induced effects on bone marrow B-cell subsets and CD4+:CD8+ T-cell ratios are reversed by smoking cessation: influence of bone mass on immune cell response to and recovery from smoke exposure.

    Science.gov (United States)

    Fusby, Jenny S; Kassmeier, Michele D; Palmer, Victoria L; Perry, Greg A; Anderson, Dirk K; Hackfort, Bryan T; Alvarez, Gwen K; Cullen, Diane M; Akhter, Mohammed P; Swanson, Patrick C

    2010-08-01

    Cigarette smoking adversely affects the immune system, and is a risk factor for developing osteoporosis. How smoking contributes to osteoporosis is unclear, but since lymphocytes help maintain bone homeostasis and lymphocyte depletion results in bone loss, one potential mechanism for how smoke exposure promotes osteoporosis is by reducing bone marrow lymphocytes. Since the risk for developing osteoporosis is reportedly greater in smokers with polymorphisms in LRP5, a gene involved in canonical Wnt signaling that regulates bone metabolism, smoking-induced effects on lymphocytes may be influenced by Lrp5 functionality. To test these possibilities, we examined how the duration and cessation of cigarette smoke exposure affects lymphocyte distribution and function in normal mice and mice predisposed to low or high bone mass due to disruption or mutation of Lrp5. We find that, independent of genotype, mice exposed to cigarette smoke for 3-12 weeks showed a significant reduction in bone marrow B220(+)CD43(-) B cells and splenic transitional T1 B cells, and exhibited a splenic CD4(+):CD8(+) T-cell ratio that was skewed toward CD8(+) T cells. Smoke exposure had little or no effect on other lymphocyte subsets or on lymphocyte function ex vivo. Interestingly, these differences were no longer apparent after 6 weeks without smoke exposure, except in mice with high bone mass where bone marrow B220(+)CD43(-) B cells failed to fully recover. These data provide the first evidence that smoke exposure reduces bone marrow B cells, providing a plausible mechanism for how smoking contributes to osteoporosis.

  16. Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification

    Science.gov (United States)

    Illien, Françoise; Rodriguez, Nicolas; Amoura, Mehdi; Joliot, Alain; Pallerla, Manjula; Cribier, Sophie; Burlina, Fabienne; Sagan, Sandrine

    2016-11-01

    The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.

  17. Immunization of rodents against Hymenolepis infections using non-viable homologous oncospheres.

    Science.gov (United States)

    Fan, Ping-Chin; Chung, Wen-Cheng; Ito, Akira

    2004-12-01

    Immunity to Taiwan Taenia infection in pigs can be stimulated using homologous or heterologous non-viable Taenia oncospheres. This study was designed to determine whether homologous non-viable oncospheres could stimulate immunity to Hymenolepis infection in rodents. Hatched oncospheres were prepared from eggs of Hymenolepis diminuta, Hymenolepis nana, and Hymenolepis microstoma and kept at -70 degrees C for more than 1 month. A mixture of 500 non-viable oncospheres of each tapeworm and complete Freund's adjuvant was injected subcutaneously in four groups of Sprague-Dawley rats or ICR mice one to four times at an interval of 1 week; controls were not immunized. After immunization, each rodent was orally inoculated with three fresh active cysticercoids of H. diminuta or H. microstoma or 500 fresh eggs of H. nana. The animals were then necropsied for adult tapeworms. No rats or mice immunized with non-viable oncospheres of H. diminuta or H. nana were infected by the challenge inoculation. However, 28 of 34 mice immunized with non-viable H. microstoma oncospheres were infected after inoculation with cysticercoids. This study demonstrated complete protection against infection by homologous parasites in rats or mice immunized with non-viable oncospheres of H. diminuta and H. nana, respectively. Repeated immunization may not be required if resistance is stimulated in rodent hosts.

  18. [Viable myocardium detecting by CARTO voltage mapping in swine model of acute myocardial infarction].

    Science.gov (United States)

    Lin, Tao; Ma, Yi-Tong; Yang, Yi-Ning; Mu, Hu-Yati; He, Peng-Yi; Yang, Yu-Chun; Chou, Ping; Liu, Fen; Zhang, Yan-Yi

    2010-08-01

    To evaluate the accuracy and practicability of detecting viable myocardium by CARTO voltage mapping in swine model of acute myocardial infarction (MI). MI was induced in 13 anesthetized swines via occluding the distal of left anterior descending coronary arteries by angioplasty balloon for 60-90 minutes. The viable myocardium detection by CARTO voltage mapping was made after reconstruction of the left ventricle using CARTO and the results were compared with TTC staining. The standard of CARTO voltage to detect viable myocardium was 0.5 - 1.5 mV while viable myocardium showed pink color by TTC staining. Eleven out of 13 swines survived the operation and 2 swines died of ventricular fibrillation at 45 and 65 minutes post ischemia. Left ventricle was divided into 16 segments and 176 segments from 11 swines were analyzed. Viable myocardium detected by CARTO voltage mapping was identical as identified by TTC staining (Kappa = 0.816, P < 0.001). Taken the TTC result as standard, the sensitivity, specificity and accuracy rate of CARTO voltage mapping are 71.8%, 96.5% and 90.9% respectively. CARTO voltage mapping could be used as a reliable tool to detect viable myocardium in this model.

  19. ELISA microplate: a viable immunocapture platform over magnetic beads for immunoaffinity-LC-MS/MS quantitation of protein therapeutics?

    Science.gov (United States)

    Yang, Wenchu; Kernstock, Robert; Simmons, Neal; Alak, Ala

    2015-01-01

    Evaluate the performance of ELISA microplates versus commonly used magnetic beads for biological sample cleanup and/or enrichment in immunoaffinity-LC-MS/MS to reduce tedious beads washing procedures and a relatively high assay cost. ELISA microplates were used as immunicapture platform and compared with magnetic beads for sample cleanup for LC-MS/MS quantitation of protein therapeutics. One unmodified and two surface-activated microplates provided comparable linear ranges and sensitivities for a therapeutic protein (mass 78 kDa) using a human serum sample of 100 µl with 1:1 dilution compared with Tosylactivated magnetic beads using 200 µl of human serum without sample dilution. The assays' precision and accuracy were all within acceptable ranges. No nonspecific binding or other selectivity issues were observed. The results suggested an ELISA microplate could be a viable immunocapture platform for immunoaffinity-LC-MS/MS quantitation of protein therapeutics.

  20. A mass-conservative adaptive FAS multigrid solver for cell-centered finite difference methods on block-structured, locally-cartesian grids

    Science.gov (United States)

    Feng, Wenqiang; Guo, Zhenlin; Lowengrub, John S.; Wise, Steven M.

    2018-01-01

    We present a mass-conservative full approximation storage (FAS) multigrid solver for cell-centered finite difference methods on block-structured, locally cartesian grids. The algorithm is essentially a standard adaptive FAS (AFAS) scheme, but with a simple modification that comes in the form of a mass-conservative correction to the coarse-level force. This correction is facilitated by the creation of a zombie variable, analogous to a ghost variable, but defined on the coarse grid and lying under the fine grid refinement patch. We show that a number of different types of fine-level ghost cell interpolation strategies could be used in our framework, including low-order linear interpolation. In our approach, the smoother, prolongation, and restriction operations need never be aware of the mass conservation conditions at the coarse-fine interface. To maintain global mass conservation, we need only modify the usual FAS algorithm by correcting the coarse-level force function at points adjacent to the coarse-fine interface. We demonstrate through simulations that the solver converges geometrically, at a rate that is h-independent, and we show the generality of the solver, applying it to several nonlinear, time-dependent, and multi-dimensional problems. In several tests, we show that second-order asymptotic (h → 0) convergence is observed for the discretizations, provided that (1) at least linear interpolation of the ghost variables is employed, and (2) the mass conservation corrections are applied to the coarse-level force term.

  1. Stable quasi-solid-state dye-sensitized solar cell using ionic gel electrolyte with low molecular mass organogelator

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Li [Key Laboratory of Novel Thin Film Solar Cells, Division of Solar Energy Materials and Engineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); Huo, Zhipeng, E-mail: zhipenghuo@163.com [Key Laboratory of Novel Thin Film Solar Cells, Division of Solar Energy Materials and Engineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); Dai, Songyuan, E-mail: sydai@ncepu.edu.cn [Key Laboratory of Novel Thin Film Solar Cells, Division of Solar Energy Materials and Engineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); Beijing Key Lab of Novel Thin Film Solar Cells, North China Electric Power University, Beijing 102206 (China); Zhu, Jun; Zhang, Changneng; Pan, Xu; Huang, Yang [Key Laboratory of Novel Thin Film Solar Cells, Division of Solar Energy Materials and Engineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); Yang, Shangfeng [Hefei National Laboratory for Physical Sciences at Microscale, Department of Materials Science and Engineering, University of Science and Technology of China (USTC), Hefei 230026 (China); Zhang, Bing; Yao, Jianxi [Beijing Key Lab of Novel Thin Film Solar Cells, North China Electric Power University, Beijing 102206 (China)

    2015-02-15

    Long-term stability is essential for the application and commercialization of dye-sensitized solar