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Sample records for affect cellular viability

  1. Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production.

    PubMed

    Vistica, D T; Skehan, P; Scudiero, D; Monks, A; Pittman, A; Boyd, M R

    1991-05-15

    The hydrogen acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) is commonly utilized to estimate cellular viability in drug screening protocols. The present investigation was prompted, in part, by observations that reduction of MTT to its colored reaction product, MTT formazan, varied between cell lines and with culture age. A correlation was established between the D-glucose concentration of the culture medium at the time of assay and the production of MTT formazan for cell lines representing seven tumor histologies. A decrease in the concentration of D-glucose from culture medium was accompanied by a decrease in MTT specific activity (MTT formazan/microgram cell protein) for a number of cell lines. Cells which extensively metabolized D-glucose exhibited the greatest reduction in MTT specific activity. Further evidence that the D-glucose concentration of the culture medium played an important role in MTT reduction was provided by experiments which demonstrated that transfer of cells to a glucose-free medium (L-15) was accompanied by an immediate decrease in MTT reduction which was pH independent. These studies suggested that cellular transport and constant metabolism of glucose were required for maximum MTT reduction. Decreases in the cellular concentration of the reduced pyridine nucleotides NADH and NADPH were accompanied by concomitant decreases in MTT formazan production. MTT formazan varied significantly among cell lines in both the kinetics of its formation and the degree of saturability exhibited. Apparent IC50 values for Adriamycin varied, in a cell line-specific manner, with MTT exposure time. These results indicate that MTT specific activity is significantly influenced by a number of parameters and suggest that assay conditions should be established which minimize their effects.

  2. A cellular viability assay to monitor drug toxicity.

    PubMed

    Hansen, Jakob; Bross, Peter

    2010-01-01

    A central part of the research in protein misfolding and its associated disorders is the development of treatment strategies based on ensuring cellular protein homeostasis. This often includes testing chemical substances or drugs for their ability to counteract protein misfolding processes and to promote correct folding. Such investigations also include assessment of how the tested chemical substances affect cellular viability, that is, their cytotoxic effect. Investigations of cytotoxicity often require testing several different concentrations and drug exposure times using cells in culture. It is therefore attractive to use a viability test that permits the analysis of many samples with little handling time. This protocol describes a simple and fast methodology to analyze viability of lymphoblastoid cells and to test putative cytotoxic effects associated with exposure to a chemical substance, here exemplified by celastrol. The natural substance celastrol has been used for many years in traditional Chinese medicine and has subsequently been shown to induce transcription of genes encoding molecular chaperones (heat shock proteins) that are involved in promoting folding of cellular proteins. The well-described colorimetric tetrazolium salt (MTT) assay, which monitors metabolic activity of cultured cells, was adapted to analyze the viability of cells exposed to celastrol. After having established a suitable cell seeding density, the dose-dependence and time-course of viability reduction of lymphoblastoid cells treated with celastrol were determined. It was found that 4- and 24-h exposure to 0.8 microM celastrol reduced the viability of lymphoblastoid cells, with the most severe effect observed at 24 h with MTT reductions approaching 30% of non-exposed cells. For a series of incubations for 24 h, it was found that concentrations as low as 0.2 microM were sufficient to affect the viability, and celastrol concentrations of 0.5 microM reduced the MTT reduction rate to

  3. 3-bromopyruvate inhibits glycolysis, depletes cellular glutathione, and compromises the viability of cultured primary rat astrocytes.

    PubMed

    Ehrke, Eric; Arend, Christian; Dringen, Ralf

    2015-07-01

    The pyruvate analogue 3-bromopyruvate (3-BP) is an electrophilic alkylator that is considered a promising anticancer drug because it has been shown to kill cancer cells efficiently while having little toxic effect on nontumor cells. To test for potential adverse effects of 3-BP on brain cells, we exposed cultured primary rat astrocytes to 3-BP and investigated the effects of this compound on cell viability, glucose metabolism, and glutathione (GSH) content. The presence of 3-BP severely compromised cell viability and slowed cellular glucose consumption and lactate production in a time- and concentration-dependent manner, with half-maximal effects observed at about 100 µM 3-BP after 4 hr of incubation. The cellular hexokinase activity was not affected in 3-BP-treated astrocytes, whereas within 30 min after application of 3-BP the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inhibited, and cellular GSH content was depleted in a concentration-dependent manner, with half-maximal effects observed at about 30 µM 3-BP. The depletion of cellular GSH after exposure to 100 µM 3-BP was not prevented by the presence of 10 mM of the monocarboxylates lactate or pyruvate, suggesting that 3-BP is not taken up into astrocytes predominantly by monocarboxylate transporters. The data suggest that inhibition of glycolysis by inactivation of GAPDH and GSH depletion contributes to the toxicity that was observed for 3-BP-treated cultured astrocytes.

  4. A versatile assay for the accurate, time-resolved determination of cellular viability.

    PubMed

    Amano, Toyoki; Hirasawa, Ken ichi; O'Donohue, Michael J; Pernolle, Jean Claude; Shioi, Yuzo

    2003-03-01

    A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5 x 10(5) to 2.0 x 10(5) cells displayed a linear response when FDA concentrations less than 12 micro M were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (V(max)) and the LD50 (half-maximal velocity or k(1/2)) were calculated as 17.2 (% death/h) and 65 nM, respectively.

  5. Silver nanoparticle toxicity effect on growth and cellular viability of the aquatic plant Lemna gibba.

    PubMed

    Oukarroum, Abdallah; Barhoumi, Lotfi; Pirastru, Laura; Dewez, David

    2013-04-01

    The toxicity effect of silver nanoparticles (AgNPs) on growth and cellular viability was investigated on the aquatic plant Lemna gibba exposed over 7 d to 0, 0.01, 0.1, 1, and 10 mg/L of AgNPs. Growth inhibition was demonstrated by a significant decrease of frond numbers dependent on AgNP concentration. Under these conditions, reduction in plant cellular viability was detected for 0.1, 1, and 10 mg/L of AgNPs within 7 d of AgNPs treatment. This effect was highly correlated with the production of intracellular reactive oxygen species (ROS). A significant increase of intracellular ROS formation was triggered by 1 and 10 mg/L of AgNP exposure. The induced oxidative stress was related to Ag accumulation within L. gibba plant cells and with the increasing concentration of AgNP exposure in the medium. The authors' results clearly suggested that AgNP suspension represented a potential source of toxicity for L. gibba plant cells. Due to the low release capacity of free soluble Ag from AgNP dissolution in the medium, it is most likely that the intracellular uptake of Ag was directly from AgNPs, triggering cellular oxidative stress that may be due to the release of free Ag inside plant cells. Therefore, the present study demonstrated that AgNP accumulation in an aquatic environment may represent a potential source of toxicity and a risk for the viability of duckweeds.

  6. Drying process strongly affects probiotics viability and functionalities.

    PubMed

    Iaconelli, Cyril; Lemetais, Guillaume; Kechaou, Noura; Chain, Florian; Bermúdez-Humarán, Luis G; Langella, Philippe; Gervais, Patrick; Beney, Laurent

    2015-11-20

    Probiotic formulations are widely used and are proposed to have a variety of beneficial effects, depending on the probiotic strains present in the product. The impact of drying processes on the viability of probiotics is well documented. However, the impact of these processes on probiotics functionality remains unclear. In this work, we investigated variations in seven different bacterial markers after various desiccation processes. Markers were composed of four different viability evaluation (combining two growth abilities and two cytometric measurements) and in three in vitro functionalities: stimulation of IL-10 and IL-12 production by PBMCs (immunomodulation) and bacterial adhesion to hexadecane. We measured the impact of three drying processes (air-drying, freeze-drying and spray-drying), without the use of protective agents, on three types of probiotic bacteria: Bifidobacterium bifidum, Lactobacillus plantarum and Lactobacillus zeae. Our results show that the bacteria respond differently to the three different drying processes, in terms of viability and functionality. Drying methods produce important variations in bacterial immunomodulation and hydrophobicity, which are correlated. We also show that adherence can be stimulated (air-drying) or inhibited (spray-drying) by drying processes. Results of a multivariate analysis show no direct correlation between bacterial survival and functionality, but do show a correlation between probiotic responses to desiccation-rewetting and the process used to dry the bacteria.

  7. CP27 affects viability, proliferation, attachment and gene expression in embryonic fibroblasts.

    PubMed

    Luan, X; Diekwisch, T G H

    2002-08-01

    CP27 is a gene that has been cloned from an E11 early embryonic library and has been suggested to mediate early organogenesis (Diekwisch et al., 1999, Gene 235, 19). We have hypothesized that CP27 exhibits its effects on organogenesis by affecting individual cell function. Based on the CP27 expression pattern we have selected the CP27 expressing embryonic fibroblast cell line BALB/c 3T3 to determine the effects of CP27 on cell function. CP27 loss of function strategies were performed by adding 5, 12.5 or 25 micro g/ml anti-CP27 antibody to cultured BALB/c 3T3 cells and comparing the results to controls in which identical concentrations of rabbit serum were added to the culture medium. Other controls included an antibody against another extracellular matrix protein amelogenin (negative control) and anti-CP27 antibodies directed against other areas of the CP27 molecule (positive control). Following cell culture, cell viability, apoptosis, cell proliferation, cell shape, cellular attachment and fibronectin matrix production were assayed using MTT colourimetric assay, BrdU staining, morphometry, immunostaining and western blot analysis. Block of CP27 function using an antibody strategy resulted in the following significant changes: (i) reduced viability, (ii) increased number of apoptotic cells, (iii) reduced proliferation, (iv) alterations in cell shape, (v) loss of attachment, and (vi) reduction in fibronectin matrix production. There was also a redistribution in fibronectin matrix organization demonstrated by immunohistochemistry. We conclude that CP27 plays an important role in the maintance of normal cell function and that CP27 block leads to significant changes in cellular behaviour.

  8. Dominance of mutations affecting viability in Drosophila melanogaster.

    PubMed Central

    Fry, James D; Nuzhdin, Sergey V

    2003-01-01

    There have been several attempts to estimate the average dominance (ratio of heterozygous to homozygous effects) of spontaneous deleterious mutations in Drosophila melanogaster, but these have given inconsistent results. We investigated whether transposable element (TE) insertions have higher average dominance for egg-to-adult viability than do point mutations, a possibility suggested by the types of fitness-depressing effects that TEs are believed to have. If so, then variation in dominance estimates among strains and crosses would be expected as a consequence of variation in TE activity. As a first test, we estimated the average dominance of all mutations and of copia insertions in a set of lines that had accumulated spontaneous mutations for 33 generations. A traditional regression method gave a dominance estimate for all mutations of 0.17, whereas average dominance of copia insertions was 0.51; the difference between these two estimates approached significance (P = 0.08). As a second test, we reanalyzed Ohnishi 1974 data on dominance of spontaneous and EMS-induced mutations. Because a considerable fraction of spontaneous mutations are caused by TE insertions, whereas EMS induces mainly point mutations, we predicted that average dominance would decline with increasing EMS concentration. This pattern was observed, but again fell short of formal significance (P = 0.07). Taken together, however, the two results give modest support for the hypothesis that TE insertions have greater average dominance in their viability effects than do point mutations, possibly as a result of deleterious effects of expression of TE-encoded genes. PMID:12702680

  9. FAK and HAS Inhibition Synergistically Decrease Colon Cancer Cell Viability and Affect Expression of Critical Genes

    PubMed Central

    Heffler, Melissa; Golubovskaya, Vita; Conroy, Jeffrey; Liu, Song; Wang, Dan; Cance, William; Dunn, Kelli B.

    2013-01-01

    Focal adhesion kinase (FAK), hyaluronan (HA), and hyaluronan synthase-3 (HAS3) have been implicated in cancer growth and progression. FAK inhibition with the small molecule inhibitor Y15 decreases colon cancer cell growth in vitro and in vivo. HAS3 inhibition in colon cancer cells decreases FAK expression and activation, and exogenous HA increases FAK activation. We sought to determine the genes affected by HAS and FAK inhibition and hypothesized that dual inhibition would synergistically inhibit viability. Y15 (FAK inhibitor) and the HAS inhibitor 4-methylumbelliferone (4-MU) decreased viability in a dose dependent manner; viability was further inhibited by treatment with Y15 and 4-MU in colon cancer cells. HAS inhibited cells treated with 2μM of Y15 showed significantly decreased viability compared to HAS scrambled cells treated with the same dose (p<0.05) demonstrating synergistic inhibition of viability with dual FAK/HAS inhibition. Microarray analysis showed more than 2-fold up- or down-regulation of 121 genes by HAS inhibition, and 696 genes by FAK inhibition (p<0.05) and revealed 29 common genes affected by both signaling. Among the genes affected by FAK or HAS3 inhibition were genes, playing role in apoptosis, cell cycle regulation, adhesion, transcription, heat-shock and WNT pathways. Thus, FAK or HAS inhibition decreases SW620 viability and affects several similar genes, which are involved in the regulation of tumor survival. Dual inhibition of FAK and HAS3 decreases viability to a greater degree than with either agent alone, and suggests that synergistic inhibition of colon cancer cell growth can result from affecting similar genetic pathways. PMID:22934709

  10. FAK and HAS inhibition synergistically decrease colon cancer cell viability and affect expression of critical genes.

    PubMed

    Heffler, Melissa; Golubovskaya, Vita M; Conroy, Jeffrey; Liu, Song; Wang, Dan; Cance, William G; Dunn, Kelli B

    2013-05-01

    Focal adhesion kinase (FAK), hyaluronan (HA), and hyaluronan synthase-3 (HAS3) have been implicated in cancer growth and progression. FAK inhibition with the small molecule inhibitor Y15 decreases colon cancer cell growth in vitro and in vivo. HAS3 inhibition in colon cancer cells decreases FAK expression and activation, and exogenous HA increases FAK activation. We sought to determine the genes affected by HAS and FAK inhibition and hypothesized that dual inhibition would synergistically inhibit viability. Y15 (FAK inhibitor) and the HAS inhibitor 4-methylumbelliferone (4-MU) decreased viability in a dose dependent manner; viability was further inhibited by treatment with Y15 and 4-MU in colon cancer cells. HAS inhibited cells treated with 2 μM of Y15 showed significantly decreased viability compared to HAS scrambled cells treated with the same dose (p < 0.05) demonstrating synergistic inhibition of viability with dual FAK/HAS inhibition. Microarray analysis showed more than 2-fold up- or down-regulation of 121 genes by HAS inhibition, and 696 genes by FAK inhibition (p < 0.05) and revealed 29 common genes affected by both signaling. Among the genes affected by FAK or HAS3 inhibition were genes, playing role in apoptosis, cell cycle regulation, adhesion, transcription, heatshock and WNT pathways. Thus, FAK or HAS inhibition decreases SW620 viability and affects several similar genes, which are involved in the regulation of tumor survival. Dual inhibition of FAK and HAS3 decreases viability to a greater degree than with either agent alone, and suggests that synergistic inhibition of colon cancer cell growth can result from affecting similar genetic pathways.

  11. Water-triacylglycerol interactions affect oil body structure and seed viability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are investigating interactions between water and triacylglycerols (TAG) that appear to affect oil body stability and viability of seeds. Dried seeds are usually stored at freezer temperatures (-20oC) for long-term conservation of genetic resources. This globally accepted genebanking practice is...

  12. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

    PubMed

    Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

    2015-08-21

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

  13. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

    PubMed Central

    Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

    2015-01-01

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

  14. A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations.

    PubMed

    Ichii, Hirohito; Inverardi, Luca; Pileggi, Antonello; Molano, R Damaris; Cabrera, Over; Caicedo, Alejandro; Messinger, Shari; Kuroda, Yoshikazu; Berggren, Per-Olof; Ricordi, Camillo

    2005-07-01

    Current methodologies to evaluate islet cell viability are largely based on tests that assess the exclusion of DNA-binding dyes. While these tests identify cells that have lost selective membrane permeability, they do not allow us to recognize apoptotic cells, which do not yet stain with DNA-binding dyes. Furthermore, current methods of analysis do not discriminate between cell subsets in the preparation and, in particular, they do not allow for selectively defining beta-cell viability. For these reasons we have developed novel methods for the specific assessment of beta-cell content and viability in human islets based on cellular composition analysis through laser scanning cytometry (LSC) coupled with identification of beta-cell-specific apoptosis at the mitochondrial level. Our novel analytical methods hold promise to prospectively analyze clinical islet transplantation preparations and predict functional performance, as suggested by the observed correlation with in vivo analysis of islet potency in immunodeficient rodents.

  15. Yeast mutant affected for viability upon nutrient starvation: characterization and cloning of the RVS161 gene.

    PubMed

    Crouzet, M; Urdaci, M; Dulau, L; Aigle, M

    1991-10-01

    In yeast, nutrient starvation leads to entry into stationary phase. Mutants that do not respond properly to starvation conditions have been isolated in Saccharomyces cerevisiae. Among them the rvs161 mutant (RVS for Reduced Viability upon Starvation) is sensitive to carbon, nitrogen and sulphur starvation. When these nutrients are depleted in the medium, mutant cells show cellular viability loss with morphological changes. The mutation rvs161-1 is very pleiotropic, and besides the defects in stationary phase entry, the mutant strain presents other alterations: sensitivity to high salt concentrations, hypersensitivity to amino acid analogs, no growth on lactate or acetate medium. The addition of salts or amino acid analogs leads to the same morphological defects observed in starved cells, suggesting that the gene could be implicated mainly in the control of cellular viability. The gene RVS161 was cloned; it codes for a 30,252 daltons protein. No homology was detected with the proteins contained in the databases. Moreover, Southern analysis revealed the presence of other sequences homologous to the RVS161 gene in the yeast genome.

  16. The influence of size and charge of chitosan/polyglutamic acid hollow spheres on cellular internalization, viability and blood compatibility.

    PubMed

    Dash, Biraja C; Réthoré, Gildas; Monaghan, Michael; Fitzgerald, Kathleen; Gallagher, William; Pandit, Abhay

    2010-11-01

    Polymeric hollow spheres can be tailored as efficient carriers of various therapeutic molecules due to their tunable properties. However, the entry of these synthetic vehicles into cells, their cell viability and blood compatibility depend on their physical and chemical properties e.g. size, surface charge. Herein, we report the effect of size and surface charge on cell viability and cellular internalization behaviour and their effect on various blood components using chitosan/polyglutamic acid hollow spheres as a model system. Negatively charged chitosan/polyglutamic acid hollow spheres of various sizes 100, 300, 500 and 1000 nm were fabricated using a template based method and covalently surface modified using linear polyethylene glycol and methoxyethanol amine to create a gradient of surface charge from negative to neutrally charged spheres respectively. The results here suggest that both size and surface charge have a significant influence on the sphere's behaviour, most prominently on haemolysis, platelet activation, plasma recalcification time, cell viability and internalization over time. Additionally, cellular internalization behaviour and viability was found to vary with different cell types. These results are in agreement with those of inorganic spheres and liposomes, and can serve as guidelines for tailoring polymeric solid spheres for specific desired applications in biological and pharmaceutical fields, including the design of nanometer to submicron-sized delivery vehicles.

  17. The Interaction between Selection, Demography and Selfing and How It Affects Population Viability

    PubMed Central

    Awad, Diala Abu; Gallina, Sophie; Bonamy, Cyrille; Billiard, Sylvain

    2014-01-01

    Population extinction due to the accumulation of deleterious mutations has only been considered to occur at small population sizes, large sexual populations being expected to efficiently purge these mutations. However, little is known about how the mutation load generated by segregating mutations affects population size and, eventually, population extinction. We propose a simple analytical model that takes into account both the demographic and genetic evolution of populations, linking population size, density dependence, the mutation load, and self-fertilisation. Analytical predictions were found to be relatively good predictors of population size and probability of population viability when verified using an explicit individual based stochastic model. We show that initially large populations do not always reach mutation-selection balance and can go extinct due to the accumulation of segregating deleterious mutations. Population survival depends not only on the relative fitness and demographic stochasticity, but also on the interaction between the two. When deleterious mutations are recessive, self-fertilisation affects viability non-monotonically and genomic cold-spots could favour the viability of outcrossing populations. PMID:24465911

  18. The quality of sperm preparation medium affects the motility, viability, and DNA integrity of human spermatozoa

    PubMed Central

    Anbari, Fatemeh; Halvaei, Iman; Nabi, Ali; Ghazali, Shahin; Khalili, Mohammad Ali; Johansson, Lars

    2016-01-01

    AIM: The goal was to compare the effects of three different sperm preparation media on sperm motility, viability, and DNA integrity of semen samples from normozoospermic men. METHODS: A total of 15 normozoospermic males were included in the study. The semen analysis (SA) was performed in accordance with the WHO guidelines (2010). After SA, each sample was divided into three aliquots, and swim-up was performed with three different sperm preparation media (Sperm Preparation Media, Origio, Denmark; Ham's F10, Biochrome, Berlin, Germany; and VitaSperm™, Innovative Biotech, Iran). Sperm motility, viability, and DNA fragmentation were evaluated at 0, 1, 2, and 24 h after swim-up. RESULTS: There were no significant differences, at any time intervals, in the total sperm motility between the different sperm preparation media. However, the rate of progressive motility was significantly higher in spermatozoa prepared using the media from Origio in comparison with VitaSperm™ (P = 0.03), whereas no significant difference was found against Ham's F10 medium. No significant differences in sperm viability were seen between the media products. However, 1 h after swim-up, the extent of sperm DNA fragmentation was lower in the medium from Origio versus VitaSperm™ (P = 0.02). CONCLUSIONS: The data showed that the quality of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoa in assisted conception programs. PMID:28216914

  19. Assessment of cellular viability on calcium sulphate/hydroxyapatite injectable scaffolds

    PubMed Central

    Naudi, Kurt; Dalby, Matthew J; Tanner, K Elizabeth; McMahon, Jeremy D; Ayoub, Ashraf

    2013-01-01

    Cements for maxillofacial reconstruction of jaw defects through calcification of rotated muscle have been tested. The objective of this study was to investigate the visibility of loading of two types of commercially available cements, Cerament™ Spine Support and Cerament Bone Void Filler with mesenchymal cells and cytokines (bone morphogenetic protein) to act as a biomimetic scaffolding for future clinical application. Determination of basic biocompatibility (cell viability) using methyl thiazolyl tetrazolium and live/dead assay was carried out using MG-63 cells at various time points. Next, in order to inform potential subsequent in vivo experiments, a collagen tissue mimic was used for characterization of rabbit mesenchymal stromal cells using immunofluorescent cytoskeleton staining, and simultaneous and then sequential injection of Cerament Spine Support cement and cells into collagen gels. Results indicated that Cerament Spine Support was more biocompatible and that sequential injection of cement and then rabbit mesenchymal stromal cells into the tissue mimics is an optimal approach for clinical applications. PMID:24555009

  20. Global Deletion of TSPO Does Not Affect the Viability and Gene Expression Profile

    PubMed Central

    Wang, Huaishan; Yang, Jia; Yang, Qi; Fu, Yi; Hu, Yu; Liu, Fang; Wang, Weiqing; Cui, Lianxian; Chen, Hui; Zhang, Jianmin; He, Wei

    2016-01-01

    Translocator Protein (18kDa, TSPO) is a mitochondrial outer membrane transmembrane protein. Its expression is elevated during inflammation and injury. However, the function of TSPO in vivo is still controversial. Here, we constructed a TSPO global knockout (KO) mouse with a Cre-LoxP system that abolished TSPO protein expression in all tissues and showed normal phenotypes in the physiological condition. The birth rates of TSPO heterozygote (Het) x Het or KO x KO breeding were consistent with Mendel’s Law, suggesting a normal viability of TSPO KO mice at birth. RNA-seq analysis showed no significant difference in the gene expression profile of lung tissues from TSPO KO mice compared with wild type mice, including the genes associated with bronchial alveoli immune homeostasis. The alveolar macrophage population was not affected by TSPO deletion in the physiological condition. Our findings contradict the results of Papadopoulos, but confirmed Selvaraj’s findings. This study confirms TSPO deficiency does not affect viability and bronchial alveolar immune homeostasis. PMID:27907096

  1. Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays.

    PubMed

    Rampersad, Sephra N

    2012-01-01

    Accurate prediction of the adverse effects of test compounds on living systems, detection of toxic thresholds, and expansion of experimental data sets to include multiple toxicity end-point analysis are required for any robust screening regime. Alamar Blue is an important redox indicator that is used to evaluate metabolic function and cellular health. The Alamar Blue bioassay has been utilized over the past 50 years to assess cell viability and cytotoxicity in a range of biological and environmental systems and in a number of cell types including bacteria, yeast, fungi, protozoa and cultured mammalian and piscine cells. It offers several advantages over other metabolic indicators and other cytotoxicity assays. However, as with any bioassay, suitability must be determined for each application and cell model. This review seeks to highlight many of the important considerations involved in assay use and design in addition to the potential pitfalls.

  2. Assessing the viability of bacterial species in drinking water by combined cellular and molecular analyses.

    PubMed

    Kahlisch, Leila; Henne, Karsten; Gröbe, Lothar; Brettar, Ingrid; Höfle, Manfred G

    2012-02-01

    The question which bacterial species are present in water and if they are viable is essential for drinking water safety but also of general relevance in aquatic ecology. To approach this question we combined propidium iodide/SYTO9 staining ("live/dead staining" indicating membrane integrity), fluorescence-activated cell sorting (FACS) and community fingerprinting for the analysis of a set of tap water samples. Live/dead staining revealed that about half of the bacteria in the tap water had intact membranes. Molecular analysis using 16S rRNA and 16S rRNA gene-based single-strand conformation polymorphism (SSCP) fingerprints and sequencing of drinking water bacteria before and after FACS sorting revealed: (1) the DNA- and RNA-based overall community structure differed substantially, (2) the community retrieved from RNA and DNA reflected different bacterial species, classified as 53 phylotypes (with only two common phylotypes), (3) the percentage of phylotypes with intact membranes or damaged cells were comparable for RNA- and DNA-based analyses, and (4) the retrieved species were primarily of aquatic origin. The pronounced difference between phylotypes obtained from DNA extracts (dominated by Betaproteobacteria, Bacteroidetes, and Actinobacteria) and from RNA extracts (dominated by Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria) demonstrate the relevance of concomitant RNA and DNA analyses for drinking water studies. Unexpected was that a comparable fraction (about 21%) of phylotypes with membrane-injured cells was observed for DNA- and RNA-based analyses, contradicting the current understanding that RNA-based analyses represent the actively growing fraction of the bacterial community. Overall, we think that this combined approach provides an interesting tool for a concomitant phylogenetic and viability analysis of bacterial species of drinking water.

  3. The translational machinery is an optimized molecular network that affects cellular homoeostasis and disease.

    PubMed

    Kazana, Eleanna; von der Haar, Tobias

    2014-02-01

    Translation involves interactions between mRNAs, ribosomes, tRNAs and a host of translation factors. Emerging evidence on the eukaryotic translational machinery indicates that these factors are organized in a highly optimized network, in which the levels of the different factors are finely matched to each other. This optimal factor network is essential for producing proteomes that result in optimal fitness, and perturbations to the optimal network that significantly affect translational activity therefore result in non-optimal proteomes, fitness losses and disease. On the other hand, experimental evidence indicates that translation and cell growth are relatively robust to perturbations, and viability can be maintained even upon significant damage to individual translation factors. How the eukaryotic translational machinery is optimized, and how it can maintain optimization in the face of changing internal parameters, are open questions relevant to the interaction between translation and cellular disease states.

  4. Short-term exposure to engineered nanomaterials affects cellular epigenome.

    PubMed

    Lu, Xiaoyan; Miousse, Isabelle R; Pirela, Sandra V; Melnyk, Stepan; Koturbash, Igor; Demokritou, Philip

    2016-01-01

    Extensive incorporation of engineered nanomaterials (ENMs) into industrial and biomedical applications increases the risks of exposure to these potentially hazardous materials. While the geno- and cytotoxic effects of ENMs have been investigated, the potential of ENMs to target the cellular epigenome remains largely unknown. Our goal was to determine whether industry relevant ENMs can affect the epigenome at low cytotoxic doses. A panel of cells relevant to inhalation exposures such as human and murine macrophages (THP-1 and RAW264.7, respectively) and human small airway epithelial cells (SAEC) were exposed to printer-emitted engineered nanoparticles (PEPs), mild steel welding fumes (MS-WF), copper oxide (CuO) and titanium dioxide nanoparticles. Toxicological effects, including cytotoxicity, oxidative stress and inflammatory responses were assessed, taking into consideration in vitro dosimetry. The effects of ENMs on cellular epigenome were determined by addressing the global and transposable elements (TEs)-associated DNA methylation and expression of DNA methylation machinery and TEs. The percentage of ENMs-induced cytotoxicity for all cell lines was in the range of 0-15%. Oxidative stress was evident in SAEC after exposure to PEPs and in THP-1 when exposed to CuO. In addition, exposure to ENMs resulted in modest alterations in DNA methylation of two most abundant TEs in mammalian genomes, LINE-1 and Alu/SINE, their transcriptional reactivation, and decreased expression of DNA methylation machinery in a cell-, dose- and ENM-dependent manner. These results indicate that exposure to ENMs at environmentally relevant concentrations, aside from the geno- and cytotoxic effects, can also affect the epigenome of target cells.

  5. Short-term exposure to engineered nanomaterials affects cellular epigenome

    PubMed Central

    Lu, Xiaoyan; Miousse, Isabelle R.; Pirela, Sandra V.; Melnyk, Stepan; Koturbash, Igor; Demokritou, Philip

    2015-01-01

    Extensive incorporation of engineered nanomaterials (ENMs) into industrial and biomedical applications increases the risks of exposure to these potentially hazardous materials. While the geno- and cytotoxic effects of ENMs have been investigated, the potential of ENMs to target the cellular epigenome remains largely unknown. Our goal was to determine whether or not industry relevant ENMs can affect the epigenome at low cytotoxic doses. A panel of cells relevant to inhalation exposures such as human and murine macrophages (THP-1 and RAW264.7, respectively) and human small airway epithelial cells (SAEC) were exposed to printer-emitted engineered nanoparticles (PEPs), mild steel welding fumes (MS-WF), copper oxide (CuO), and titanium dioxide (TiO2) nanoparticles. Toxicological effects, including cytotoxicity, oxidative stress, and inflammatory responses were assessed, taking into consideration in-vitro dosimetry. The effects of ENMs on cellular epigenome were determined by addressing the global and transposable elements (TEs)-associated DNA methylation and expression of DNA methylation machinery and TEs. The percentage of ENMs-induced cytotoxicity for all cell lines was in the range of 0-15%. Oxidative stress was evident in SAEC after exposure to PEPs and in THP-1 when exposed to CuO. Additionally, exposure to ENMs resulted in modest alterations in DNA methylation of two most abundant TEs in mammalian genomes, LINE-1 and Alu/SINE, their transcriptional reactivation, and decreased expression of DNA methylation machinery in a cell-, dose-, and ENM-dependent manner. These results indicate that exposure to ENMs at environmentally relevant concentrations, aside from the geno- and cytotoxic effects, can also affect the epigenome of target cells. PMID:25938281

  6. Positive impact of sucrose supplementation during slow freezing of cat ovarian tissues on cellular viability, follicle morphology, and DNA integrity.

    PubMed

    Tanpradit, Nae; Comizzoli, Pierre; Srisuwatanasagul, Sayamon; Chatdarong, Kaywalee

    2015-06-01

    The objectives of the study were to (1) examine and optimize the impact of sucrose during slow freezing and (2) compare the results of two freezing methods (slow freezing and vitrification) on cellular viability (germinal and stromal cells), follicle morphology, DNA integrity, and gap junction protein expression (connexin 43 [Cx 43]). Different sucrose supplementations (0, 0.1, and 0.3 M) in standard freezing medium were compared before and after slow freezing. Ovarian tissue slow frozen using 0.1- (4.0 ± 0.4) or 0.3-M sucrose (3.9 ± 0.5) yielded better follicular viability (number of positive follicles per 0.0625 mm(2)) than the group without sucrose (1.9 ± 0.2; P < 0.05). Morphologically normal primordial follicles were higher in the sucrose-treated groups (0.1 M, 47.4% and 0.3 M, 43.5%) than the group without sucrose (0 M, 33.8%; P < 0.05). Moreover, less apoptotic primordial follicles were found in both sucrose groups (0.1 M, 1.2% and 0.3 M, 1.9%) than the group without sucrose (7.7%; P < 0.05). However, their Cx 43 expression showed no difference among the groups of different sucrose concentrations. In terms of the freezing methods used, vitrified ovarian tissues had fewer viable follicles (3.2 ± 0.6) than the slow-freezing method (4.6 ± 0.6; P < 0.05). In addition, the slow freezing resulted in more postthawed morphologically normal primordial follicles (38.8% vs. 28.3%, P < 0.05) and less apoptotic primordial follicles (3.8% vs. 8.9%, P < 0.05) than vitrification. The Cx 43 expression showed no difference between slow freezing and vitrification. The present study reported the positive effects of sucrose supplementation and slow-freezing method on the follicular viability, follicular histologic appearances of follicles, and apoptosis of the follicles and stromal cells in cat ovarian tissues.

  7. Cell viability studies and operation in cellular culture medium of n-type organic field-effect transistors

    NASA Astrophysics Data System (ADS)

    Barra, M.; Viggiano, D.; Di Capua, R.; Di Girolamo, F.; Santoro, F.; Taglialatela, M.; Cassinese, A.

    2012-02-01

    The possibility of the fabrication of organic devices suitable to be applied in bio-sensing fields depends largely on the availability of organic compounds displaying robust electrical properties even in aqueous solutions and effective biocompatibility features. In this paper, we report about the good cellular biocompatibility and the electrical response stability in an ionic medium of n-type organic transistors based on the recently developed PDI-8CN2 oligomer. The biocompatibility has been tested by analyzing the adhesion and viability of two different cell lines, human epithelial HeLa cells and murine neuronal F11 cells, on PDI-8CN2 films grown by organic molecular beam deposition (OMBD) on SiO2 substrates. The effect of film thickness on cell attachment was also tested. Uncoated SiO2 substrates were used as control surfaces and sexithiophene (T6) as device testing control. Moreover, the possible toxicity of -CN groups of PDI-8CN2 was tested on HeLa cell cultures, using PDI-8 and T6 molecules as controls. Results showed that, although at high concentration these organic compounds are toxic in solution, if they are presented in form of film, cell lines can attach and grow on them. The electrical response stability of PDI-8CN2 transistors in a cellular culture medium characterized by high concentrations of ionic species has been also investigated. For this purpose, low-voltage operation devices with VGS ranging from -5 V to 5 V, able to strongly reduce the influence of Faradaic currents coming from the electrical operation in an highly ionic environment, have been fabricated on 35 nm thick SiO2 layers and electrically characterized. These results are useful to experimentally define the main critical issues to be further addressed for the fabrication of reliable bio-sensors based on organic transistors.

  8. The Effects of Imatinib Mesylate on Cellular Viability, Platelet Derived Growth Factor and Stem Cell Factor in Mouse Testicular Normal Leydig Cells

    PubMed Central

    Kheradmand, Fatemeh; Hashemnia, Seyyed Mohammad Reza; Valizadeh, Nasim; Roshan-Milani, Shiva

    2016-01-01

    Background: Growth factors play an essential role in the development of tumor and normal cells like testicular leydig cells. Treatment of cancer with anti-cancer agents like imatinib mesylate may interfere with normal leydig cell activity, growth and fertility through failure in growth factors’ production or their signaling pathways. The purpose of the study was to determine cellular viability and the levels of, platelet derived growth factor (PDGF) and stem cell factor (SCF) in normal mouse leydig cells exposed to imatinib, and addressing the effect of imatinib on fertility potential. Methods: The mouse TM3 leydig cells were treated with 0 (control), 2.5, 5, 10 and 20 μM imatinib for 2, 4 and 6 days. Each experiment was repeated three times (15 experiments in each day).The cellular viability and growth factors levels were assessed by MTT and ELISA methods, respectively. For statistical analysis, one-way ANOVA with Tukey’s post hoc and Kruskal-Wallis test were performed. A p-value less than 0.05 was considered statistically significant. Results: With increasing drug concentration, cellular viability decreased significantly (p<0.05) and in contrast, PDGF levels increased (p<0.05). Different imatinib concentrations had no significant effect on SCF level. Increasing the duration of treatment from 2 to 6 days had no obvious effect on cellular viability, PDGF and SCF levels. Conclusion: Imatinib may reduce fertility potential especially at higher concentrations in patients treated with this drug by decreasing cellular viability. The effect of imatinib on leydig cells is associated with PDGF stimulation. Of course future studies can be helpful in exploring the long term effects of this drug. PMID:27141462

  9. Histone deacetylase (HDAC) inhibitor kinetic rate constants correlate with cellular histone acetylation but not transcription and cell viability.

    PubMed

    Lauffer, Benjamin E L; Mintzer, Robert; Fong, Rina; Mukund, Susmith; Tam, Christine; Zilberleyb, Inna; Flicke, Birgit; Ritscher, Allegra; Fedorowicz, Grazyna; Vallero, Roxanne; Ortwine, Daniel F; Gunzner, Janet; Modrusan, Zora; Neumann, Lars; Koth, Christopher M; Lupardus, Patrick J; Kaminker, Joshua S; Heise, Christopher E; Steiner, Pascal

    2013-09-13

    Histone deacetylases (HDACs) are critical in the control of gene expression, and dysregulation of their activity has been implicated in a broad range of diseases, including cancer, cardiovascular, and neurological diseases. HDAC inhibitors (HDACi) employing different zinc chelating functionalities such as hydroxamic acids and benzamides have shown promising results in cancer therapy. Although it has also been suggested that HDACi with increased isozyme selectivity and potency may broaden their clinical utility and minimize side effects, the translation of this idea to the clinic remains to be investigated. Moreover, a detailed understanding of how HDACi with different pharmacological properties affect biological functions in vitro and in vivo is still missing. Here, we show that a panel of benzamide-containing HDACi are slow tight-binding inhibitors with long residence times unlike the hydroxamate-containing HDACi vorinostat and trichostatin-A. Characterization of changes in H2BK5 and H4K14 acetylation following HDACi treatment in the neuroblastoma cell line SH-SY5Y revealed that the timing and magnitude of histone acetylation mirrored both the association and dissociation kinetic rates of the inhibitors. In contrast, cell viability and microarray gene expression analysis indicated that cell death induction and changes in transcriptional regulation do not correlate with the dissociation kinetic rates of the HDACi. Therefore, our study suggests that determining how the selective and kinetic inhibition properties of HDACi affect cell function will help to evaluate their therapeutic utility.

  10. Glufosinate does not affect floral morphology and pollen viability in glufosinate-resistant cotton (Gossypium hirsutum)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies were conducted to determine whether glufosinate treatments to glufosinate-resistant cotton caused changes in floral morphology, pollen viability, and seed set. Four glufosinate treatments were included: (1) glufosinate applied postemergence over the top (POST) at the four-leaf stage, (2) glu...

  11. Inflammation affects the viability and plasticity of equine mesenchymal stem cells: possible implications in intra-articular treatments

    PubMed Central

    Barrachina, Laura; Remacha, Ana Rosa; Romero, Antonio; Vázquez, Francisco José; Albareda, Jorge; Prades, Marta; Ranera, Beatriz; Zaragoza, Pilar; Martín-Burriel, Inmaculada

    2017-01-01

    Mesenchymal stem cells (MSCs) are gaining relevance for treating equine joint injuries because of their ability to limit inflammation and stimulate regeneration. Because inflammation activates MSC immunoregulatory function, proinflammatory priming could improve MSC efficacy. However, inflammatory molecules present in synovial fluid or added to the culture medium might have deleterious effects on MSCs. Therefore, this study was conducted to investigate the effects of inflammatory synovial fluid and proinflammatory cytokines priming on viability and plasticity of equine MSCs. Equine bone marrow derived MSCs (eBM-MSCs) from three animals were cultured for 72 h in media supplemented with: 20% inflammatory synovial fluid (SF); 50 ng/mL IFN-γ and TNF-α (CK50); and 20 ng/mL IFN-γ and TNF-α (CK20). Proliferation assay and expression of proliferation and apoptosis-related genes showed that SF exposed-eBM-MSCs maintained their viability, whereas the viability of CK primed-eBM-MSCs was significantly impaired. Tri-lineage differentiation assay revealed that exposure to inflammatory synovial fluid did not alter eBM-MSCs differentiation potential; however, eBM-MSCs primed with cytokines did not display osteogenic, adipogenic or chondrogenic phenotype. The inflammatory synovial environment is well tolerated by eBM-MSCs, whereas cytokine priming negatively affects the viability and differentiation abilities of eBM-MSCs, which might limit their in vivo efficacy. PMID:27297420

  12. Amyloid β induces adhesion of erythrocytes to endothelial cells and affects endothelial viability and functionality.

    PubMed

    Nakagawa, Kiyotaka; Kiko, Takehiro; Kuriwada, Satoko; Miyazawa, Taiki; Kimura, Fumiko; Miyazawa, Teruo

    2011-01-01

    It has been suggested that amyloid β-peptide (Aβ) might mediate the adhesion of erythrocytes to the endothelium which could disrupt the properties of endothelial cells. We provide evidence here that Aβ actually induced the binding of erythrocytes to endothelial cells and decreased endothelial viability, perhaps by the generation of oxidative and inflammatory stress. These changes are likely to contribute to the pathogenesis of Alzheimer's disease.

  13. Loss of all 3 Extended Synaptotagmins does not affect normal mouse development, viability or fertility.

    PubMed

    Tremblay, Michel G; Moss, Tom

    2016-09-01

    The extended synaptotagmins, E-Syt1, 2 and 3, are multiple C2 domain membrane proteins that are tethered to the endoplasmic reticulum and interact in a calcium dependent manner with plasma membrane phospholipids to form endoplasmic reticulum - plasma membrane junctions. These junctions have been implicated in the exchange of phospholipids between the 2 organelles. The E-Syts have further been implicated in receptor signaling and endocytosis and can interact directly with fibroblast growth factor and other cell surface receptors. Despite these multiple functions, the search for a requirement in vivo has been elusive. Most recently, we found that the genes for E-Syt2 and 3 could be inactivated without effect on mouse development, viability, fertility or morphology. We have now created insertion and deletion mutations in the last of the mouse E-Syt genes. We show that E-Syt1 is specifically expressed throughout the embryonic skeleton during the early stages of chrondrogenesis in a pattern quite distinct from that of E-Syt2 or 3. Despite this, E-Syt1 is also not required for mouse development and propagation. We further show that even the combined inactivation of all 3 E-Syt genes has no effect on mouse viability or fertility in the laboratory. However, this inactivation induces an enhancement in the expression of the genes encoding Orp5/8, Orai1, STIM1 and TMEM110, endoplasmic reticulum - plasma membrane junction proteins that potentially could compensate for E-Syt loss. Given the multiple functions suggested for the E-Syts and their evolutionary conservation, our unexpected findings suggest that they may only provide a survival advantage under specific conditions that have as yet to be identified.

  14. Dyrk1A Haploinsufficiency Affects Viability and Causes Developmental Delay and Abnormal Brain Morphology in Mice

    PubMed Central

    Fotaki, Vassiliki; Dierssen, Mara; Alcántara, Soledad; Martínez, Salvador; Martí, Eulàlia; Casas, Caty; Visa, Joana; Soriano, Eduardo; Estivill, Xavier; Arbonés, Maria L.

    2002-01-01

    DYRK1A is the human orthologue of the Drosophila minibrain (mnb) gene, which is involved in postembryonic neurogenesis in flies. Because of its mapping position on chromosome 21 and the neurobehavioral alterations shown by mice overexpressing this gene, involvement of DYRK1A in some of the neurological defects of Down syndrome patients has been suggested. To gain insight into its physiological role, we have generated mice deficient in Dyrk1A function by gene targeting. Dyrk1A−/− null mutants presented a general growth delay and died during midgestation. Mice heterozygous for the mutation (Dyrk1A+/−) showed decreased neonatal viability and a significant body size reduction from birth to adulthood. General neurobehavioral analysis revealed preweaning developmental delay of Dyrk1A+/− mice and specific alterations in adults. Brains of Dyrk1A+/− mice were decreased in size in a region-specific manner, although the cytoarchitecture and neuronal components in most areas were not altered. Cell counts showed increased neuronal densities in some brain regions and a specific decrease in the number of neurons in the superior colliculus, which exhibited a significant size reduction. These data provide evidence about the nonredundant, vital role of Dyrk1A and suggest a conserved mode of action that determines normal growth and brain size in both mice and flies. PMID:12192061

  15. Vitellogenin knockdown strongly affects cotton boll weevil egg viability but not the number of eggs laid by females.

    PubMed

    Coelho, Roberta R; de Souza Júnior, José Dijair Antonino; Firmino, Alexandre A P; de Macedo, Leonardo L P; Fonseca, Fernando C A; Terra, Walter R; Engler, Gilbert; de Almeida Engler, Janice; da Silva, Maria Cristina M; Grossi-de-Sa, Maria Fatima

    2016-09-01

    Vitellogenin (Vg), a yolk protein precursor, is the primary egg nutrient source involved in insect reproduction and embryo development. The Cotton Boll weevil (CBW) Anthonomus grandis Boheman, the most important cotton pest in Americas, accumulates large amounts of Vg during reproduction. However, the precise role of this protein during embryo development in this insect remains unknown. Herein, we investigated the effects of vitellogenin (AgraVg) knockdown on the egg-laying and egg viability in A. grandis females, and also characterized morphologically the unviable eggs. AgraVg transcripts were found during all developmental stages of A. grandis, with highest abundance in females. Silencing of AgraVg culminated in a significant reduction in transcript amount, around 90%. Despite this transcriptional reduction, egg-laying was not affected in dsRNA-treated females but almost 100% of the eggs lost their viability. Eggs from dsRNA-treated females showed aberrant embryos phenotype suggesting interference at different stages of embryonic development. Unlike for other insects, the AgraVg knockdown did not affect the egg-laying ability of A. grandis, but hampered A. grandis reproduction by perturbing embryo development. We concluded that the Vg protein is essential for A. grandis reproduction and a good candidate to bio-engineer the resistance against this devastating cotton pest.

  16. Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability.

    PubMed

    Godbey, W T; Wu, K K; Mikos, A G

    2001-03-01

    Poly(ethylenimine) (PEI) was used to transfect the endothelial cell line EA.hy 926, and the secreted levels of three gene products, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), and von Willebrand Factor (vWF), were assessed via ELISA. We found that the levels of these gene products in cell supernatants increased by factors up to 16.3 (tPA), 8.3 (PAI-1), or 6.7 (vWF) times the levels recorded for untreated cells, and roughly correlated with the percentage of cells that expressed the reporter plasmid. Transfections carried out using promotorless constructs of the same reporter plasmid also yielded increases in tPA, PAI-1, and vWF to similar extents. Additionally, data regarding cell viability were gathered and found to inversely relate to both the effectiveness of the PEI used for transfection and the secreted levels of the three mentioned products. There appeared to be two distinct types of cell death, resulting from the use of either free PEI (which acts within 2 h) or PEI/DNA complexes (which cause death 7-9 h after transfection). Cells were also transfected by poly(L-lysine) and liposomal carriers, and increases in secreted tPA similar to those seen with PEI-mediated transfection were observed for positively transfected cells. The results of these investigations indicate that non-viral gene delivery can induce a state of endothelial cell dysfunction, and that PEI-mediated transfection can lead to two distinct types of cell death.

  17. Novel vascular endothelial growth factor blocker improves cellular viability and reduces hypobaric hypoxia-induced vascular leakage and oedema in rat brain.

    PubMed

    Saraswat, Deepika; Nehra, Sarita; Chaudhary, Kamal; CVS, Siva Prasad

    2015-05-01

    Vascular endothelial growth factor (VEGF) is an important cerebral angiogenic and permeability factor under hypoxia. There is a need to find effective molecules that may ameliorate hypoxia-induced cerebral oedema. In silico identification of novel candidate molecules that block VEGF-A site were identified and validated with a Ramachandran plot. The active site residues of VEGF-A were detected by Pocketfinder, CASTp, and DogSiteScorer. Based on in silico data, three VEGF-A blocker (VAB) candidate molecules (VAB1, VAB2, and VAB3) were checked for improvement in cellular viability and regulation of VEGF levels in N2a cells under hypoxia (0.5% O2 ). Additionally, the best candidate molecule's efficacy was assessed in male Sprague-Dawley rats for its ameliorative effect on cerebral oedema and vascular leakage under hypobaric hypoxia 7260 m. All experimental results were compared with the commercially available VEGF blocker sunitinib. Vascular endothelial growth factor-A blocker 1 was found most effective in increasing cellular viability and maintaining normal VEGF levels under hypoxia (0.5% oxygen) in N2a cells. Vascular endothelial growth factor-A blocker 1 effectively restored VEGF levels, decreased cerebral oedema, and reduced vascular leakage under hypobaric hypoxia when compared to sunitinib-treated rats. Vascular endothelial growth factor-A blocker 1 may be a promising candidate molecule for ameliorating hypobaric hypoxia-induced vasogenic oedema by regulating VEGF levels.

  18. Factors affecting settling, survival, and viability of black bears reintroduced to Felsenthal National Wildlife Refuge, Arkansas

    USGS Publications Warehouse

    Wear, B.J.; Eastridge, R.; Clark, J.D.

    2005-01-01

    We used radiotelemetry and population modeling techniques to examine factors related to population establishment of black bears (Ursus americanus) reintroduced to Felsenthal National Wildlife Refuge (NWR), Arkansas. Our objectives were to determine whether settling (i.e., establishment of a home range at or near the release site), survival, recruitment, and population viability were related to age class of reintroduced bears, presence of cubs, time since release, or number of translocated animals. We removed 23 adult female black bears with 56 cubs from their winter dens at White River NWR and transported them 160 km to man-made den structures at Felsenthal NWR during spring 2000–2002. Total movement and average circuity of adult females decreased from 1 month, 6 months, and 1 year post-emergence (F2,14 =19.7, P < 0.001 and F2,14 =5.76, P=0.015, respectively). Mean first-year post-release survival of adult female bears was 0.624 (SE = 0.110, SEinterannual = 0.144), and the survival rate of their cubs was 0.750 (SE = 0.088, SEinterannual = 0.109). The homing rate (i.e., the proportion of bears that returned to White River NWR) was 13%. Annual survival for female bears that remained at the release site and survived >1-year post-release increased to 0.909 (SE = 0.097, SEinterannual=0.067; Z=3.5, P < 0.001). Based on stochastic population growth simulations, the average annual growth rate (λ) was 1.093 (SD = 0.053) and the probability of extinction with no additional stockings ranged from 0.56-1.30%. The bear population at Felsenthal NWR is at or above the number after which extinction risk declines dramatically, although additional releases of bears could significantly decrease time to population reestablishment. Poaching accounted for at least 3 of the 8 adult mortalities that we documented; illegal kills could be a significant impediment to population re-establishment at Felsenthal NWR should poaching rates escalate.

  19. Seed viability and functional properties of broccoli sprouts during germination and postharvest storage as affected by irradiation of seeds.

    PubMed

    Waje, Catherine K; Jun, So-Yun; Lee, Yeon-Kyung; Moon, Kwang-Deog; Choi, Yong Hee; Kwon, Joong-Ho

    2009-06-01

    The viability of broccoli seeds and functional properties, such as ascorbic acid, carotenoid, chlorophyll, and total phenol contents, of broccoli sprouts grown from irradiated seeds were evaluated. The seeds were irradiated using electron beam and gamma ray at doses up to 8 kGy. High germination percentages (>90%) were observed in seeds irradiated at < or =4 kGy, but the yield ratio and sprout length decreased with increased irradiation dose. Irradiation at > or =6 kGy resulted in curling of the sprout roots. Germinated seeds contained higher amounts of nutrients than raw seeds but the nutritional quality of sprouts decreased during postharvest storage. Radiation treatment hampered the growth of irradiated seeds resulting in underdeveloped sprouts with decreased ascorbic acid, carotenoid, and chlorophyll contents. In addition, the decrease in functional content of sprouts was more substantial in samples grown from high-dose (5 kGy) irradiated seeds than that of the low-dose (1 kGy) treated ones. Seed irradiation did not negatively affect the total phenol content of sprouts. In general, electron beam and gamma irradiation of broccoli seeds showed similar effects on the viability and functional properties of sprouts.

  20. Histopathology of Growth Anomaly Affecting the Coral, Montipora capitata: Implications on Biological Functions and Population Viability

    PubMed Central

    Burns, John H. R.; Takabayashi, Misaki

    2011-01-01

    Growth anomalies (GAs) affect the coral, Montipora capitata, at Wai'ōpae, southeast Hawai'i Island. Our histopathological analysis of this disease revealed that the GA tissue undergoes changes which compromise anatomical machinery for biological functions such as defense, feeding, digestion, and reproduction. GA tissue exhibited significant reductions in density of ova (66.1–93.7%), symbiotic dinoflagellates (38.8–67.5%), mesenterial filaments (11.2–29.0%), and nematocytes (28.8–46.0%). Hyperplasia of the basal body wall but no abnormal levels of necrosis and algal or fungal invasion was found in GA tissue. Skeletal density along the basal body wall was significantly reduced in GAs compared to healthy or unaffected sections. The reductions in density of the above histological features in GA tissue were collated with disease severity data to quantify the impact of this disease at the colony and population level. Resulting calculations showed this disease reduces the fecundity of M. capitata colonies at Wai'ōpae by 0.7–49.6%, depending on GA severity, and the overall population fecundity by 2.41±0.29%. In sum, GA in this M. capitata population reduces the coral's critical biological functions and increases susceptibility to erosion, clearly defining itself as a disease and an ecological threat. PMID:22205976

  1. APEH Inhibition Affects Osteosarcoma Cell Viability via Downregulation of the Proteasome

    PubMed Central

    Palumbo, Rosanna; Gogliettino, Marta; Cocca, Ennio; Iannitti, Roberta; Sandomenico, Annamaria; Ruvo, Menotti; Balestrieri, Marco; Rossi, Mosè; Palmieri, Gianna

    2016-01-01

    The proteasome is a multienzymatic complex that controls the half-life of the majority of intracellular proteins, including those involved in apoptosis and cell-cycle progression. Recently, proteasome inhibition has been shown to be an effective anticancer strategy, although its downregulation is often accompanied by severe undesired side effects. We previously reported that the inhibition of acylpeptide hydrolase (APEH) by the peptide SsCEI 4 can significantly affect the proteasome activity in A375 melanoma or Caco-2 adenocarcinoma cell lines, thus shedding new light on therapeutic strategies based on downstream regulation of proteasome functions. In this work, we investigated the functional correlation between APEH and proteasome in a panel of cancer cell lines, and evaluated the cell proliferation upon SsCEI 4-treatments. Results revealed that SsCEI 4 triggered a proliferative arrest specifically in osteosarcoma U2OS cells via downregulation of the APEH–proteasome system, with the accumulation of the typical hallmarks of proteasome: NF-κB, p21Waf1, and polyubiquitinylated proteins. We found that the SsCEI 4 anti-proliferative effect involved a senescence-like growth arrest without noticeable cytotoxicity. These findings represent an important step toward understanding the mechanism(s) underlying the APEH-mediated downregulation of proteasome in order to design new molecules able to efficiently regulate the proteasome system for alternative therapeutic strategies. PMID:27669226

  2. Perfluorinated compounds differentially affect steroidogenesis and viability in the human adrenocortical carcinoma (H295R) in vitro cell assay.

    PubMed

    Kraugerud, Marianne; Zimmer, Karin E; Ropstad, Erik; Verhaegen, Steven

    2011-08-10

    Perfluorinated compounds (PFCs) comprise a large class of man-made chemicals of which some are persistent and present throughout the ecosystem. This raises concerns about potential harmful effects of such PFCs on humans and the environment. In order to investigate the effects of potentially harmful PFCs on steroid hormone production, human adrenocortical H295R cells were exposed to three persistent PFCs including perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) at six different concentrations (6nM to 600μM) for 48h. Exposure to 600μM PFOS resulted in a dose-responsive increase in oestradiol as well as a smaller dose-responsive increase in progesterone and testosterone secretion measured using radioimmunoassay. The aromatase activity was not significantly altered by PFOS. Only small changes in hormone secretion were detected following exposure to PFOA and PFNA. Gene expression of CYP11A, quantified using qRT-PCR was decreased by all exposure doses of PFOA, whereas HMGR expression was decreased by 60nM PFNA. The viability markedly decreased by exposure to 600μM of PFOA or PFNA, but not PFOS. Flow cytometric analysis demonstrated a significant increase in apoptosis following exposure to PFNA at the highest concentration. We conclude that PFOS is capable of altering steroidogenesis in the H295R in vitro model by a mechanism other than changes in gene expression or activity of aromatase. Additionally, PFCs appear to differentially affect cell viability with induction of cell death via apoptosis at high doses of PFNA.

  3. Cell viability, adhesion and function of RAW 264.7 macrophages on fluorinated xerogel-derived nitric oxide permeable membrane for the application of cellular sensing.

    PubMed

    Kang, Wook Sung; Seo, Bochan; Kim, Ji-Hye; Kim, Ok-Kyun; Shin, Jae Ho; Lee, Gi-Ja; Park, Hun-Kuk

    2014-11-01

    Organically modified xerogels have an advantage over gas sensing applications due to their open, rigid structure and hydrophobicity. Here we evaluated the biocompatibility of xerogel-derived nitric oxide (NO) permeable membranes modified with fluorinated functional groups for application in cellular sensing by growing RAW 264.7 macrophages on them. We examined the cell viability, adhesion and growth of RAW 264.7 macrophages on NO permselective membrane and other cell-adhesive matrices, poly L-lysine and collagen. The surface roughness of each membrane was obtained from topographic atomic force microscopy (AFM) images. In addition, we measured the level of NO release of RAW 264.7 macrophages by lipopolysaccharide (LPS) stimulation using a Griess assay to confirm the function of cells. The fluorinated xerogel-derived membrane had a very smooth surface with rms roughness 2.1 Å and did not show cytotoxic effects in RAW 264.7 macrophages. As a result, the morphology and function of adhering RAW 264.7 macrophage showed no differences from those of other cell-adhesive membranes. Finally, we successfully detected NO release in RAW 264.7 macrophages stimulated by LPS, using a planar-type xerogel-derived NO sensor. Therefore, we suggest that fluorinated xerogel-derived membrane could be used as both a NO permeable and cell-adhesive membrane for cellular sensing applications.

  4. Mechanical dissociation of human embryonic stem cell colonies by manual scraping after collagenase treatment is much more detrimental to cellular viability than is trypsinization with gentle pipetting.

    PubMed

    Heng, Boon Chin; Liu, Hua; Ge, Zigang; Cao, Tong

    2007-05-01

    Because hESC (human embryonic stem cells) are 'social cells' that require co-operative interactions and intimate physical contact with each other, it is absolutely essential to dissociate hESC colonies into cellular clumps rather than into a single-cell suspension during serial passage. The present study compared two commonly used protocols for dissociating hESC colonies. The first protocol involved mild enzymatic treatment with collagenase type IV (1 mg/ml) for approx. 5-10 min, prior to mechanical dissociation into cellular clumps through manual scraping with a plastic pipette tip. The second protocol involved a short duration of exposure (2-3 min) to low concentrations of trypsin (0.05%), followed by gentle pipetting. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay was used to compare the recovery of viable cells after dissociating hESC colonies with these two protocols, before and after conventional freeze-thawing with 10% (v/v) DMSO. Besides undifferentiated hESC, the randomly differentiated fibroblastic progenies of hESC at various passages (P0-P4), together with an immortalized cell line (CRL-1486), were also utilized to compare the two protocols. The results demonstrated that the second protocol (trypsinization with gentle pipetting) is much less detrimental to cellular viability than is the first protocol (collagenase treatment with scratching). This in turn translated to higher freeze-thaw survival rates. It is hypothesized that scratching after collagenase treatment (first protocol) somehow induces physical damage to the cells, thereby leading to a lower recovery of viable cells, both before and after freeze-thawing.

  5. Rice stripe virus affects the viability of its vector offspring by changing developmental gene expression in embryos

    PubMed Central

    Li, Shuo; Wang, Shijuan; Wang, Xi; Li, Xiaoli; Zi, Jinyan; Ge, Shangshu; Cheng, Zhaobang; Zhou, Tong; Ji, Yinghua; Deng, Jinhua; Wong, Sek-Man; Zhou, Yijun

    2015-01-01

    Plant viruses may affect the viability and development process of their herbivore vectors. Small brown planthopper (SBPH) is main vector of Rice stripe virus (RSV), which causes serious rice stripe disease. Here, we reported the effects of RSV on SBPH offspring by crossing experiments between viruliferous and non-viruliferous strains. The life parameters of offspring from different cross combinations were compared. The hatchability of F1 progeny from viruliferous parents decreased significantly, and viruliferous rate was completely controlled by viruliferous maternal parent. To better elucidate the underlying biological mechanisms, the morphology of eggs, viral propagation and distribution in the eggs and expression profile of embryonic development genes were investigated. The results indicated that RSV replicated and accumulated in SBPH eggs resulting in developmental stunt or delay of partial eggs; in addition, RSV was only able to infect ovum but not sperm. According to the expression profile, expression of 13 developmental genes was regulated in the eggs from viruliferous parents, in which two important regulatory genes (Ls-Dorsal and Ls-CPO) were most significantly down-regulated. In general, RSV exerts an adverse effect on SBPH, which is unfavourable for the expansion of viruliferous populations. The viewpoint is also supported by systematic monitoring of SBPH viruliferous rate. PMID:25601039

  6. Characterization of cspB, a Bacillus subtilis inducible cold shock gene affecting cell viability at low temperatures.

    PubMed Central

    Willimsky, G; Bang, H; Fischer, G; Marahiel, M A

    1992-01-01

    A new class of cold shock-induced proteins that may be involved in an adaptive process required for cell viability at low temperatures or may function as antifreeze proteins in Escherichia coli and Saccharomyces cerevisiae has been identified. We purified a small Bacillus subtilis cold shock protein (CspB) and determined its amino-terminal sequence. By using mixed degenerate oligonucleotides, the corresponding gene (cspB) was cloned on two overlapping fragments of 5 and 6 kb. The gene encodes an acidic 67-amino-acid protein (pI 4.31) with a predicted molecular mass of 7,365 Da. Nucleotide and deduced amino acid sequence comparisons revealed 61% identity to the major cold shock protein of E. coli and 43% identity to a family of eukaryotic DNA binding proteins. Northern RNA blot and primer extension studies indicated the presence of one cspB transcript that was initiated 119 bp upstream of the initiation codon and was found to be induced severalfold when exponentially growing B. subtilis cell cultures were transferred from 37 degrees C to 10 degrees C. Consistent with this cold shock induction of cspB mRNA, a six- to eightfold induction of a cspB-directed beta-galactosidase synthesis was observed upon downshift in temperature. To investigate the function of CspB, we inactivated the cold shock protein by replacing the cspB gene in the B. subtilis chromosome with a cat-interrupted copy (cspB::cat) by marker replacement recombination. The viability of cells of this mutant strain, GW1, at freezing temperatures was strongly affected. However, the effect of having no CspB in GW1 could be slightly compensated for when cells were preincubated at 10 degrees C before freezing. These results indicate that CspB belongs to a new type of stress-inducible proteins that might be able to protect B. subtilis cells from damage caused by ice crystal formation during freezing. Images PMID:1400185

  7. A systematic review of the key factors affecting tissue viability and rehabilitation outcomes of the residual limb in lower extremity traumatic amputees.

    PubMed

    Butler, Katrina; Bowen, Catherine; Hughes, Ann-Marie; Torah, Russel; Ayala, Ivo; Tudor, John; Metcalf, Cheryl D

    2014-08-01

    Most traumatic lower limb amputees ambulate using a prosthetic limb. Comfort, appearance of the missing limb and function are confirmed as being important during rehabilitation post-amputation. Emerging evidence suggests that impaired tissue viability of the stump affects rehabilitation and thus clinical ability to provide optimum care. The primary objective of this systematic review was to identify key factors relating to tissue viability of the residual limb in lower extremity traumatic amputees. A secondary objective was to identify factors that affect rehabilitation post-amputation. In total, 218 studies were assessed; 37 met pre-determined criteria. Studies were classified according to the WHO ICF framework and the NHMRC level of evidence. Five key themes emerged; Prosthetic Fit; The Residuum; Quality of Life; Amputee Care and Prosthetic Use. The evidence indicates that high frequencies of skin problems affecting tissue viability within this population are inherently linked to intolerance of the prosthesis. Stump integrity, amputee care regimen and pain were also identified as impacting on quality of life, affecting rehabilitation and the ability to become independently mobile. Levels of evidence within all studies were low and indicative of the majority being non-randomised cohort studies or case-control studies. As there are a limited number of interventional studies, further development of robust outcome measures, clinical trials and prospective studies are of utmost importance to unravel the links between tissue viability and the other key factors. This will inform clinical management strategies and help develop targeted therapies and care pathways.

  8. Effect of Procyanidin-rich Extract from Natural Cocoa Powder on Cellular Viability, Cell Cycle Progression, and Chemoresistance in Human Epithelial Ovarian Carcinoma Cell Lines

    PubMed Central

    Taparia, Shruti; Khanna, Aparna

    2016-01-01

    Background: Over the last 400 years, cocoa and chocolate have been described as having potential medicinal value, being consumed as a beverage or eaten as food. Concentration–dependant, antiproliferation, and cytotoxic effects of some of their polyphenolic constituents have been demonstrated against various cancers. Such an effect remains to be demonstrated in ovarian cancer Objective: To investigate the effect of cocoa procyanidins against ovarian cancer in vitro using OAW42 and OVCAR3 cell lines. Materials and Methods: Cocoa procyanidins were extracted and enriched from non alkalized cocoa powder. The polyphenolic content and antioxidant activity were determined. Effect on cell viability was determined after the treatment with ≤1000 μg/mL cocoa procyanidin-rich extract on OAW42 and OVCAR3 and normal human dermal fibroblasts. Similarly, chemosensitization effect was determined by pretreating cancer cell lines with extract followed by doxorubicin hydrochloride treatment. The effect of treatment on cell cycle and P-glycoprotein (P-gp) expression was determined using flow cytometry. Results: The cocoa extract showed high polyphenolic content and antioxidant activity. Treatment with extract caused cytotoxicity and chemosensitization in OAW42 and OVCAR3 cell lines. Normal dermal fibroblasts showed an increase in cell viability post treatment with extract. Treatment with extract affected the cell cycle and an increasing percentage of cells in hypodiploid sub-G1/G0 phase was observed. Treatment of OVCAR3 with the extract caused reduction of P-gp expression. Conclusion: Cocoa procyanidins were found to be selectively cytotoxic against epithelial ovarian cancer, interfered with the normal cell cycle and sensitized cells to subsequent chemotherapeutic treatment. Chemosensitization was found to be associated with P-gp reduction in OVCAR3 cells. SUMMARY Among the naturally occurring flavonoids, procyanidins have been shown to be effective against cancersNon alkalized

  9. A new type of quinoxalinone derivatives affects viability, invasion, and intracellular growth of Toxoplasma gondii tachyzoites in vitro.

    PubMed

    Rivera Fernández, Norma; Mondragón Castelán, Mónica; González Pozos, Sirenia; Ramírez Flores, Carlos J; Mondragón González, Ricardo; Gómez de León, Carmen T; Castro Elizalde, Kitzia N; Marrero Ponce, Yovani; Arán, Vicente J; Martins Alho, Miriam A; Mondragón Flores, Ricardo

    2016-05-01

    Quinoxalinone derivatives, identified as VAM2 compounds (7-nitroquinoxalin-2-ones), were evaluated against Toxoplasma gondii tachyzoites of the RH strain. The VAM2 compounds were previously synthesized based on the design obtained from an in silico prediction with the software TOMOCOMD-CARDD. From the ten VAM2 drugs tested, several showed a deleterious effect on tachyzoites. However, VAM2-2 showed the highest toxoplasmicidal activity generating a remarkable decrease in tachyzoite viability (in about 91 %) and a minimal alteration in the host cell. An evident inhibition of host cell invasion by tachyzoites previously treated with VAM2-2 was observed in a dose-dependent manner. In addition, remarkable alterations were observed in the pellicle parasite, such as swelling, roughness, and blebbing. Toxoplasma motility was inhibited, and subpellicular cytoskeleton integrity was altered, inducing a release of its components to the soluble fraction. VAM2-2 showed a clear and specific deleterious effect on tachyzoites viability, structural integrity, and invasive capabilities with limited effects in host cells morphology and viability. VAM2-2 minimum inhibitory concentration (MIC50) was determined as 3.3 μM ± 1.8. Effects of quinoxalinone derivatives on T. gondii provide the basis for a future therapeutical alternative in the treatment of toxoplasmosis.

  10. Mutations Affecting Potassium Import Restore the Viability of the Escherichia coli DNA Polymerase III holD Mutant

    PubMed Central

    Durand, Adeline

    2016-01-01

    Mutants lacking the ψ (HolD) subunit of the Escherichia coli DNA Polymerase III holoenzyme (Pol III HE) have poor viability, but a residual growth allows the isolation of spontaneous suppressor mutations that restore ΔholD mutant viability. Here we describe the isolation and characterization of two suppressor mutations in the trkA and trkE genes, involved in the main E. coli potassium import system. Viability of ΔholD trk mutants is abolished on media with low or high K+ concentrations, where alternative K+ import systems are activated, and is restored on low K+ concentrations by the inactivation of the alternative Kdp system. These findings show that the ΔholD mutant is rescued by a decrease in K+ import. The effect of trk inactivation is additive with the previously identified ΔholD suppressor mutation lexAind that blocks the SOS response indicating an SOS-independent mechanism of suppression. Accordingly, although lagging-strand synthesis is still perturbed in holD trkA mutants, the trkA mutation allows HolD-less Pol III HE to resist increased levels of the SOS-induced bypass polymerase DinB. trk inactivation is also partially additive with an ssb gene duplication, proposed to stabilize HolD-less Pol III HE by a modification of the single-stranded DNA binding protein (SSB) binding mode. We propose that lowering the intracellular K+ concentration stabilizes HolD-less Pol III HE on DNA by increasing electrostatic interactions between Pol III HE subunits, or between Pol III and DNA, directly or through a modification of the SSB binding mode; these three modes of action are not exclusive and could be additive. To our knowledge, the holD mutant provides the first example of an essential protein-DNA interaction that strongly depends on K+ import in vivo. PMID:27280472

  11. Bioactive Volatiles from an Endophytic Daldinia cf. concentrica Isolate Affect the Viability of the Plant Parasitic Nematode Meloidogyne javanica

    PubMed Central

    Braun Miyara, Sigal; Ezra, David

    2016-01-01

    Plant-parasitic nematodes form one of the largest sources of biotic stress imposed on plants, and are very difficult to control; among them are the obligate parasites, the sedentary root-knot nematodes (RKNs)–Meloidogyne spp.–which are extremely polyphagous and exploit a very wide range of hosts. Endophytic fungi are organisms that spend most of their life cycle within plant tissue without causing visible damage to the host plant. Many endophytes secrete specialized metabolites and/or emit volatile organic compounds (VOCs) that exhibit biological activity. Recently, we demonstrated that the endophytic fungus Daldinia cf. concentrica secrets biologically active VOCs. Here we examined the ability of the fungus and its VOCs to control the RKN M. javanica both in vitro and greenhouse experiments. The D. cf. concentrica VOCs showed bionematicidal activity against the second-stage juveniles (J2s) of M. javanica. We found that exposure of J2s to fungal volatiles caused 67% reduction in viability, and that application of a synthetic volatile mixture (SVM), comprising 3-methyl-1-butanol, (±)-2-methyl-1-butanol, 4-heptanone, and isoamyl acetate, in volumetric ratio of 1:1:2:1 further reduced J2s viability by 99%. We demonstrated that, although each of the four VOCs significantly reduced the viability of J2s relative to the control, only 4-heptanone elicited the same effect as the whole mixture, with nematicidal activity of 90% reduction in viability of the J2s. Study of the effect of the SVM on egg hatching demonstrated that it decreased eggs hatching by 87%. Finally, application of the SVM to soil inoculated with M. javanica eggs or J2s prior to planting susceptible tomato plants resulted in a significantly reduced galling index and fewer eggs produced on each root system, with no effect on root weight. Thus, D. cf. concentrica and/or SVM based on fungal VOCs may be considered as a novel alternative approach to controlling the RKN M. javanica. PMID:27997626

  12. Cadmium affects the mitochondrial viability and the acid soluble thiols concentration in liver, kidney, heart and gills of Ancistrus brevifilis (Eigenmann, 1920)

    PubMed Central

    Velasquez-Vottelerd, P.; Anton, Y.; Salazar-Lugo, R.

    2015-01-01

    The freshwater fish Ancistrus brevifilis, which is found in Venezuelan rivers, is considered a potential sentinel fish in ecotoxicological studies. The cadmium (Cd) effect on the mitochondrial viability (MV) and acid soluble thiols levels (AST) in A. brevifilis tissues (liver, kidney, heart, and gill) was evaluated. Forty-two fish with similar sizes and weights were randomly selected, of which 7 fish (with their respective replicate) were exposed for 7 and 30 days to a Cd sublethal concentration (0.1 mg.l-1). We determined the MV through a Janus Green B colorimetric assay and we obtained the concentration of AST by Ellman’s method. Mitochondrial viability decreased in fish exposed to Cd for 30 days with the liver being the most affected tissue. We also detected a significant decrease in AST levels was in fishes exposed to Cd for 7 days in liver and kidney tissues; these results suggests that AST levels are elevated in some tissues may act as cytoprotective and adaptive alternative mechanism related to the ROS detoxification, maintenance redox status and mitochondrial viability. Organ-specifics variations were observed in both assays. We conclude that the Cd exposure effect on AST levels and MV, vary across fish tissues and is related to the exposure duration, the molecule dynamics in different tissues, the organism and environmental conditions. PMID:26623384

  13. Contaminant loading in remote Arctic lakes affects cellular stress-related proteins expression in feral charr.

    USGS Publications Warehouse

    Wiseman, Steve; Jorgensen, Even H.; Maule, Alec G.; Vijayan, Mathilakath M.

    2011-01-01

    The remote Arctic lakes on Bjornoya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasjoen has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake Oyangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasjoen was approximately 25-fold higher than in individuals from Oyangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasjoen compared with Oyangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasjoen compared with Oyangen. Also, liver GR protein expression was significantly higher in the Ellasjoen charr compared with Oyangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.

  14. In situ CUTANEOUS CELLULAR IMMUNE RESPONSE IN DOGS NATURALLY AFFECTED BY VISCERAL LEISHMANIASIS

    PubMed Central

    ROSSI, Claudio Nazaretian; TOMOKANE, Thaise Yumie; BATISTA, Luis Fábio da Silva; MARCONDES, Mary; LARSSON, Carlos Eduardo; LAURENTI, Márcia Dalastra

    2016-01-01

    SUMMARY Thirty-eight dogs naturally affected by visceral leishmaniasis were recruited in Araçatuba, São Paulo State, Brazil - an endemic area for visceral leishmaniasis. The animals were distributed into one of two groups, according to their clinical and laboratory features, as either symptomatic or asymptomatic dogs. Correlations between clinical features and inflammatory patterns, cellular immune responses, and parasitism in the macroscopically uninjured skin of the ear were investigated. Histological skin patterns were similar in both groups, and were generally characterized by a mild to intense inflammatory infiltrate in the dermis, mainly consisting of mononuclear cells. There was no difference in the number of parasites in the skin (amastigotes/mm²) between the two groups. Concerning the characterization of the cellular immune response, the number of positive inducible nitric oxide synthase (iNOS+) cells was higher in the dermis of symptomatic than in asymptomatic dogs (p = 0.0368). A positive correlation between parasite density and macrophages density (p = 0.031), CD4+ T-cells (p = 0.015), and CD8+ T-cells (p = 0.023) was observed. Furthermore, a positive correlation between density of iNOS+ cells and CD3+ T-cells (p = 0.005), CD4+ T-cells (p = 0.001), and CD8+ T-cells (p = 0.0001) was also found. The results showed the existence of a non-specific chronic inflammatory infiltrate in the dermis of dogs affected by visceral leishmaniasis, characterized by the presence of activated macrophages and T-lymphocytes, associated to cutaneous parasitism, independent of clinical status. PMID:27410908

  15. Keratin-containing inclusions affect cell morphology and distribution of cytosolic cellular components.

    PubMed

    Hanada, Shinichiro; Harada, Masaru; Kumemura, Hiroto; Omary, M Bishr; Kawaguchi, Takumi; Taniguchi, Eitaro; Koga, Hironori; Yoshida, Takafumi; Maeyama, Michiko; Baba, Shinji; Ueno, Takato; Sata, Michio

    2005-04-01

    Many neurodegenerative diseases are characterized by the presence of protein aggregates bundled with intermediate filaments (IFs) and similar structures, known as Mallory bodies (MBs), are observed in various liver diseases. IFs are anchored at desmosomes and hemidesmosomes, however, interactions with other intercellular junctions have not been determined. We investigated the effect of IF inclusions on junction-associated and cytosolic proteins in various cultured cells. We performed gene transfection of the green fluorescent protein (GFP)-tagged cytokeratin (CK) 18 mutant arg89cys (GFP-CK18 R89C) in cultured cells and observed CK aggregations as well as loss of IF networks. Among various junction-associated proteins, zonula occludens-1 and beta-catenin were colocalized with CK aggregates on immunofluorescent analyses. Similar results were obtained on immunostaining for cytosolic proteins, 14-3-3 zeta protein, glucose-6-phosphate dehydrogenase and DsRed. E-cadherin, a basolateral membrane protein in polarized epithelia, was present on both the apical and basolateral domains in GFP-CK18 R89C-transfected cells. Furthermore, cells containing CK aggregates were significantly larger than GFP-tagged wild type CK18 (GFP-WT CK18)-transfected or non-transfected cells (P < 0.01) and sometimes their morphology was significantly altered. Our data indicate that CK aggregates affect not only cell morphology but also the localization of various cytosolic components, which may affect the cellular function.

  16. Dinaciclib potently suppresses MCL-1 and selectively induces the cell death in human iPS cells without affecting the viability of cardiac tissue

    PubMed Central

    Alsayegh, Khaled; Matsuura, Katsuhisa; Sekine, Hidekazu; Shimizu, Tatsuya

    2017-01-01

    Induced pluripotent stem (iPS) cells hold great potential for being a major source of cells for regenerative medicine. One major issue that hinders their advancement to clinic is the persistence of undifferentiated iPS cells in iPS-derived tissue. In this report, we show that the CDKs inhibitor, Dinaciclib, selectively eliminates iPS cells without affecting the viability of cardiac cells. We found that low nanomolar concentration of dinaciclib increased DNA damage and p53 protein levels in iPSCs. This was accompanied by negative regulation of the anti-apoptotic protein MCL-1. Gene knockdown experiments revealed that p53 downregulation only increased the threshold of dinaciclib induced apoptosis in iPS cells. Dinaciclib also inhibited the phosphorylation of Serine 2 of the C-terminal domain of RNA Polyemrase II through CDK9 inhibition. This resulted in the inhibition of transcription of MCL-1 and the pluripotency genes, NANOG and c-MYC. Even though dinaciclib caused a slight downregulation of MCL-1 in iPS-derived cardiac cells, the viability of the cells was not significantly affected, and beating iPS-derived cardiac cell sheet could still be fabricated. These findings suggest a difference in tolerance of MCL-1 downregulation between iPSCs and iPS-derived cardiac cells which could be exploited to eliminate remaining iPS cells in bioengineered cell sheet tissues. PMID:28361959

  17. Meal size and frequency affect neuronal plasticity and vulnerability to disease: cellular and molecular mechanisms.

    PubMed

    Mattson, Mark P; Duan, Wenzhen; Guo, Zhihong

    2003-02-01

    Although all cells in the body require energy to survive and function properly, excessive calorie intake over long time periods can compromise cell function and promote disorders such as cardiovascular disease, type-2 diabetes and cancers. Accordingly, dietary restriction (DR; either caloric restriction or intermittent fasting, with maintained vitamin and mineral intake) can extend lifespan and can increase disease resistance. Recent studies have shown that DR can have profound effects on brain function and vulnerability to injury and disease. DR can protect neurons against degeneration in animal models of Alzheimer's, Parkinson's and Huntington's diseases and stroke. Moreover, DR can stimulate the production of new neurons from stem cells (neurogenesis) and can enhance synaptic plasticity, which may increase the ability of the brain to resist aging and restore function following injury. Interestingly, increasing the time interval between meals can have beneficial effects on the brain and overall health of mice that are independent of cumulative calorie intake. The beneficial effects of DR, particularly those of intermittent fasting, appear to be the result of a cellular stress response that stimulates the production of proteins that enhance neuronal plasticity and resistance to oxidative and metabolic insults; they include neurotrophic factors such as brain-derived neurotrophic factor (BDNF), protein chaperones such as heat-shock proteins, and mitochondrial uncoupling proteins. Some beneficial effects of DR can be achieved by administering hormones that suppress appetite (leptin and ciliary neurotrophic factor) or by supplementing the diet with 2-deoxy-d-glucose, which may act as a calorie restriction mimetic. The profound influences of the quantity and timing of food intake on neuronal function and vulnerability to disease have revealed novel molecular and cellular mechanisms whereby diet affects the nervous system, and are leading to novel preventative and

  18. Mugil cephalus roe oil obtained by supercritical fluid extraction affects the lipid profile and viability in cancer HeLa and B16F10 cells.

    PubMed

    Rosa, A; Piras, A; Nieddu, M; Putzu, D; Cesare Marincola, F; Falchi, A M

    2016-09-14

    We explored the changes in viability and lipid profile occurring in cancer cells, murine melanoma cells (B16F10 cells) and human cervical carcinoma cells (HeLa cells), when exposed to 24 h-treatments with an n-3 PUFA-rich oil obtained by supercritical extraction with CO2 from Mugil cephalus processed roe (bottarga). The composition of the major lipid classes of bottarga oil was determined by the (13)C NMR technique. Reversed-phase HPLC with DAD/ELSD detection was performed to analyze cells' total fatty acid profile and the levels of phospholipids, total/free cholesterol, triacylglycerols, and cholesteryl esters. Cell-based fluorescent measurements of intracellular membranes and lipid droplets were performed on bottarga oil-treated cells using the Nile red staining technique. The treatments of cancer cells with bottarga oil reduced the viability and affected the fatty acid profile, with a significant n-3 PUFA increase in treated cells. Mullet roe oil uptake modulated the cancer cell lipid composition, inducing a remarkable incorporation of health beneficial n-3 PUFA in the polar and neutral lipid fractions. Bottarga oil treatment influenced the synthesis of intracellular membranes and accumulation of cytoplasmic lipid droplets in cancer cells.

  19. Investigation of microstructure, mechanical properties and cellular viability of poly(L-lactic acid) tissue engineering scaffolds prepared by different thermally induced phase separation protocols.

    PubMed

    Molladavoodi, Sara; Gorbet, Maud; Medley, John; Kwon, Hyock Ju

    2013-01-01

    Two thermally induced phase separation (TIPS) methods have been used to fabricate biodegradable poly(L-lactic acid) (PLLA) tissue engineering scaffolds each with fibrous (F-TIPS) and porous (P-TIPS) microstructures. Three levels of PLLA concentration (3, 5 and 7 wt%) were employed in each fabrication method and both wet and dry specimens were studied. Simple compression testing revealed that an elastic-plastic representation of the mechanical behavior was possible for all specimens. Both elastic and plastic moduli were higher for the P-TIPS, for higher polymer concentration, and might be somewhat higher for dry as opposed to wet specimens. For F-TIPS specimens, permanent deformation occurred successively during cyclic deformation but a "memory effect" simplified the behavior. Although F-TIPS microstructure better resembled the natural extracellular matrix, human osteosarcoma fibroblast cells showed more consistent viability in the P-TIPS scaffolds under our unloaded test protocols. Biodegradation in cell culture medium resulted in a decreased elastic moduli for F-TIPS specimens. Information presented regarding the microstructure, mechanical properties and cell viability of these PLLA scaffolds that should help reduce the number of iterations involved in developing tissue engineering products.

  20. Incompatibility of silver nanoparticles with lactate dehydrogenase leakage assay for cellular viability test is attributed to protein binding and reactive oxygen species generation.

    PubMed

    Oh, Seok-Jeong; Kim, Hwa; Liu, Yingqiu; Han, Hyo-Kyung; Kwon, Kyenghee; Chang, Kyung-Hwa; Park, Kwangsik; Kim, Younghun; Shim, Kyuhwan; An, Seong Soo A; Lee, Moo-Yeol

    2014-03-21

    A growing number of studies report that conventional cytotoxicity assays are incompatible with certain nanoparticles (NPs) due to artifacts caused by the distinctive characteristics of NPs. Lactate dehydrogenase (LDH) leakage assays have inadequately detected cytotoxicity of silver nanoparticles (AgNPs), leading to research into the underlying mechanism. When ECV304 endothelial-like umbilical cells were treated with citrate-capped AgNPs (cAgNPs) or bare AgNPs (bAgNPs), the plasma membrane was disrupted, but the LDH leakage assay failed to detect cytotoxicity, indicating interference with the assay by AgNPs. Both cAgNPs and bAgNPs inactivated LDH directly when treated to cell lysate as expected. AgNPs adsorbed LDH and thus LDH, together with AgNPs, was removed from assay reactants during sample preparation, with a resultant underestimation of LDH leakage from cells. cAgNPs, but not bAgNPs, generated reactive oxygen species (ROS), which were successfully scavenged by N-acetylcysteine or ascorbic acid. LDH inhibition by cAgNPs could be restored partially by simultaneous treatment with those antioxidants, suggesting the contribution of ROS to LDH inactivation. Additionally, the composition of the protein corona surrounding AgNPs was identified employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In sum, the LDH leakage assay, a conventional cell viability test method, should be employed with caution when assessing cytotoxicity of AgNPs.

  1. Molecular and cellular targets affected by green tea extracts in vascular cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Consumption of green or black tea has been associated with a lower risk for the development of cardiovascular diseases, but despite many studies, a firm connection has not been delineated. Several molecular and cellular mechanisms may play a role in the preventive activity of tea. As reviewed here, ...

  2. Polycyclic Aromatic Hydrocarbon Affects Acetic Acid Production during Anaerobic Fermentation of Waste Activated Sludge by Altering Activity and Viability of Acetogen.

    PubMed

    Luo, Jingyang; Chen, Yinguang; Feng, Leiyu

    2016-07-05

    Till now, almost all the studies on anaerobic fermentation of waste activated sludge (WAS) for bioproducts generation focused on the influences of operating conditions, pretreatment methods and sludge characteristics, and few considered those of widespread persistent organic pollutants (POPs) in sludge, for example, polycyclic aromatic hydrocarbons (PAHs). Herein, phenanthrene, which was a typical PAH and widespread in WAS, was selected as a model compound to investigate its effect on WAS anaerobic fermentation for short-chain fatty acids (SCFAs) accumulation. Experimental results showed that the concentration of SCFAs derived from WAS was increased in the presence of phenanthrene during anaerobic fermentation. The yield of acetic acid which was the predominant SCFA in the fermentation reactor with the concentration of 100 mg/kg dry sludge was 1.8 fold of that in the control. Mechanism exploration revealed that the present phenanthrene mainly affected the acidification process of anaerobic fermentation and caused the shift of the microbial community to benefit the accumulation of acetic acid. Further investigation showed that both the activities of key enzymes (phosphotransacetylase and acetate kinase) involved in acetic acid production and the quantities of their corresponding encoding genes were enhanced in the presence of phenanthrene. Viability tests by determining the adenosine 5'-triphosphate content and membrane potential confirmed that the acetogens were more viable in anaerobic fermentation systems with phenanthrene, which resulted in the increased production of acetic acid.

  3. Topical ceramides neither enhance UVB-induced apoptosis in normal human keratinocytes nor affect viability in UVB-irradiated reconstructed human epidermis.

    PubMed

    Grether-Beck, Susanne; Felsner, Ingo; Koehler, Tim; Farwick, Mike; Lersch, Peter; Rawlings, Anthony V; Krutmann, Jean

    2014-11-01

    Ceramides are the major lipid of lamellar sheets present in intercellular spaces of the stratum corneum contributing to epidermal barrier properties. Therefore, ceramides and their analogues have been studied for barrier enhancing and water-holding properties for decades. In vitro studies have indicated cytotoxic potential for cell-permeable ceramides thereby raising the question whether topical ceramide application might contribute to UVB-induced apoptosis. Phytosphingosine, N-hexanoyl-phytosphingosine and N-stearoylphytosphingosine (ceramide III) in concentrations ≤5 μm have been used for co-stimulation with low (160 J/m(2) ) or high (600 J/m(2) ) UVB doses in subconfluent basal and confluent differentiating keratinocytes. Significantly, increased caspase-3 activity was observed in basal keratinocytes irradiated with 600 J/m(2) UVB and in differentiating keratinocytes with both UVB doses. Co-stimulation with the named ceramides did not further increase (i) caspase-3 activity and (ii) nucleosomal fragmentation in differentiating keratinocytes. Moreover, co-stimulation with 1-mm ceramides did not further affect viability/lactate dehydrogenase release in UVB-irradiated reconstructed human epidermis corroborating the safety of these ceramides.

  4. Irradiation affects cellular properties and Eph receptor expression in human melanoma cells

    PubMed Central

    Mosch, Birgit; Pietzsch, Doreen; Pietzsch, Jens

    2012-01-01

    X-ray irradiation influences metastatic properties of tumor cells and, moreover, metastasis and cellular motility can be modified by members of the Eph receptor/ephrin family of receptor tyrosine kinases. We hypothesized that irradiation-induced changes in cellular properties relevant for metastasis in melanoma cells could be mediated by Eph receptor/ephrin signaling. In this pilot study, we analyzed one pre-metastatic (Mel-Juso) and three metastatic human melanoma (Mel-Juso-L3, A375, and A2058) cells lines and predominantly found anti-metastatic effects of X-ray irradiation with impaired cell growth, clonal growth and motility. Additionally, we observed an irradiation-induced increase in adhesion paralleled by a decrease in migration in Mel-Juso and Mel-Juso-L3 cells and, in part, also in A375 cells. We further demonstrate a decrease of EphA2 both in expression and activity at 7 d after irradiation paralleled by an upregulation of EphA3. Analyzing downstream signaling after irradiation, we detected decreased Src kinase phosphorylation, but unchanged focal adhesion kinase (FAK) phosphorylation, indicating, in part, irradiation-induced downregulation of signaling via the EphA2-Src-FAK axis in melanoma cells. However, to which extent this finding contributes to the modification of metastasis-relevant cellular properties remains to be elucidated. PMID:22568947

  5. Clostridium botulinum type C hemagglutinin affects the morphology and viability of cultured mammalian cells via binding to the ganglioside GM3.

    PubMed

    Sugawara, Yo; Iwamori, Masao; Matsumura, Takuhiro; Yutani, Masahiro; Amatsu, Sho; Fujinaga, Yukako

    2015-09-01

    Botulinum neurotoxin is conventionally divided into seven serotypes, designated A-G, and is produced as large protein complexes through associations with non-toxic components, such as hemagglutinin (HA) and non-toxic non-HA. These non-toxic proteins dramatically enhance the oral toxicity of the toxin complex. HA is considered to have a role in toxin transport through the intestinal epithelium by carbohydrate binding and epithelial barrier-disrupting activity. Type A and B HAs disrupt E-cadherin-mediated cell adhesion, and, in turn, the intercellular epithelial barrier. Type C HA (HA/C) disrupts the barrier function by affecting cell morphology and viability, the mechanism of which remains unknown. In this study, we identified GM3 as the target molecule of HA/C. We found that sialic acid binding of HA is essential for the activity. It was abolished when cells were pre-treated with an inhibitor of ganglioside synthesis. Consistent with this, HA/C bound to a-series gangliosides in a glycan array. In parallel, we isolated clones resistant to HA/C activity from a susceptible mouse fibroblast strain. These cells lacked expression of ST-I, the enzyme that transfers sialic acid to lactosylceramide to yield GM3. These clones became sensitive to HA/C activity when GM3 was expressed by transfection with the ST-I gene. The sensitivity of fibroblasts to HA/C was reduced by expressing ganglioside synthesis genes whose products utilize GM3 as a substrate and consequently generate other a-series gangliosides, suggesting a GM3-specific mechanism. Our results demonstrate that HA/C affects cells in a GM3-dependent manner.

  6. Functional characterization of calliphorid cell death genes and cellularization gene promoters for controlling gene expression and cell viability in early embryos.

    PubMed

    Edman, R M; Linger, R J; Belikoff, E J; Li, F; Sze, S-H; Tarone, A M; Scott, M J

    2015-02-01

    The New World screwworm fly, Cochliomyia hominivorax, and the Australian sheep blow fly, Lucilia cuprina, are major pests of livestock. The sterile insect technique was used to eradicate C. hominivorax from North and Central America. This involved area-wide releases of male and female flies that had been sterilized by radiation. Genetic systems have been developed for making 'male-only' strains that would improve the efficiency of genetic control of insect pests. One system involves induction of female lethality in embryos through activation of a pro-apoptotic gene by the tetracycline-dependent transactivator. Sex-specific expression is achieved using an intron from the transformer gene, which we previously isolated from several calliphorids. In the present study, we report the isolation of the promoters from the C. hominivorax slam and Lucilia sericata bnk cellularization genes and show that these promoters can drive expression of a GFP reporter gene in early embryos of transgenic L. cuprina. Additionally, we report the isolation of the L. sericata pro-apoptotic hid and rpr genes, identify conserved motifs in the encoded proteins and determine the relative expression of these genes at different stages of development. We show that widespread expression of the L. sericata pro-apoptotic genes was lethal in Drosophila melanogaster. The isolated gene promoters and pro-apoptotic genes could potentially be used to build transgenic embryonic sexing strains of calliphorid livestock pests.

  7. How Might the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 Affect the Financial Viability of Rural Pharmacies? An Analysis of Preimplementation Prescription Volume and Payment Sources in Rural and Urban Areas

    ERIC Educational Resources Information Center

    Fraher, Erin P.; Slifkin, Rebecca T.; Smith, Laura; Randolph, Randy; Rudolf, Matthew; Holmes, George M.

    2005-01-01

    Passage of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (MMA) has created interest in how the legislation will affect access to prescription drugs among rural beneficiaries. Policy attention has focused to a much lesser degree on the implications of the MMA for the financial viability of rural pharmacies. This article…

  8. Slight temperature changes affect protein affinity and cellular uptake/toxicity of nanoparticles

    NASA Astrophysics Data System (ADS)

    Mahmoudi, Morteza; Shokrgozar, Mohammad A.; Behzadi, Shahed

    2013-03-01

    It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular/organ temperature.It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular

  9. BmNHR96 participate BV entry of BmN-SWU1 cells via affecting the cellular cholesterol level.

    PubMed

    Dong, Xiao-Long; Liu, Tai-Hang; Wang, Wei; Pan, Cai-Xia; Du, Guo-Yu; Wu, Yun-Fei; Pan, Min-Hui; Lu, Cheng

    2017-01-22

    B.mori nucleopolyhedrovirus (BmNPV), which produces BV and ODV two virion phenotypes in its life cycle, caused the amount of economic loss in sericulture. But the mechanism of its infection was still unclear. In this study we characterized B.mori nuclear hormone receptor 96 (BmNHR96) as a NHR96 family member, which was localized in the nucleus. We also found BmNHR96 over-expression could enhance the entry of BV as well as cellular cholesterol level. Furthermore, we validated that BmNHR96 increased membrane fusion mediated by GP64, which could probably promote BV-infection. In summary, our study suggested that BmNHR96 plays an important role in BV infection and this function probably actualized by affecting cellular cholesterol level, and our results provided insights to the mechanisms of BV-infection of B.mori.

  10. Docosahexaenoic Acid Sensitizes Leukemia Lymphocytes to Barasertib and Everolimus by ROS-dependent Mechanism Without Affecting the Level of ROS and Viability of Normal Lymphocytes.

    PubMed

    Zhelev, Zhivko; Ivanova, Donika; Lazarova, Desislava; Aoki, Ichio; Bakalova, Rumiana; Saga, Tsuneo

    2016-04-01

    The aim of the present study was: (i) to investigate the possibility of sensitizing leukemia lymphocytes to anticancer drugs using docosahexaenoic acid (DHA); (ii) to find combinations with synergistic cytotoxic effect on leukemia lymphocytes, without or with only very low cytotoxicity towards normal lymphocytes; (iii) and to clarify the role of reactive oxygen species (ROS) in the induction of apoptosis and cytotoxicity by such combinations. The study covered 15 anticancer drugs, conventional and new-generation. Well-expressed synergistic cytotoxic effects were observed after treatment of leukemia lymphocytes (Jurkat) with DHA in combination with: barasertib, lonafarnib, everolimus, and palbociclib. We selected two synergistic combinations, DHA with everolimus or barasertib, and investigated their effects on viability of normal lymphocytes, as well as on the production of ROS and induction of apoptosis in both cell lines (leukemia and normal). At the selected concentrations, DHA, everolimus and barasertib (applied separately) were cytotoxic towards leukemia lymphocytes, but not normal lymphocytes. In leukemia cells, the cytotoxicity of combinations was accompanied by strong induction of apoptosis and production of ROS. In normal lymphocytes, drugs alone and in combination with DHA did not affect the level of ROS and did not induce apoptosis. To our knowledge, the present study is the first to report synergistic ROS-dependent cytotoxicity between DHA and new-generation anticancer drugs, such as everolimus and barasertib, that is cancer cell-specific (particularly for acute lymphoblastic leukemia cells Jurkat). These combinations are harmless to normal lymphocytes and do not induce abnormal production of ROS in these cells. The data suggest that DHA could be used as a supplementary component in anticancer chemotherapy, allowing therapeutic doses of everolimus and barasertib to be reduced, minimizing their side-effects.

  11. Ablation of CBP in forebrain principal neurons causes modest memory and transcriptional defects and a dramatic reduction of histone acetylation but does not affect cell viability.

    PubMed

    Valor, Luis M; Pulopulos, Matias M; Jimenez-Minchan, Maria; Olivares, Roman; Lutz, Beat; Barco, Angel

    2011-02-02

    Rubinstein-Taybi syndrome (RSTS) is an inheritable disease associated with mutations in the gene encoding the CREB (cAMP response element-binding protein)-binding protein (CBP) and characterized by growth impairment, learning disabilities, and distinctive facial and skeletal features. Studies in mouse models for RSTS first suggested a direct role for CBP and histone acetylation in cognition and memory. Here, we took advantage of the genetic tools for generating mice in which the CBP gene is specifically deleted in postmitotic principal neurons of the forebrain to investigate the consequences of the loss of CBP in the adult brain. In contrast to the conventional CBP knock-out mice, which exhibit very early embryonic lethality, postnatal forebrain-restricted CBP mutants were viable and displayed no overt abnormalities. We identified the dimer of histones H2A and H2B as the preferred substrate of the histone acetyltransferase domain of CBP. Surprisingly, the loss of CBP and subsequent histone hypoacetylation had a very modest impact in the expression of a number of immediate early genes and did not affect neuronal viability. In addition, the behavioral characterization of these mice dissociated embryonic and postnatal deficits caused by impaired CBP function, narrowed down the anatomical substrate of specific behavioral defects, and confirmed the special sensitivity of object recognition memory to CBP deficiency. Overall, our study provides novel insights into RSTS etiology and clarifies some of the standing questions concerning the role of CBP and histone acetylation in activity-driven gene expression, memory formation, and neurodegeneration.

  12. Ocean acidification affects competition for space: projections of community structure using cellular automata

    PubMed Central

    2016-01-01

    Historical ecological datasets from a coastal marine community of crustose coralline algae (CCA) enabled the documentation of ecological changes in this community over 30 years in the Northeast Pacific. Data on competitive interactions obtained from field surveys showed concordance between the 1980s and 2013, yet also revealed a reduction in how strongly species interact. Here, we extend these empirical findings with a cellular automaton model to forecast ecological dynamics. Our model suggests the emergence of a new dominant competitor in a global change scenario, with a reduced role of herbivory pressure, or trophic control, in regulating competition among CCA. Ocean acidification, due to its energetic demands, may now instead play this role in mediating competitive interactions and thereby promote species diversity within this guild. PMID:26936244

  13. Ocean acidification affects competition for space: projections of community structure using cellular automata.

    PubMed

    McCoy, Sophie J; Allesina, Stefano; Pfister, Catherine A

    2016-03-16

    Historical ecological datasets from a coastal marine community of crustose coralline algae (CCA) enabled the documentation of ecological changes in this community over 30 years in the Northeast Pacific. Data on competitive interactions obtained from field surveys showed concordance between the 1980s and 2013, yet also revealed a reduction in how strongly species interact. Here, we extend these empirical findings with a cellular automaton model to forecast ecological dynamics. Our model suggests the emergence of a new dominant competitor in a global change scenario, with a reduced role of herbivory pressure, or trophic control, in regulating competition among CCA. Ocean acidification, due to its energetic demands, may now instead play this role in mediating competitive interactions and thereby promote species diversity within this guild.

  14. Myocardial viability.

    PubMed Central

    Birnbaum, Y; Kloner, R A

    1996-01-01

    Left ventricular function is a major predictor of outcome in patients with coronary artery disease. Acute ischemia, postischemic dysfunction (stunning), myocardial hibernation, or a combination of these 3 are among the reversible forms of myocardial dysfunction. In myocardial stunning, dysfunction occurs despite normal myocardial perfusion, and function recovers spontaneously over time. In acute ischemia and hibernation, there is regional hypoperfusion. Function improves only after revascularization. Evidence of myocardial viability usually relies on the demonstration of uptake of various metabolic tracers, such as thallium (thallous chloride TI 201) or fludeoxyglucose F 18, by dysfunctional myocardium or by the demonstration of contractile reserve in a dysfunctional region. This can be shown as an augmentation of function during the infusion of various sympathomimetic agents. The response of ventricular segments to increasing doses of dobutamine may indicate the underlying mechanism of dysfunction. Stunned segments that have normal perfusion show dose-dependent augmentation of function. If perfusion is reduced as in hibernating myocardium, however, a biphasic response usually occurs: function improves at low doses of dobutamine, whereas higher doses may induce ischemia and, hence, dysfunction. But in patients with severely impaired perfusion, even low doses may cause ischemia. Myocardial regions with subendocardial infarction or diffuse scarring may also have augmented contractility during catecholamine infusion due to stimulation of the subepicardial layers. In these cases, augmentation of function after revascularization is not expected. Because the underlying mechanism, prognosis, and therapy may differ among these conditions, it is crucial to differentiate among dysfunctional myocardial segments that are nonviable and have no potential to regain function, hibernating or ischemic segments in which recovery of function occurs only after revascularization, and

  15. Can common adhesion molecules and microtopography affect cellular elasticity? A combined atomic force microscopy and optical study.

    PubMed

    McPhee, Gordon; Dalby, Matthew J; Riehle, Mathis; Yin, Huabing

    2010-10-01

    The phenomenon that cells respond to chemical and topographic cues in their surroundings has been widely examined and exploited in many fields ranging from basic life science research to biomedical therapeutics. Adhesion promoting molecules such as poly-L-lysine (PLL) and fibronectin (Fn) are commonly used for in vitro cell assays to promote cell spreading/proliferation on tissue culture plastic and to enhance the biocompatibility of biomedical devices. Likewise, engineered topography is often used to guide cell growth and differentiation. Little is known about how these cues affect the biomechanical properties of cells and subsequent cell function. In this study we have applied atomic force microscopy (AFM) to investigate these biomechanical properties. In the first stage of the study we formulated a rigorous approach to quantify cellular elasticity using AFM. Operational factors, including indentation depth and speed, and mathematical models for data fitting have been systematically evaluated. We then quantified how PLL, Fn and microtopography affected cellular elasticity and the organization of the cytoskeleton. Cellular elasticity after 1 day in culture was greater on a Fn-coated surface as compared to PLL or glass. These statistically significant differences disappeared after two more days in culture. In contrast, the significantly higher elasticity associated with cells grown on micrometric grooves remained for at least 3 days. This work sheds light on the apparently simple but debatable questions: "Are engineered chemical cues eventually masked by a cell's own matrix proteins and so only exert short-term influence? Does engineered topography as well as engineered chemistry affect cell elasticity?"

  16. Protein source and choice of anticoagulant decisively affect nanoparticle protein corona and cellular uptake

    NASA Astrophysics Data System (ADS)

    Schöttler, S.; Klein, Katja; Landfester, K.; Mailänder, V.

    2016-03-01

    Protein adsorption on nanoparticles has been a focus of the field of nanocarrier research in the past few years and more and more papers are dealing with increasingly detailed lists of proteins adsorbed to a plethora of nanocarriers. While there is an urgent need to understand the influence of this protein corona on nanocarriers' interactions with cells the strong impact of the protein source on corona formation and the consequence for interaction with different cell types are factors that are regularly neglected, but should be taken into account for a meaningful analysis. In this study, the importance of the choice of protein source used for in vitro protein corona analysis is concisely investigated. Major and decisive differences in cellular uptake of a polystyrene nanoparticle incubated in fetal bovine serum, human serum, human citrate and heparin plasma are reported. Furthermore, the protein compositions are determined for coronas formed in the respective incubation media. A strong influence of heparin, which is used as an anticoagulant for plasma generation, on cell interaction is demonstrated. While heparin enhances the uptake into macrophages, it prevents internalization into HeLa cells. Taken together we can give the recommendation that human plasma anticoagulated with citrate seems to give the most relevant results for in vitro studies of nanoparticle uptake.Protein adsorption on nanoparticles has been a focus of the field of nanocarrier research in the past few years and more and more papers are dealing with increasingly detailed lists of proteins adsorbed to a plethora of nanocarriers. While there is an urgent need to understand the influence of this protein corona on nanocarriers' interactions with cells the strong impact of the protein source on corona formation and the consequence for interaction with different cell types are factors that are regularly neglected, but should be taken into account for a meaningful analysis. In this study, the importance

  17. Fetal radiofrequency radiation exposure from 800-1900 mhz-rated cellular telephones affects neurodevelopment and behavior in mice.

    PubMed

    Aldad, Tamir S; Gan, Geliang; Gao, Xiao-Bing; Taylor, Hugh S

    2012-01-01

    Neurobehavioral disorders are increasingly prevalent in children, however their etiology is not well understood. An association between prenatal cellular telephone use and hyperactivity in children has been postulated, yet the direct effects of radiofrequency radiation exposure on neurodevelopment remain unknown. Here we used a mouse model to demonstrate that in-utero radiofrequency exposure from cellular telephones does affect adult behavior. Mice exposed in-utero were hyperactive and had impaired memory as determined using the object recognition, light/dark box and step-down assays. Whole cell patch clamp recordings of miniature excitatory postsynaptic currents (mEPSCs) revealed that these behavioral changes were due to altered neuronal developmental programming. Exposed mice had dose-responsive impaired glutamatergic synaptic transmission onto layer V pyramidal neurons of the prefrontal cortex. We present the first experimental evidence of neuropathology due to in-utero cellular telephone radiation. Further experiments are needed in humans or non-human primates to determine the risk of exposure during pregnancy.

  18. Fetal Radiofrequency Radiation Exposure From 800-1900 Mhz-Rated Cellular Telephones Affects Neurodevelopment and Behavior in Mice

    PubMed Central

    Aldad, Tamir S.; Gan, Geliang; Gao, Xiao-Bing; Taylor, Hugh S.

    2012-01-01

    Neurobehavioral disorders are increasingly prevalent in children, however their etiology is not well understood. An association between prenatal cellular telephone use and hyperactivity in children has been postulated, yet the direct effects of radiofrequency radiation exposure on neurodevelopment remain unknown. Here we used a mouse model to demonstrate that in-utero radiofrequency exposure from cellular telephones does affect adult behavior. Mice exposed in-utero were hyperactive and had impaired memory as determined using the object recognition, light/dark box and step-down assays. Whole cell patch clamp recordings of miniature excitatory postsynaptic currents (mEPSCs) revealed that these behavioral changes were due to altered neuronal developmental programming. Exposed mice had dose-responsive impaired glutamatergic synaptic transmission onto layer V pyramidal neurons of the prefrontal cortex. We present the first experimental evidence of neuropathology due to in-utero cellular telephone radiation. Further experiments are needed in humans or non-human primates to determine the risk of exposure during pregnancy. PMID:22428084

  19. Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore.

    PubMed

    Zhang, Zhifen; Park, Minyoung; Tam, John; Auger, Anick; Beilhartz, Greg L; Lacy, D Borden; Melnyk, Roman A

    2014-03-11

    Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues--clustered between amino acids 1,035 and 1,107--that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming "hotspots" of TcdB and the well-characterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.

  20. Cellular senescence induced by prolonged subculture adversely affects glutamate uptake in C6 lineage.

    PubMed

    Pereira, Mery Stéfani Leivas; Zenki, Kamila; Cavalheiro, Marcela Mendonça; Thomé, Chairini Cássia; Filippi-Chiela, Eduardo Cremonese; Lenz, Guido; de Souza, Diogo Onofre Gomes; de Oliveira, Diogo Losch

    2014-05-01

    Several researchers have recently used C6 cells to evaluate functional properties of high-affinity glutamate transporters. However, it has been demonstrated that this lineage suffers several morphological and biochemical alterations according to the number of passages in culture. Currently, there are no reports showing whether functional properties of high-affinity glutamate transporters comply with these sub culturing-dependent modifications. The present study aimed to compare the functional properties of high-affinity glutamate transporters expressed in early (EPC6) and late (LPC6) passage C6 cells through a detailed pharmacological and biochemical characterization. Between 60-180 min of L-[(3)H]glu incubation, LPC6 presented an intracellular [(3)H] 55% lower than EPC6. Both cultures showed a time-dependent increase of intracellular [(3)H] reaching maximal levels at 120 min. Cultures incubated with D-[(3)H]asp showed a time-dependent increase of [(3)H] until 180 min. Moreover, LPC6 have a D-[(3)H]asp-derived intracellular [(3)H] 30-45% lower than EPC6 until 120 min. Only EAAT3 was immunodetected in cultures and its total content was equal between them. PMA-stimulated EAAT3 trafficking to membrane increased 50% of L-[(3)H]glu-derived intracellular [(3)H] in EPC6 and had no effect in LPC6. LPC6 displayed characteristics that resemble senescence, such as high β-Gal staining, cell enlargement and increase of large and regular nuclei. Our results demonstrated that LPC6 exhibited glutamate uptake impairment, which may have occurred due to its inability to mobilize EAAT3 to cell membrane. This profile might be related to senescent process observed in this culture. Our results suggest that LPC6 cells are an inappropriate glial cellular model to investigate the functional properties of high-affinity glutamate transporters.

  1. Mutations in MCT8 in patients with Allan-Herndon-Dudley-syndrome affecting its cellular distribution.

    PubMed

    Kersseboom, Simone; Kremers, Gert-Jan; Friesema, Edith C H; Visser, W Edward; Klootwijk, Wim; Peeters, Robin P; Visser, Theo J

    2013-05-01

    Monocarboxylate transporter 8 (MCT8) is a thyroid hormone (TH)-specific transporter. Mutations in the MCT8 gene are associated with Allan-Herndon-Dudley Syndrome (AHDS), consisting of severe psychomotor retardation and disturbed TH parameters. To study the functional consequences of different MCT8 mutations in detail, we combined functional analysis in different cell types with live-cell imaging of the cellular distribution of seven mutations that we identified in patients with AHDS. We used two cell models to study the mutations in vitro: 1) transiently transfected COS1 and JEG3 cells, and 2) stably transfected Flp-in 293 cells expressing a MCT8-cyan fluorescent protein construct. All seven mutants were expressed at the protein level and showed a defect in T3 and T4 transport in uptake and metabolism studies. Three mutants (G282C, P537L, and G558D) had residual uptake activity in Flp-in 293 and COS1 cells, but not in JEG3 cells. Four mutants (G221R, P321L, D453V, P537L) were expressed at the plasma membrane. The mobility in the plasma membrane of P537L was similar to WT, but the mobility of P321L was altered. The other mutants studied (insV236, G282C, G558D) were predominantly localized in the endoplasmic reticulum. In essence, loss of function by MCT8 mutations can be divided in two groups: mutations that result in partial or complete loss of transport activity (G221R, P321L, D453V, P537L) and mutations that mainly disturb protein expression and trafficking (insV236, G282C, G558D). The cell type-dependent results suggest that MCT8 mutations in AHDS patients may have tissue-specific effects on TH transport probably caused by tissue-specific expression of yet unknown MCT8-interacting proteins.

  2. Histological Lesions and Cellular Response in the Skin of Alpine Chamois (Rupicapra r. rupicapra) Spontaneously Affected by Sarcoptic Mange

    PubMed Central

    Salvadori, Claudia; Lazzarotti, Camilla; Trogu, Tiziana; Lanfranchi, Paolo

    2016-01-01

    Population dynamics of chamois (genus Rupicapra, subfamily Caprinae) can be influenced by infectious diseases epizootics, of which sarcoptic mange is probably the most severe in the Alpine chamois (Rupicapra rupicapra rupicapra). In this study, skin lesions and cellular inflammatory infiltrates were characterized in 44 Alpine chamois affected by sarcoptic mange. Dermal cellular responses were evaluated in comparison with chamois affected by trombiculosis and controls. In both sarcoptic mange and trombiculosis, a significantly increase of eosinophils, mast cells, T and B lymphocytes, and macrophages was detected. Moreover, in sarcoptic mange significant higher numbers of T lymphocytes and macrophages compared to trombiculosis were observed. Lesions in sarcoptic mange were classified in three grades, according to crusts thickness, correlated with mite counts. Grade 3 represented the most severe form with crust thickness more than 3.5 mm, high number of mites, and severe parakeratosis with diffuse bacteria. Evidence of immediate and delayed hypersensitivity was detected in all three forms associated with diffuse severe epidermal hyperplasia. In grade 3, a significant increase of B lymphocytes was evident compared to grades 1 and 2, while eosinophil counts were significantly higher than in grade 1, but lower than in grade 2 lesions. An involvement of nonprotective Th2 immune response could in part account for severe lesions of grade 3. PMID:27403422

  3. Early-life adversity accelerates cellular ageing and affects adult inflammation: Experimental evidence from the European starling

    PubMed Central

    Nettle, Daniel; Andrews, Clare; Reichert, Sophie; Bedford, Tom; Kolenda, Claire; Parker, Craig; Martin-Ruiz, Carmen; Monaghan, Pat; Bateson, Melissa

    2017-01-01

    Early-life adversity is associated with accelerated cellular ageing during development and increased inflammation during adulthood. However, human studies can only establish correlation, not causation, and existing experimental animal approaches alter multiple components of early-life adversity simultaneously. We developed a novel hand-rearing paradigm in European starling nestlings (Sturnus vulgaris), in which we separately manipulated nutritional shortfall and begging effort for a period of 10 days. The experimental treatments accelerated erythrocyte telomere attrition and increased DNA damage measured in the juvenile period. For telomere attrition, amount of food and begging effort exerted additive effects. Only the combination of low food amount and high begging effort increased DNA damage. We then measured two markers of inflammation, high-sensitivity C-reactive protein and interleukin-6, when the birds were adults. The experimental treatments affected both inflammatory markers, though the patterns were complex and different for each marker. The effect of the experimental treatments on adult interleukin-6 was partially mediated by increased juvenile DNA damage. Our results show that both nutritional input and begging effort in the nestling period affect cellular ageing and adult inflammation in the starling. However, the pattern of effects is different for different biomarkers measured at different time points. PMID:28094324

  4. Modeling physicochemical interactions affecting in vitro cellular dosimetry of engineered nanomaterials: application to nanosilver

    NASA Astrophysics Data System (ADS)

    Mukherjee, Dwaipayan; Leo, Bey Fen; Royce, Steven G.; Porter, Alexandra E.; Ryan, Mary P.; Schwander, Stephan; Chung, Kian Fan; Tetley, Teresa D.; Zhang, Junfeng; Georgopoulos, Panos G.

    2014-10-01

    Engineered nanomaterials (ENMs) possess unique characteristics affecting their interactions in biological media and biological tissues. Systematic investigation of the effects of particle properties on biological toxicity requires a comprehensive modeling framework which can be used to predict ENM particokinetics in a variety of media. The Agglomeration-diffusion-sedimentation-reaction model (ADSRM) described here is stochastic, using a direct simulation Monte Carlo method to study the evolution of nanoparticles in biological media, as they interact with each other and with the media over time. Nanoparticle diffusion, gravitational settling, agglomeration, and dissolution are treated in a mechanistic manner with focus on silver ENMs (AgNPs). The ADSRM model utilizes particle properties such as size, density, zeta potential, and coating material, along with medium properties like density, viscosity, ionic strength, and pH, to model evolving patterns in a population of ENMs along with their interaction with associated ions and molecules. The model predictions for agglomeration and dissolution are compared with in vitro measurements for various types of ENMs, coating materials, and incubation media, and are found to be overall consistent with measurements. The model has been implemented for an in vitro case in cell culture systems to inform in vitro dosimetry for toxicology studies, and can be directly extended to other biological systems, including in vivo tissue sub-systems by suitably modifying system geometry.

  5. Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factors

    PubMed Central

    Peiris, Diluka; Spector, Alexander F.; Lomax-Browne, Hannah; Azimi, Tayebeh; Ramesh, Bala; Loizidou, Marilena; Welch, Hazel; Dwek, Miriam V.

    2017-01-01

    Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 μM DXR P < 0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P < 0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer. PMID:28223691

  6. In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution.

    PubMed

    Gasperini, Lisa; Meneghetti, Elisa; Legname, Giuseppe; Benetti, Federico

    2016-01-01

    Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments.

  7. In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution

    PubMed Central

    Gasperini, Lisa; Meneghetti, Elisa; Legname, Giuseppe; Benetti, Federico

    2016-01-01

    Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments. PMID:27729845

  8. Obatoclax, saliphenylhalamide and gemcitabine inhibit Zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism.

    PubMed

    Kuivanen, Suvi; Bespalov, Maxim M; Nandania, Jatin; Ianevski, Aleksandr; Velagapudi, Vidya; De Brabander, Jef K; Kainov, Denis E; Vapalahti, Olli

    2017-03-01

    An epidemic of Zika virus (ZIKV) infection associated with congenital abnormalities such as microcephaly, is ongoing in the Americas and the Pacific. Currently there are no approved therapies to treat this emerging viral disease. Here, we tested three cell-directed broad-spectrum antiviral compounds against ZIKV replication using human retinal pigment epithelial (RPE) cells and a low-passage ZIKV strain isolated from fetal brain. We found that obatoclax, SaliPhe, and gemcitabine inhibited ZIKV infections at noncytotoxic concentrations. Moreover, all three compounds prevented production of viral RNA and proteins as well as activation of cellular caspase 8, 3 and 7. However, these compounds differentially affected ZIKV-mediated transcription, translation and posttranslational modifications of cellular factors as well as metabolic pathways indicating that these agents possess different mechanisms of action. Interestingly, combination of obatoclax and SaliPhe at nanomolar concentrations had a synergistic effect against ZIKV infection. Thus, our results provided the foundation for development of broad-spectrum cell-directed antivirals or their combinations for treatment of ZIKV and other emerging viral diseases.

  9. Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

    PubMed Central

    de Moraes, Vanessa C S; Bernardinelli, Emanuele; Zocal, Nathalia; Fernandez, Jhonathan A; Nofziger, Charity; Castilho, Arthur M; Sartorato, Edi L; Paulmichl, Markus; Dossena, Silvia

    2016-01-01

    Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants. PMID:26752218

  10. CD147 mediates chemoresistance in breast cancer via ABCG2 by affecting its cellular localization and dimerization.

    PubMed

    Zhou, Shuangyuan; Liao, Liqiu; Chen, Chen; Zeng, Weiqi; Liu, Shuang; Su, Juan; Zhao, Shuang; Chen, Mingliang; Kuang, Yehong; Chen, Xiang; Li, Jie

    2013-09-01

    CD147 and ABCG2 both have been reported to mediate Multidrug resistance (MDR) in breast cancer. Recent study demonstrates that CD147 could form a complex with ABCG2 on the cell membrane in primary effusion lymphoma. However, whether these two molecules regulate each other in breast cancer and result in MDR is not clear. We established four MCF-7 cell lines transfected with CD147 and/or ABCG2 and found that CD147 could increase the expression and dimerization of ABCG2, affect its cellular localization and regulate its drug transporter function. The findings derived from cells were confirmed subsequently in clinic samples of chemotherapy-sensitive/resistant breast cancer.

  11. Identification of multiple cellular uptake pathways of polystyrene nanoparticles and factors affecting the uptake: relevance for drug delivery systems.

    PubMed

    Firdessa, Rebuma; Oelschlaeger, Tobias A; Moll, Heidrun

    2014-01-01

    Nanoparticles may address challenges by human diseases through improving diagnosis, vaccination and treatment. The uptake mechanism regulates the type of threat a particle poses on the host cells and how a cell responds to it. Hence, understanding the uptake mechanisms and cellular interactions of nanoparticles at the cellular and subcellular level is a prerequisite for their effective biomedical applications. The present study shows the uptake mechanisms of polystyrene nanoparticles and factors affecting their uptake in bone marrow-derived macrophages, 293T kidney epithelial cells and L929 fibroblasts. Labeling with the endocytic marker FM4-64 and transmission electron microscopy studies show that the nanoparticles were internalized rapidly via endocytosis and accumulated in intracellular vesicles. Soon after their internalizations, nanoparticles trafficked to organelles with acidic pH. Analysis of the ultrastructural morphology of the plasma membrane invaginations or extravasations provides clear evidence for the involvement of several uptake routes in parallel to internalize a given type of nanoparticles by mammalian cells, highlighting the complexity of the nanoparticle-cell interactions. Blocking the specific endocytic pathways by different pharmacological inhibitors shows similar outcomes. The potential to take up nanoparticles varies highly among different cell types in a particle sizes-, time- and energy-dependent manner. Furthermore, infection and the activation status of bone marrow-derived macrophages significantly affect the uptake potential of the cells, indicating the need to understand the diseases' pathogenesis to establish effective and rational drug-delivery systems. This study enhances our understanding of the application of nanotechnology in biomedical sciences.

  12. Short-term acute hypercapnia affects cellular responses to trace metals in the hard clams Mercenaria mercenaria.

    PubMed

    Ivanina, Anna V; Beniash, Elia; Etzkorn, Markus; Meyers, Tiffany B; Ringwood, Amy H; Sokolova, Inna M

    2013-09-15

    Estuarine and coastal habitats experience large fluctuations of environmental factors such as temperature, salinity, partial pressure of CO2 ( [Formula: see text] ) and pH; they also serve as the natural sinks for trace metals. Benthic filter-feeding organisms such as bivalves are exposed to the elevated concentrations of metals in estuarine water and sediments that can strongly affect their physiology. The effects of metals on estuarine organisms may be exacerbated by other environmental factors. Thus, a decrease in pH caused by high [Formula: see text] (hypercapnia) can modulate the effects of trace metals by affecting metal bioavailability, accumulation or binding. To better understand the cellular mechanisms of interactions between [Formula: see text] and trace metals in marine bivalves, we exposed isolated mantle cells of the hard clams (Mercenaria mercenaria) to different levels of [Formula: see text] (0.05, 1.52 and 3.01 kPa) and two major trace metal pollutants - cadmium (Cd) and copper (Cu). Elevated [Formula: see text] resulted in a decrease in intracellular pH (pHi) of the isolated mantle cells from 7.8 to 7.4. Elevated [Formula: see text] significantly but differently affected the trace metal accumulation by the cells. Cd uptake was suppressed at elevated [Formula: see text] levels while Cu accumulation has greatly accelerated under hypercapnic conditions. Interestingly, at higher extracellular Cd levels, labile intracellular Cd(2+) concentration remained the same, while intracellular levels of free Zn(2+) increased suggesting that Cd(2+) substitutes bound Zn(2+) in these cells. In contrast, Cu exposure did not affect intracellular Zn(2+) but led to a profound increase in the intracellular levels of labile Cu(2+) and Fe(2+). An increase in the extracellular concentrations of Cd and Cu led to the elevated production of reactive oxygen species under the normocapnic conditions (0.05 kPa [Formula: see text] ); surprisingly, this effect was mitigated in

  13. Stress, Inflammation, and Cellular Vulnerability during Early Stages of Affective Disorders: Biomarker Strategies and Opportunities for Prevention and Intervention

    PubMed Central

    Walker, Adam J.; Kim, Yesul; Price, J. Blair; Kale, Rajas P.; McGillivray, Jane A.; Berk, Michael; Tye, Susannah J.

    2014-01-01

    The mood disorder prodrome is conceptualized as a symptomatic, but not yet clinically diagnosable stage of an affective disorder. Although a growing area, more focused research is needed in the pediatric population to better characterize psychopathological symptoms and biological markers that can reliably identify this very early stage in the evolution of mood disorder pathology. Such information will facilitate early prevention and intervention, which has the potential to affect a person’s disease course. This review focuses on the prodromal characteristics, risk factors, and neurobiological mechanisms of mood disorders. In particular, we consider the influence of early-life stress, inflammation, and allostatic load in mediating neural mechanisms of neuroprogression. These inherently modifiable factors have known neuroadaptive and neurodegenerative implications, and consequently may provide useful biomarker targets. Identification of these factors early in the course of the disease will accordingly allow for the introduction of early interventions which augment an individual’s capacity for psychological resilience through maintenance of synaptic integrity and cellular resilience. A targeted and complementary approach to boosting both psychological and physiological resilience simultaneously during the prodromal stage of mood disorder pathology has the greatest promise for optimizing the neurodevelopmental potential of those individuals at risk of disabling mood disorders. PMID:24782789

  14. Oxidative stress affects FET proteins localization and alternative pre-mRNA processing in cellular models of ALS.

    PubMed

    Svetoni, Francesca; Caporossi, Daniela; Paronetto, Maria Paola

    2014-10-01

    FUS/TLS, EWS and TAF15 are members of the FET family of DNA and RNA binding proteins, involved in multiple steps of DNA and RNA processing and implicated in the regulation of gene expression and cell-signaling. All members of the FET family contribute to human pathologies, as they are involved in sarcoma translocations and neurodegenerative diseases. Mutations in FUS/TLS, in EWSR1 and in TAF15 genescause Amyotrophic Lateral Sclerosis (ALS), a fatal human neurodegenerative disease that affects primarily motor neurons and is characterized by the progressive loss of motor neurons and degradation of the neuromuscular junctions.ALS-associated FET mutations cause FET protein relocalization into cytoplasmic aggregates, thus impairing their normal function. Protein aggregation has been suggested as a co-opting factor during the disease pathogenesis. Cytoplasmic mislocalization of FET proteins contributes to the formation of cytoplasmic aggregates that may alter RNA processing and initiate motor neuron degeneration. Interestingly, oxidative stress, which is implicated in the pathogenesis of ALS, triggers the accumulation of mutant FUS in cytoplasmic stress granules where it binds and sequester wild-type FUS.In order to evaluate the role of FET proteins in ALS and their involvement in the response to oxidative stress, we have developed cellular models of ALS expressing ALS-related FET mutants in neuroblastoma cell lines. Upon treatment with sodium arsenite, cells were analysed by immunofluorescence to monitor the localization of wild-type and mutated FET proteins. Furthermore, we have characterized signal transduction pathways and cell survival upon oxidative stress in our cellular models of ALS. Interestingly, we found that EWS mutant proteins display a different localization from FUS mutants and neither wild-type nor mutated EWS protein translocate into stress granules upon oxidative stress treatment. Collectively, our data provide a new link between the oxidative stress

  15. Inefficiency in macromolecular transport of SCS-based microcapsules affects viability of primary human mesenchymal stem cells but not of immortalized cells.

    PubMed

    Sanz-Nogués, Clara; Horan, Jason; Thompson, Kerry; Howard, Linda; Ryan, Gerard; Kassem, Moustapha; O'Brien, Timothy

    2015-11-01

    Microcapsules made of sodium cellulose sulphate (SCS) and poly-diallyl-dimethyl-ammonium chloride (pDADMAC) have been employed to encapsulate a wide range of established cell lines for several applications. However, little is known about the encapsulation of primary cells including human mesenchymal stem cells (hMSCs). Human MSCs are of interest in regenerative medicine applications due to pro-angiogenic, anti-inflammatory and immunomodulatory properties, which result from paracrine effects of this cell type. In the present work we have encapsulated primary hMSCs and hMSC-TERT immortalized cells and compared their behavior and in vitro angiogenic potential. We found that, although both cell types were able to secret angiogenic factors such as VEGF, there was a marked reduction of primary hMSC viability compared to hMSC-TERT cells when cultured in these microcapsules. Moreover, this applied to other primary cell cultures such as primary human fibroblasts but not to other cell lines such as human embryonic kidney 293 (HEK293) cells. We found that the microcapsule membrane had a molecular weight cut-off below a critical size, which caused impairment in the diffusion of essential nutrients and had a more detrimental effect on the viability of primary cell cultures compared to cell lines and immortalized cells.

  16. Photosynthesis, water use, and root viability under water stress as affected by expression of SAG12-ipt controlling cytokinin synthesis in Agrostis stolonifera.

    PubMed

    Merewitz, Emily B; Gianfagna, Thomas; Huang, Bingru

    2011-01-01

    Water stress reduces endogenous cytokinin (CK) content and may inhibit CK production. Maintenance of endogenous CK levels by genetic transformation with ipt in leaves and roots undergoing senescence may promote stress tolerance. This study was designed to determine the physiological effects of ipt expression on immature and mature leaves and in roots for plants exposed to different levels of water stress for creeping bentgrass (Agrostis stolonifera). Plants containing the ipt gene, encoding the enzyme adenine isopentenyl phosphotransferase for CK synthesis ligated to a senescence-activated promoter (SAG12), and wild-type 'Penncross' (WT) were grown hydroponically in a growth chamber and exposed to water stress by weekly additions of polyethylene glycol 8000 to reduce the growing solution osmotic potential from -0.05 to -0.3, -0.5, -0.7, -1.0, and -1.4 MPa. Immature and mature leaves and roots of SAG12-ipt creeping bentgrass were evaluated for ipt expression, CK content, leaf relative water content (RWC), chlorophyll content (Chl), photochemical efficiency (F(v)F(m)), osmotic adjustment (OA), photosynthesis rate (Pn), stomatal conductance (g(s)), transpiration (E), water use efficiency (WUE), carbon isotope discrimination (Δ), and root viability. Expression of ipt was detected in all plant parts and a higher CK content, primarily in the form of isopentyladenine (iPa), was found in SAG12-ipt plants but not in the WT plants under water stress. Immature leaves exhibited higher iPa and OA at all treatment levels. Mature leaves of SAG12-ipt plants maintained higher OA, Pn, Chl, WUE, and Δ, whereas g(s) and E were relatively unaffected compared to the WT. Roots of SAG12-ipt plants had higher levels of iPa and greater root viability than the WT. The results demonstrate that expression of ipt enhanced the tolerance of creeping bentgrass to water stress, which could be attributed to the positive effects on osmotic adjustment, efficient water use, and maintaining higher

  17. Superoxide dismutase affects the viability of thawed European mouflon (Ovis g. musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection.

    PubMed

    Berlinguer, Fiammetta; Ledda, Sergio; Rosati, Irma; Bogliolo, Luisa; Leoni, Giovanni; Naitana, Salvatore

    2003-01-01

    This study evaluated the effects of superoxide dismutase (SOD) on viability and acrosome integrity of European mouflon spermatozoa after cryopreservation and on the fertilization rates of sheep oocytes after i.v.f. or intracytoplasmatic sperm injection (i.c.s.i.). Frozen semen was thawed and washed with synthetic oviduct fluid supplemented with 0.6% bovine serum albumin. After centrifugation, the spermatozoa pellet was split into two culture systems: (i) without SOD; and (ii) in the presence of 1500 IU mL(-1) SOD. Sperm viability and acrosome integrity were evaluated simultaneously, immediately after thawing and after 3, 6 and 9 h of culture (5% CO2, 39 degrees C, 90% humidity), by incubating sperm with propidium iodide and fluorescein isothiocyanate-labelled Pisum sativum agglutinin. At the same time, sperm were assessed for motility using a standard scoring system (independent operators' observation of sperm) that graded degree of motility (i.e. 1 = immotile to 10 = maximum motility, as observed at the moment of thawing). For i.v.f., frozen-thawed semen derived from the two culture systems was placed in culture together with in vitro-matured sheep oocytes. For i.c.s.i., semen derived from the same culture systems as that for i.v.f. was used, and incubated for 1 h under standard conditions. The results showed a marked difference (P < 0.01) between the percentages of live spermatozoa in medium with SOD and those obtained in medium alone, after 3, 6 and 9 h of culture. The percentages of intact acrosome spermatozoa were higher in medium with SOD after 6 h (P = 0.05) of culture. Spermatozoa motility decreased significantly in SOD containing medium at 3 and 6 h of culture compared with motility in control medium. Fertilization rates were significantly lower in medium with SOD than in medium alone, whereas in the i.c.s.i. system fertilization rates were significantly higher in the presence of SOD. The results indicate that the addition of SOD to the culture media

  18. How the knowledge of genetic "makeup" and cellular data can affect the analysis of repolarization in surface electrocardiogram.

    PubMed

    Shimizu, Wataru

    2010-01-01

    This review article sought to describe patterns of repolarization on the surface electrocardiogram in inherited cardiac arrhythmias and to discuss how the knowledge of genetic makeup and cellular data can affect the analysis based on the data derived from the experimental studies using arterially perfused canine ventricular wedge preparations. Molecular genetic studies have established a link between a number of inherited cardiac arrhythmia syndromes and mutations in genes encoding cardiac ion channels or membrane components during the past 2 decades. Twelve forms of congenital long QT syndrome have been so far identified, and genotype-phenotype correlations have been investigated especially in the 3 major genotypes-LQT1, LQT2, and LQT3. Abnormal T waves are reported in the LQT1, LQT2, and LQT3, and the differences in the time course of repolarization of the epicardial, midmyocardial, and endocardial cells give rise to voltage gradients responsible for the manifestation of phenotypic appearance of abnormal T waves. Brugada syndrome is characterized by ST-segment elevation in leads V1 to V3 and an episode of ventricular fibrillation, in which 7 genotypes have been reported. An intrinsically prominent transient outward current (I(to))-mediated action potential notch and a subsequent loss of action potential dome in the epicardium, but not in the endocardium of the right ventricular outflow tract, give rise to a transmural voltage gradient, resulting in ST-segment elevation, and a subsequent phase 2 reentry-induced ventricular fibrillation. In conclusion, transmural electrical heterogeneity of repolarization across the ventricular wall profoundly affects the phenotypic manifestation of repolarization patterns on the surface electrocardiogram in inherited cardiac arrhythmias.

  19. High-throughput screening of Australian marine organism extracts for bioactive molecules affecting the cellular storage of neutral lipids.

    PubMed

    Rae, James; Fontaine, Frank; Salim, Angela A; Lo, Harriet P; Capon, Robert J; Parton, Robert G; Martin, Sally

    2011-01-01

    Mammalian cells store excess fatty acids as neutral lipids in specialised organelles called lipid droplets (LDs). Using a simple cell-based assay and open-source software we established a high throughput screen for LD formation in A431 cells in order to identify small bioactive molecules affecting lipid storage. Screening an n-butanol extract library from Australian marine organisms we identified 114 extracts that produced either an increase or a decrease in LD formation in fatty acid-treated A431 cells with varying degrees of cytotoxicity. We selected for further analysis a non-cytotoxic extract derived from the genus Spongia (Heterofibria). Solvent partitioning, HPLC fractionation and spectroscopic analysis (NMR, MS) identified a family of related molecules within this extract with unique structural features, a subset of which reduced LD formation. We selected one of these molecules, heterofibrin A1, for more detailed cellular analysis. Inhibition of LD biogenesis by heterofibrin A1 was observed in both A431 cells and AML12 hepatocytes. The activity of heterofibrin A1 was dose dependent with 20 µM inhibiting LD formation and triglyceride accumulation by ∼50% in the presence of 50 µM oleic acid. Using a fluorescent fatty acid analogue we found that heterofibrin A1 significantly reduces the intracellular accumulation of fatty acids and results in the formation of distinct fatty acid metabolites in both cultured cells and in embryos of the zebrafish Danio rerio. In summary we have shown using readily accessible software and a relatively simple assay system that we can identify and isolate bioactive molecules from marine extracts, which affect the formation of LDs and the metabolism of fatty acids both in vitro and in vivo.

  20. Noncatalytic, N-terminal Domains of DNA Polymerase Lambda Affect Its Cellular Localization and DNA Damage Response.

    PubMed

    Stephenson, Anthony A; Taggart, David J; Suo, Zucai

    2017-04-13

    Specialized DNA polymerases, such as DNA polymerase lambda (Polλ), are important players in DNA damage tolerance and repair pathways. Knowing how DNA polymerases are regulated and recruited to sites of DNA damage is imperative to understanding these pathways. Recent work has suggested that Polλ plays a role in several distinct DNA damage tolerance and repair pathways. In this paper, we report previously unknown roles of the N-terminal domains of human Polλ for modulating its involvement in DNA damage tolerance and repair. By using Western blot analysis, fluorescence microscopy, and cell survival assays, we found that the BRCA1 C-terminal (BRCT) and proline/serine-rich (PSR) domains of Polλ affect its cellular localization and DNA damage responses. The nuclear localization signal (NLS) of Polλ was necessary to overcome the impediment of its nuclear localization caused by its BRCT and PSR domains. Induction of DNA damage resulted in recruitment of Polλ to chromatin, which was controlled by its BRCT and PSR domains. In addition, the presence of both domains was required for Polλ-mediated tolerance of oxidative DNA damage but not DNA methylation damage. These findings suggest that the N-terminal domains of Polλ are important for regulating its responses to DNA damage.

  1. Nonsteroidal anti-inflammatory drugs modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways

    PubMed Central

    Mozolewski, Paweł; Moskot, Marta; Jakóbkiewicz-Banecka, Joanna; Węgrzyn, Grzegorz; Bocheńska, Katarzyna; Banecki, Bogdan; Gabig-Cimińska, Magdalena

    2017-01-01

    In this report, selected non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin and nimesulide, and analgesics acetaminophen, alone, as well as in combination with isoflavone genistein as potential glycosaminoglycan (GAG) metabolism modulators were considered for the treatment of mucopolysaccharidoses (MPSs) with neurological symptoms due to the effective blood-brain barrier (BBB) penetration properties of these compounds. We found that indomethacin and nimesulide, but not acetaminophen, inhibited GAG synthesis in fibroblasts significantly, while the most pronounced impairment of glycosaminoglycan production was observed after exposure to the mixture of nimesulide and genistein. Phosphorylation of the EGF receptor (EGFR) was inhibited even more effective in the presence of indomethacin and nimesulide than in the presence of genistein. When examined the activity of phosphatidylinositol-3-kinase (PI3K) production, we observed its most significant decrease in the case of fibroblast exposition to nimesulide, and afterwards to indomethacin and genistein mix, rather than indomethacin used alone. Some effects on expression of individual GAG metabolism-related and lysosomal function genes, and significant activity modulation of a number of genes involved in intracellular signal transduction pathways and metabolism of DNA and proteins were detected. This study documents that NSAIDs, and their mixtures with genistein modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways. PMID:28240227

  2. KIR/HLA interactions negatively affect rituximab- but not GA101 (obinutuzumab)-induced antibody-dependent cellular cytotoxicity.

    PubMed

    Terszowski, Grzegorz; Klein, Christian; Stern, Martin

    2014-06-15

    Ab-dependent cellular cytotoxicity (ADCC) mediated by NK cells is regulated by inhibitory killer cell Ig-like receptors (KIRs), which interact with target cell HLA class I. We analyzed how KIR/HLA interactions influence ADCC induced by rituximab and by GA101, a novel type II CD20 Ab glycoengineered for increased FcgRIII binding and ADCC capacity. We found that KIR/HLA interactions strongly and selectively inhibit rituximab-induced in vitro ADCC toward target cells expressing cognate HLA KIR ligands. NK cells of donors carrying all three ligands to inhibitory KIR showed weak activation and target cell depletion capacity when incubated with rituximab and KIR-ligand matched target B cells. In contrast, NK cells from individuals missing one or more KIR ligands activated more strongly and depleted KIR ligand-matched target B cells more efficiently in the presence of rituximab. NK cells expressing a KIR for which the ligand was absent were the main effectors of ADCC in these donors. Notably, the influence of KIR/HLA interactions on NK cell activation was synergistic with the effect of the V158F FCGR3A single nucleotide polymorphism. In contrast, GA101 induced activation of NK cells irrespective of inhibitory KIR expression, and efficiency of target cell depletion was not negatively affected by KIR/HLA interactions. These data show that modification of the Fc fragment to enhance ADCC can be an effective strategy to augment the efficacy of therapeutic mAbs by recruiting NK cells irrespective of their inhibitory KIR expression.

  3. Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

    PubMed Central

    Díaz, Rommy; Boguen, Rodrigo; Martins, Simone Maria Massami Kitamura; Ravagnani, Gisele Mouro; Leal, Diego Feitosa; Oliveira, Melissa de Lima; Muro, Bruno Bracco Donatelli; Parra, Beatriz Martins; Meirelles, Flávio Vieira; Papa, Frederico Ozanan; Dell’Aqua, José Antônio; Alvarenga, Marco Antônio; Moretti, Aníbal de Sant’Anna; Sepúlveda, Néstor

    2016-01-01

    Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility. PMID:27529819

  4. Partial XBP1 knockdown does not affect viability of oligodendrocyte precursor cells exposed to new models of hypoxia and ischemia in vitro.

    PubMed

    Kraskiewicz, Honorata; FitzGerald, Una

    2011-05-01

    The endoplasmic reticulum (ER) stress signaling pathway has been implicated in tissue injury in several rodent models of brain ischemia. To understand better the effects of ischemia on white matter in particular, we developed several in vitro models of hypoxia and ischemia in oligodendrocyte precursors. For the first time, we present data showing that exposure of rat oligodendrocyte precursor cells (OPCs) to cobalt chloride (CoCl(2)), antimycin A (AA), or oxygen, glucose and nutrient deprivation (OGND) causes up-regulation of glucose-regulated protein 78/B-cell immunoglobulin-binding protein (Grp78/BiP), C/EBP homologous binding protein (CHOP), and spliced X-box-binding protein 1 (XBP1). To mimic the effects of ischemia, OPCs supplemented with 5% normal growth medium and 95% Hank's balanced salt solution were incubated in a hypoxia chamber set at 0.1% oxygen. Because the toxic effects of AA on OPCs more closely resembled those seen when OPCs were subjected to OGND, we found AA treatment preferable to CoCl(2) as an in vitro model. To investigate the role of XBP1 in survival following an ischemic insult, we generated a stable XBP1 knockdown OPC cell line and subjected it to simulated hypoxia or ischemia. Surprisingly, 65% XBP1 knockdown had no effect on viability following chemical treatment or OGND. These data strengthen the case for targeting the ER stress signalling pathway in an effort to develop new early treatments for ischemic stroke patients but at the same time demonstrate that partial knockdown of XBP1 is not sufficient to protect precursor oligodendrocytes from ischemic damage.

  5. Red Maca (Lepidium meyenii) did not affect cell viability despite increased androgen receptor and prostate-specific antigen gene expression in the human prostate cancer cell line LNCaP.

    PubMed

    Díaz, P; Cardenas, H; Orihuela, P A

    2016-10-01

    We examined whether aqueous extract of Lepidium meyenii (red Maca) could inhibit growth, potentiate apoptotic activity of two anticancer drugs Taxol and 2-methoxyestradiol (2ME) or change mRNA expression for the androgen target genes, androgen receptor (Ar) and prostate-specific antigen (Psa) in the human prostate cancer cell line LNCaP. Red Maca aqueous extract at 0, 10, 20, 40 or 80 μg/ml was added to LNCaP cells, and viability was evaluated by the MTS assay at 24 or 48 hr after treatment. Furthermore, LNCaP cells were treated with 80 μg/ml of red Maca plus Taxol or 2ME 5 μM and viability was assessed 48 hr later. Finally, LNCaP cells were treated with red Maca 0, 20, 40 or 80 μg/ml, and 12 hr later, mRNA level for Ar or Psa was assessed by real-time PCR. Treatment with red Maca did not affect viability of LNCaP cells. Apoptotic activity induced by Taxol and 2ME in LNCaP cells was not altered with red Maca treatment. Relative expression of the mRNA for Ar and Psa increased with red Maca 20 and 40 μg/ml, but not at 80 μg/ml. We conclude that red Maca aqueous extract does not have toxic effects, but stimulates androgen signalling in LNCaP cells.

  6. Deletion or overexpression of mitochondrial NAD+ carriers in Saccharomyces cerevisiae alters cellular NAD and ATP contents and affects mitochondrial metabolism and the rate of glycolysis.

    PubMed

    Agrimi, Gennaro; Brambilla, Luca; Frascotti, Gianni; Pisano, Isabella; Porro, Danilo; Vai, Marina; Palmieri, Luigi

    2011-04-01

    The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.

  7. Electrochemical sensing of hepatocyte viability.

    PubMed

    Tsai, Hweiyan; Tsai, Shang-heng; Ting, Wei-Jen; Hu, Chao-Chin; Fuh, C Bor

    2014-05-21

    We investigated the use of amperometric and chronoamperometric methods with a double mediator system and screen-printed electrodes (SPEs) for the electrochemical sensing of hepatocyte viability. Cell counts were determined based on measuring cellular respiration via interaction of electroactive redox mediators. The oxidation currents of chronoamperometric measurement were proportional to the concentrations of ferrocyanide which was produced via interaction of cellular respiration, succinate and ferricyanide. The integrated oxidation charges increased linearly with the density of the cultured primary rat hepatocytes over a range of 1 × 10(5) to 5 × 10(5) cells per well (slope = 1.98 (±0.08) μC per 10(5) cells; R(2) = 0.9969), and the detection limit was 7600 (±300) cells per well based on S/N = 3. Each density of cells was cultured in triple replicates and individual cell samples were evaluated. The results of the cytotoxic effect of the chronoamperometric method are comparable to those of the tetrazolium-based colorimetric assay. The chronoamperometric method with ferricyanide and succinate mediators is an efficient, alternative method for assessing the viability of primary hepatocytes which can be completed in 20 min. Succinate did not provide an efficient electron shuttle between cytosolic respiratory redox activity of cancer cells and extracellular ferricyanide, an effect that may be useful for distinguishing hepatocarcinoma cells from healthy hepatocytes.

  8. The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

    SciTech Connect

    Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela

    2013-09-15

    Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs. Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.

  9. Sublethal pesticide doses negatively affect survival and the cellular responses in American foulbrood-infected honeybee larvae

    NASA Astrophysics Data System (ADS)

    López, Javier Hernández; Krainer, Sophie; Engert, Antonia; Schuehly, Wolfgang; Riessberger-Gallé, Ulrike; Crailsheim, Karl

    2017-02-01

    Disclosing interactions between pesticides and bee infections is of most interest to understand challenges that pollinators are facing and to which extent bee health is compromised. Here, we address the individual and combined effect that three different pesticides (dimethoate, clothianidin and fluvalinate) and an American foulbrood (AFB) infection have on mortality and the cellular immune response of honeybee larvae. We demonstrate for the first time a synergistic interaction when larvae are exposed to sublethal doses of dimethoate or clothianidin in combination with Paenibacillus larvae, the causative agent of AFB. A significantly higher mortality than the expected sum of the effects of each individual stressor was observed in co-exposed larvae, which was in parallel with a drastic reduction of the total and differential hemocyte counts. Our results underline that characterizing the cellular response of larvae to individual and combined stressors allows unmasking previously undetected sublethal effects of pesticides in colony health.

  10. Sublethal pesticide doses negatively affect survival and the cellular responses in American foulbrood-infected honeybee larvae

    PubMed Central

    López, Javier Hernández; Krainer, Sophie; Engert, Antonia; Schuehly, Wolfgang; Riessberger-Gallé, Ulrike; Crailsheim, Karl

    2017-01-01

    Disclosing interactions between pesticides and bee infections is of most interest to understand challenges that pollinators are facing and to which extent bee health is compromised. Here, we address the individual and combined effect that three different pesticides (dimethoate, clothianidin and fluvalinate) and an American foulbrood (AFB) infection have on mortality and the cellular immune response of honeybee larvae. We demonstrate for the first time a synergistic interaction when larvae are exposed to sublethal doses of dimethoate or clothianidin in combination with Paenibacillus larvae, the causative agent of AFB. A significantly higher mortality than the expected sum of the effects of each individual stressor was observed in co-exposed larvae, which was in parallel with a drastic reduction of the total and differential hemocyte counts. Our results underline that characterizing the cellular response of larvae to individual and combined stressors allows unmasking previously undetected sublethal effects of pesticides in colony health. PMID:28145462

  11. Sublethal pesticide doses negatively affect survival and the cellular responses in American foulbrood-infected honeybee larvae.

    PubMed

    López, Javier Hernández; Krainer, Sophie; Engert, Antonia; Schuehly, Wolfgang; Riessberger-Gallé, Ulrike; Crailsheim, Karl

    2017-02-01

    Disclosing interactions between pesticides and bee infections is of most interest to understand challenges that pollinators are facing and to which extent bee health is compromised. Here, we address the individual and combined effect that three different pesticides (dimethoate, clothianidin and fluvalinate) and an American foulbrood (AFB) infection have on mortality and the cellular immune response of honeybee larvae. We demonstrate for the first time a synergistic interaction when larvae are exposed to sublethal doses of dimethoate or clothianidin in combination with Paenibacillus larvae, the causative agent of AFB. A significantly higher mortality than the expected sum of the effects of each individual stressor was observed in co-exposed larvae, which was in parallel with a drastic reduction of the total and differential hemocyte counts. Our results underline that characterizing the cellular response of larvae to individual and combined stressors allows unmasking previously undetected sublethal effects of pesticides in colony health.

  12. Chlamydomonas reinhardtii cells adjust the metabolism to maintain viability in response to atrazine stress.

    PubMed

    Esperanza, Marta; Seoane, Marta; Rioboo, Carmen; Herrero, Concepción; Cid, Ángeles

    2015-08-01

    Chlamydomonas reinhardtii cells were exposed to a sublethal concentration of the widespread herbicide atrazine for 3 and 24h. Physiological parameters related to cellular energy status, such as cellular activity and mitochondrial and cytoplasmic membrane potentials, monitored by flow cytometry, were altered in microalgal cells exposed to 0.25μM of atrazine. Transcriptomic analyses, carried out by RNA-Seq technique, displayed 12 differentially expressed genes between control cultures and atrazine-exposed cultures at both tested times. Many cellular processes were affected, but the most significant changes were observed in genes implicated in amino acid catabolism and respiratory cellular process. Obtained results suggest that photosynthesis inhibition by atrazine leads cells to get energy through a heterotrophic metabolism to maintain their viability.

  13. Use of a generalized additive model to investigate key abiotic factors affecting microcystin cellular quotas in heavy bloom areas of Lake Taihu.

    PubMed

    Tao, Min; Xie, Ping; Chen, Jun; Qin, Boqiang; Zhang, Dawen; Niu, Yuan; Zhang, Meng; Wang, Qing; Wu, Laiyan

    2012-01-01

    Lake Taihu is the third largest freshwater lake in China and is suffering from serious cyanobacterial blooms with the associated drinking water contamination by microcystin (MC) for millions of citizens. So far, most studies on MCs have been limited to two small bays, while systematic research on the whole lake is lacking. To explain the variations in MC concentrations during cyanobacterial bloom, a large-scale survey at 30 sites across the lake was conducted monthly in 2008. The health risks of MC exposure were high, especially in the northern area. Both Microcystis abundance and MC cellular quotas presented positive correlations with MC concentration in the bloom seasons, suggesting that the toxic risks during Microcystis proliferations were affected by variations in both Microcystis density and MC production per Microcystis cell. Use of a powerful predictive modeling tool named generalized additive model (GAM) helped visualize significant effects of abiotic factors related to carbon fixation and proliferation of Microcystis (conductivity, dissolved inorganic carbon (DIC), water temperature and pH) on MC cellular quotas from recruitment period of Microcystis to the bloom seasons, suggesting the possible use of these factors, in addition to Microcystis abundance, as warning signs to predict toxic events in the future. The interesting relationship between macrophytes and MC cellular quotas of Microcystis (i.e., high MC cellular quotas in the presence of macrophytes) needs further investigation.

  14. The Interaction of the Gammaherpesvirus 68 orf73 Protein with Cellular BET Proteins Affects the Activation of Cell Cycle Promoters▿

    PubMed Central

    Ottinger, Matthias; Pliquet, Daniel; Christalla, Thomas; Frank, Ronald; Stewart, James P.; Schulz, Thomas F.

    2009-01-01

    Infection of mice with murine gammaherpesvirus 68 (MHV-68) provides a valuable animal model for gamma-2 herpesvirus (rhadinovirus) infection and pathogenesis. The MHV-68 orf73 protein has been shown to be required for the establishment of viral latency in vivo. This study describes a novel transcriptional activation function of the MHV-68 orf73 protein and identifies the cellular bromodomain containing BET proteins Brd2/RING3, Brd3/ORFX, and BRD4 as interaction partners for the MHV-68 orf73 protein. BET protein members are known to interact with acetylated histones, and Brd2 and Brd4 have been implicated in fundamental cellular processes, including cell cycle regulation and transcriptional regulation. Using MHV-68 orf73 peptide array assays, we identified Brd2 and Brd4 interaction sites in the orf73 protein. Mutation of one binding site led to a loss of the interaction with Brd2/4 but not the retinoblastoma protein Rb, to impaired chromatin association, and to a decreased ability to activate the BET-responsive cyclin D1, D2, and E promoters. The results therefore pinpoint the binding site for Brd2/4 in a rhadinoviral orf73 protein and suggest that the recruitment of a member of the BET protein family allows the MHV-68 orf73 protein to activate the promoters of G1/S cyclins. These findings point to parallels between the transcriptional activator functions of rhadinoviral orf73 proteins and papillomavirus E2 proteins. PMID:19244327

  15. The cellular energization state affects peripheral stalk stability of plant vacuolar H+-ATPase and impairs vacuolar acidification.

    PubMed

    Schnitzer, Daniel; Seidel, Thorsten; Sander, Tim; Golldack, Dortje; Dietz, Karl-Josef

    2011-05-01

    The plant vacuolar H(+)-ATPase takes part in acidifying compartments of the endomembrane system including the secretory pathway and the vacuoles. The structural variability of the V-ATPase complex as well as its presence in different compartments and tissues involves multiple isoforms of V-ATPase subunits. Furthermore, a versatile regulation is essential to allow for organelle- and tissue-specific fine tuning. In this study, results from V-ATPase complex disassembly with a chaotropic reagent, immunodetection and in vivo fluorescence resonance energy transfer (FRET) analyses point to a regulatory mechanism in plants, which depends on energization and involves the stability of the peripheral stalks as well. Lowering of cellular ATP by feeding 2-deoxyglucose resulted in structural alterations within the V-ATPase, as monitored by changes in FRET efficiency between subunits VHA-E and VHA-C. Potassium iodide-mediated disassembly revealed a reduced stability of V-ATPase after 2-deoxyglucose treatment of the cells, but neither the complete V(1)-sector nor VHA-C was released from the membrane in response to 2-deoxyglucose treatment, precluding a reversible dissociation mechanism like in yeast. These data suggest the existence of a regulatory mechanism of plant V-ATPase by modification of the peripheral stator structure that is linked to the cellular energization state. This mechanism is distinct from reversible dissociation as reported for the yeast V-ATPase, but might represent an evolutionary precursor of reversible dissociation.

  16. Enterococcus faecalis plasmid pAD1-encoded Fst toxin affects membrane permeability and alters cellular responses to lantibiotics.

    PubMed

    Weaver, Keith E; Weaver, Dariel M; Wells, Carol L; Waters, Christopher M; Gardner, Marshall E; Ehli, Erik A

    2003-04-01

    Fst is a peptide toxin encoded by the par toxin-antitoxin stability determinant of Enterococcus faecalis plasmid pAD1. Intracellular overproduction of Fst resulted in simultaneous inhibition of all cellular macromolecular synthesis concomitant with cell growth inhibition and compromised the integrity of the cell membrane. Cells did not lyse or noticeably leak intracellular contents but had specific defects in chromosome partitioning and cell division. Extracellular addition of synthetic Fst had no effect on cell growth. Spontaneous Fst-resistant mutants had a phenotype consistent with changes in membrane composition. Interestingly, overproduction of Fst sensitized cells to the lantibiotic nisin, and Fst-resistant mutants were cross-resistant to nisin and the pAD1-encoded cytolysin.

  17. Cellular Location and Expression of Na+, K+-ATPase α Subunits Affect the Anti-Proliferative Activity of Oleandrin

    PubMed Central

    Yang, Peiying; Cartwright, Carrie; Efuet, Ekem; Hamilton, Stanley R.; Wistuba, Ignacio Ivan; Menter, David; Addington, Crandell; Shureiqi, Imad; Newman, Robert A.

    2015-01-01

    The purpose of this study was to investigate whether intracellular distribution of Na+, K+-ATPase α3 subunit, a receptor for cardiac glycosides including oleandrin, is differentially altered in cancer versus normal cells and whether this altered distribution can be therapeutically targeted to inhibit cancer cell survival. The cellular distribution of Na+, K+-ATPase α3 isoform was investigated in paired normal and cancerous mucosa biopsy samples from patients with lung and colorectal cancers by immunohistochemical staining. The effects of oleandrin on α3 subunit intracellular distribution, cell death, proliferation, and EKR phosphorylation were examined in differentiated and undifferentiated human colon cancer CaCO-2 cells. While Na+, K+-ATPase α3 isoform was predominantly located near the cytoplasmic membrane in normal human colon and lung epithelia, the expression of this subunit in their paired cancer epithelia was shifted to a peri-nuclear position in both a qualitative and quantitative manner. Similarly, distribution of α3 isoform was also shifted from a cytoplasmic membrane location in differentiated human colon cancer CaCO-2 cells to a peri-nuclear position in undifferentiated CaCO-2 cells. Intriguingly, oleandrin exerted threefold stronger anti-proliferative activity in undifferentiated CaCO-2 cells (IC50, 8.25 nM) than in differentiated CaCO-2 cells (IC50, >25 nM). Oleandrin (10 to 20 nM) caused an autophagic cell death and altered ERK phosphorylation in undifferentiated but not in differentiated CaCO-2 cells. These data demonstrate that the intracellular location of Na+, K+-ATPase α3 isoform is altered in human cancer versus normal cells. These changes in α3 cellular location and abundance may indicate a potential target of opportunity for cancer therapy. PMID:23073998

  18. Mps1 (Monopolar Spindle 1) Protein Inhibition Affects Cellular Growth and Pro-Embryogenic Masses Morphology in Embryogenic Cultures of Araucaria angustifolia (Araucariaceae)

    PubMed Central

    Douétts-Peres, Jackellinne C.; Cruz, Marco Antônio L.; Reis, Ricardo S.; Heringer, Angelo S.; de Oliveira, Eduardo A. G.; Elbl, Paula M.; Floh, Eny I. S.; Silveira, Vanildo

    2016-01-01

    Somatic embryogenesis has been shown to be an efficient tool for studying processes based on cell growth and development. The fine regulation of the cell cycle is essential for proper embryo formation during the process of somatic embryogenesis. The aims of the present work were to identify and perform a structural and functional characterization of Mps1 and to analyze the effects of the inhibition of this protein on cellular growth and pro-embryogenic mass (PEM) morphology in embryogenic cultures of A. angustifolia. A single-copy Mps1 gene named AaMps1 was retrieved from the A. angustifolia transcriptome database, and through a mass spectrometry approach, AaMps1 was identified and quantified in embryogenic cultures. The Mps1 inhibitor SP600125 (10 μM) inhibited cellular growth and changed PEMs, and these effects were accompanied by a reduction in AaMps1 protein levels in embryogenic cultures. Our work has identified the Mps1 protein in a gymnosperm species for the first time, and we have shown that inhibiting Mps1 affects cellular growth and PEM differentiation during A. angustifolia somatic embryogenesis. These data will be useful for better understanding cell cycle control during somatic embryogenesis in plants. PMID:27064899

  19. Equine Metabolic Syndrome Affects Viability, Senescence, and Stress Factors of Equine Adipose-Derived Mesenchymal Stromal Stem Cells: New Insight into EqASCs Isolated from EMS Horses in the Context of Their Aging.

    PubMed

    Marycz, Krzysztof; Kornicka, Katarzyna; Basinska, Katarzyna; Czyrek, Aleksandra

    2016-01-01

    Currently, equine metabolic syndrome (EMS), an endocrine disease linked to insulin resistance, affects an increasing number of horses. However, little is known about the effect of EMS on mesenchymal stem cells that reside in adipose tissue (ASC). Thus it is crucial to evaluate the viability and growth kinetics of these cells, particularly in terms of their application in regenerative medicine. In this study, we investigated the proliferative capacity, morphological features, and accumulation of oxidative stress factors in mesenchymal stem cells isolated from healthy animals (ASCN) and horses suffering from EMS (ASCEMS). ASCEMS displayed senescent phenotype associated with β-galactosidase accumulation, enlarged cell bodies and nuclei, increased apoptosis, and reduced heterochromatin architecture. Moreover, we observed increased amounts of nitric oxide (NO) and reactive oxygen species (ROS) in these cells, accompanied by reduced superoxide dismutase (SOD) activity. We also found in ASCEMS an elevated number of impaired mitochondria, characterized by membrane raptures, disarrayed cristae, and vacuole formation. Our results suggest that the toxic compounds, accumulating in the mitochondria under oxidative stress, lead to alternations in their morphology and may be partially responsible for the senescent phenotype and decreased proliferation potential of ASCEMS.

  20. Equine Metabolic Syndrome Affects Viability, Senescence, and Stress Factors of Equine Adipose-Derived Mesenchymal Stromal Stem Cells: New Insight into EqASCs Isolated from EMS Horses in the Context of Their Aging

    PubMed Central

    Marycz, Krzysztof; Kornicka, Katarzyna; Basinska, Katarzyna; Czyrek, Aleksandra

    2016-01-01

    Currently, equine metabolic syndrome (EMS), an endocrine disease linked to insulin resistance, affects an increasing number of horses. However, little is known about the effect of EMS on mesenchymal stem cells that reside in adipose tissue (ASC). Thus it is crucial to evaluate the viability and growth kinetics of these cells, particularly in terms of their application in regenerative medicine. In this study, we investigated the proliferative capacity, morphological features, and accumulation of oxidative stress factors in mesenchymal stem cells isolated from healthy animals (ASCN) and horses suffering from EMS (ASCEMS). ASCEMS displayed senescent phenotype associated with β-galactosidase accumulation, enlarged cell bodies and nuclei, increased apoptosis, and reduced heterochromatin architecture. Moreover, we observed increased amounts of nitric oxide (NO) and reactive oxygen species (ROS) in these cells, accompanied by reduced superoxide dismutase (SOD) activity. We also found in ASCEMS an elevated number of impaired mitochondria, characterized by membrane raptures, disarrayed cristae, and vacuole formation. Our results suggest that the toxic compounds, accumulating in the mitochondria under oxidative stress, lead to alternations in their morphology and may be partially responsible for the senescent phenotype and decreased proliferation potential of ASCEMS. PMID:26682006

  1. Naphthalene Acetic Acid Potassium Salt (NAA-K(+)) Affects Conidial Germination, Sporulation, Mycelial Growth, Cell Surface Morphology, and Viability of Fusarium oxysporum f. sp. radici-lycopersici and F. oxysporum f. sp. cubense in Vitro.

    PubMed

    Manzo-Valencia, María Karina; Valdés-Santiago, Laura; Sánchez-Segura, Lino; Guzmán-de-Peña, Dora Linda

    2016-11-09

    The response to exogenous addition of naphthalene acetic acid potassium salt (NAA-K(+)) to Fusarium oxysporum f. sp radici-lycopersici ATCC 60095 and F. oxysporum f. sp. cubense isolated from Michoacan Mexico soil is reported. The in vitro study showed that NAA-K(+) might be effective in the control of Fusarium oxysporum. Exogenous application of NAA-K(+) affected both spores and mycelium stages of the fungi. Viability testing using acridine orange and propidium iodide showed that NAA-K(+) possesses fungal killing properties, doing it effectively in the destruction of conidia of this phytopathogenic fungi. Analysis of treated spores by scanning electron microscopy showed changes in the shape factor and fractal dimension. Moreover, NAA-K(+) repressed the expression of brlA and fluG genes. The results disclosed here give evidence of the use of this synthetic growth factor as a substance of biocontrol that presents advantages, and the methods of application in situ should be explored.

  2. Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts.

    PubMed

    Ferizi, Mehrije; Aneja, Manish K; Balmayor, Elizabeth R; Badieyan, Zohreh Sadat; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2016-12-15

    Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5'- and 3'-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5'-UTR and/or downstream 3'-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.

  3. Cellular Lipid Composition Affects Sensitivity of Plant Pathogens to Fengycin, an Antifungal Compound Produced by Bacillus subtilis Strain CU12.

    PubMed

    Wise, Cody; Falardeau, Justin; Hagberg, Ingrid; Avis, Tyler J

    2014-10-01

    Fengycin is an antimicrobial cyclic lipopeptide produced by various Bacillus subtilis strains, including strain CU12. Direct effects of fengycin include membrane pore formation and efflux of cellular contents leading to cell death in sensitive microorganisms. In this study, four plant pathogens were studied in order to elucidate the role of membrane lipids in their relative sensitivity to fengycin. Inhibition of mycelial growth in these pathogens varied considerably. Analysis of membrane lipids in these microorganisms indicated that sensitivity correlated with low ergosterol content and shorter phospholipid fatty acyl chains. Sensitivity to fengycin also correlated with a lower anionic/zwitterionic phospholipid ratio. Our data suggest that decreased fluidity buffering capacity, as a result of low ergosterol content, and higher intrinsic fluidity afforded by short fatty acyl chain length may increase the sensitivity of microbial membranes to fengycin. Our results also suggest that lower content in anionic phospholipids may increase fengycin insertion into the membrane through reduced electrostatic repulsion with the negatively charged fengycin. The intrinsic membrane lipid composition may contribute, in part, to the observed level of antimicrobial activity of fengycin in various plant pathogens.

  4. Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts

    PubMed Central

    Ferizi, Mehrije; Aneja, Manish K.; Balmayor, Elizabeth R.; Badieyan, Zohreh Sadat; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

    2016-01-01

    Modified nucleotide chemistries that increase the half-life (T1/2) of transfected recombinant mRNA and the use of non-native 5′- and 3′-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T1/2 were synthesized and cloned as five different recombinant sequence set combinations as upstream 5′-UTR and/or downstream 3′-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs. PMID:27974853

  5. Antibody-dependent-cellular-cytotoxicity-inducing antibodies significantly affect the post-exposure treatment of Ebola virus infection

    PubMed Central

    Liu, Qiang; Fan, Changfa; Li, Qianqian; Zhou, Shuya; Huang, Weijin; Wang, Lan; Sun, Chunyun; Wang, Meng; Wu, Xi; Ma, Jian; Li, Baowen; Xie, Liangzhi; Wang, Youchun

    2017-01-01

    Passive immunotherapy with monoclonal antibodies (mAbs) is an efficacious treatment for Ebola virus (EBOV) infections in animal models and humans. Understanding what constitutes a protective response is critical for the development of novel therapeutic strategies. We generated an EBOV-glycoprotein-pseudotyped Human immunodeficiency virus to develop sensitive neutralizing and antibody-dependent cellular cytotoxicity (ADCC) assays as well as a bioluminescent-imaging-based mouse infection model that does not require biosafety level 4 containment. The in vivo treatment efficiencies of three novel anti-EBOV mAbs at 12 h post-infection correlated with their in vitro anti-EBOV ADCC activities, without neutralizing activity. When they were treated with these mAbs, natural killer cell (NK)-deficient mice had lower viral clearance than WT mice, indicating that the anti-EBOV mechanism of the ADCC activity of these mAbs is predominantly mediated by NK cells. One potent anti-EBOV mAb (M318) displayed unprecedented neutralizing and ADCC activities (neutralization IC50, 0.018 μg/ml; ADCC EC50, 0.095 μg/ml). These results have important implications for the efficacy of antiviral drugs and vaccines as well as for pathogenicity studies of EBOV. PMID:28358050

  6. Cell viability assays: introduction.

    PubMed

    Stoddart, Martin J

    2011-01-01

    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  7. Mimicking the phosphorylation of Rsp5 in PKA site T761 affects its function and cellular localization.

    PubMed

    Jastrzebska, Zaneta; Kaminska, Joanna; Chelstowska, Anna; Domanska, Anna; Rzepnikowska, Weronika; Sitkiewicz, Ewa; Cholbinski, Piotr; Gourlay, Campbell; Plochocka, Danuta; Zoladek, Teresa

    2015-12-01

    Rsp5 ubiquitin ligase belongs to the Nedd4 family of proteins, which affect a wide variety of processes in the cell. Here we document that Rsp5 shows several phosphorylated variants of different mobility and the migration of the phosphorylated forms of Rsp5 was faster for the tpk1Δ tpk3Δ mutant devoid of two alternative catalytic subunits of protein kinase A (PKA), indicating that PKA possibly phosphorylates Rsp5 in vivo. We demonstrated by immunoprecipitation and Western blot analysis of GFP-HA-Rsp5 protein using the anti-phospho PKA substrate antibody that Rsp5 is phosphorylated in PKA sites. Rsp5 contains the sequence 758-RRFTIE-763 with consensus RRXS/T in the catalytic HECT domain and four other sites with consensus RXXS/T, which might be phosphorylated by PKA. The strain bearing the T761D substitution in Rsp5 which mimics phosphorylation grew more slowly at 28°C and did not grow at 37°C, and showed defects in pre-tRNA processing and protein sorting. The rsp5-T761D strain also demonstrated a reduced ability to form colonies, an increase in the level of reactive oxygen species (ROS) and hypersensitivity to ROS-generating agents. These results indicate that PKA may downregulate many functions of Rsp5, possibly affecting its activity. Rsp5 is found in the cytoplasm, nucleus, multivesicular body and cortical patches. The rsp5-T761D mutation led to a strongly increased cortical localization while rsp5-T761A caused mutant Rsp5 to locate more efficiently in internal spots. Rsp5-T761A protein was phosphorylated less efficiently in PKA sites under specific growth conditions. Our data suggests that Rsp5 may be phosphorylated by PKA at position T761 and that this regulation is important for its localization and function.

  8. Salmonella Adhesion, Invasion and Cellular Immune Responses Are Differentially Affected by Iron Concentrations in a Combined In Vitro Gut Fermentation-Cell Model

    PubMed Central

    Dostal, Alexandra; Gagnon, Mélanie; Chassard, Christophe; Zimmermann, Michael Bruce; O'Mahony, Liam; Lacroix, Christophe

    2014-01-01

    In regions with a high infectious disease burden, concerns have been raised about the safety of iron supplementation because higher iron concentrations in the gut lumen may increase risk of enteropathogen infection. The aim of this study was to investigate interactions of the enteropathogen Salmonella enterica ssp. enterica Typhimurium with intestinal cells under different iron concentrations encountered in the gut lumen during iron deficiency and supplementation using an in vitro colonic fermentation system inoculated with immobilized child gut microbiota combined with Caco-2/HT29-MTX co-culture monolayers. Colonic fermentation effluents obtained during normal, low (chelation by 2,2'-dipyridyl) and high iron (26.5 mg iron/L) fermentation conditions containing Salmonella or pure Salmonella cultures with similar iron conditions were applied to cellular monolayers. Salmonella adhesion and invasion capacity, cellular integrity and immune response were assessed. Under high iron conditions in pure culture, Salmonella adhesion was 8-fold increased compared to normal iron conditions while invasion was not affected leading to decreased invasion efficiency (−86%). Moreover, cellular cytokines IL-1β, IL-6, IL-8 and TNF-α secretion as well as NF-κB activation in THP-1 cells were attenuated under high iron conditions. Low iron conditions in pure culture increased Salmonella invasion correlating with an increase in IL-8 release. In fermentation effluents, Salmonella adhesion was 12-fold and invasion was 428-fold reduced compared to pure culture. Salmonella in high iron fermentation effluents had decreased invasion efficiency (−77.1%) and cellular TNF-α release compared to normal iron effluent. The presence of commensal microbiota and bacterial metabolites in fermentation effluents reduced adhesion and invasion of Salmonella compared to pure culture highlighting the importance of the gut microbiota as a barrier during pathogen invasion. High iron concentrations as

  9. Diversification of the AlpB Outer Membrane Protein of Helicobacter pylori Affects Biofilm Formation and Cellular Adhesion

    PubMed Central

    Osaki, Takako; Fukutomi, Toshiyuki; Hanawa, Tomoko; Kurata, Satoshi; Zaman, Cynthia; Hojo, Fuhito; Kamiya, Shigeru

    2016-01-01

    ABSTRACT Helicobacter pylori is one of the most common causes of bacterial infection in humans, and it forms biofilms on human gastric mucosal epithelium as well as on in vitro abiotic surfaces. Bacterial biofilm is critical not only for environmental survival but also for successful infection. We previously demonstrated that strain TK1402, which was isolated from a Japanese patient with duodenal and gastric ulcers, has high biofilm-forming ability in vitro relative to other strains. In addition, we showed that outer membrane vesicles (OMV) play an important role in biofilm formation. The aim of this study was to analyze which protein(s) in the OMV contributes to biofilm formation in TK1402. We obtained a spontaneous mutant strain derived from TK1402 lacking biofilm-forming ability. The protein profiles of the OMV were compared between this mutant strain and the wild type, and it was found that AlpB, an outer membrane protein in the OMV of the mutant strain, was markedly decreased compared to that of the wild type. Restoration of TK1402 alpB to the mutant strain fully recovered the ability to form biofilm. However, restoration with alpB from other strains demonstrated incomplete recovery of biofilm-forming ability. We therefore inferred that the variable region of AlpB (amino acid positions 121 to 146) was involved in TK1402 biofilm formation. In addition, diversification of the AlpB sequence was shown to affect the ability to adhere to AGS cells. These results demonstrate a new insight into the molecular mechanisms of host colonization by H. pylori. IMPORTANCE Bacterial biofilm is critical not only for environmental survival but also for successful infection. The mechanism of Helicobacter pylori adherence to host cells mediated by cell surface adhesins has been the focus of many studies, but little is known regarding factors involved in H. pylori biofilm formation. Our study demonstrated that AlpB plays an important role in biofilm formation and that this property

  10. Diversification of the AlpB Outer Membrane Protein of Helicobacter pylori Affects Biofilm Formation and Cellular Adhesion.

    PubMed

    Yonezawa, Hideo; Osaki, Takako; Fukutomi, Toshiyuki; Hanawa, Tomoko; Kurata, Satoshi; Zaman, Cynthia; Hojo, Fuhito; Kamiya, Shigeru

    2017-03-15

    Helicobacter pylori is one of the most common causes of bacterial infection in humans, and it forms biofilms on human gastric mucosal epithelium as well as on in vitro abiotic surfaces. Bacterial biofilm is critical not only for environmental survival but also for successful infection. We previously demonstrated that strain TK1402, which was isolated from a Japanese patient with duodenal and gastric ulcers, has high biofilm-forming ability in vitro relative to other strains. In addition, we showed that outer membrane vesicles (OMV) play an important role in biofilm formation. The aim of this study was to analyze which protein(s) in the OMV contributes to biofilm formation in TK1402. We obtained a spontaneous mutant strain derived from TK1402 lacking biofilm-forming ability. The protein profiles of the OMV were compared between this mutant strain and the wild type, and it was found that AlpB, an outer membrane protein in the OMV of the mutant strain, was markedly decreased compared to that of the wild type. Restoration of TK1402 alpB to the mutant strain fully recovered the ability to form biofilm. However, restoration with alpB from other strains demonstrated incomplete recovery of biofilm-forming ability. We therefore inferred that the variable region of AlpB (amino acid positions 121 to 146) was involved in TK1402 biofilm formation. In addition, diversification of the AlpB sequence was shown to affect the ability to adhere to AGS cells. These results demonstrate a new insight into the molecular mechanisms of host colonization by H. pyloriIMPORTANCE Bacterial biofilm is critical not only for environmental survival but also for successful infection. The mechanism of Helicobacter pylori adherence to host cells mediated by cell surface adhesins has been the focus of many studies, but little is known regarding factors involved in H. pylori biofilm formation. Our study demonstrated that AlpB plays an important role in biofilm formation and that this property depends

  11. Cartilage viability after trochleoplasty.

    PubMed

    Schöttle, Philip B; Schell, Hanna; Duda, Georg; Weiler, Andreas

    2007-02-01

    Trochleoplasty is an established and accepted technique for the treatment of patellar instability because of a missing trochlear groove. In this technique, a flap of cartilage over the trochlea is carefully removed and a new trochlear groove is created in the underlying bone before the cartilaginous flap is reattached with sutures. The mid-term clinical and radiological results of this operation are promising but no information about the viability of the reattached cartilage has been reported. To evaluate cartilage viability and quality after trochleoplasty and to verify the healing process, two osteochondral biopsies were harvested from three patients 6, 8, and 9 months after trochleoplasty. One cylinder was evaluated histologically to assess cartilage, calcified cartilage (cc), and subchondral bone quality, while the other one was examined by confocal microscopy to evaluate cell viability. The histological examination showed a normal matrix and cell distribution of the cartilage, while the cc showed lacunae ingrowing from the underlying bone. The subchondral bone showed normal lamellae and histology, and the healing of the flap. Confocal microscopy showed almost exclusively viable chondrocytes. This demonstration of non-injured cartilage at short-term follow-up together with promising clinical and radiological 2- and 5-year follow-up results indicate a potential promising outlook for the long term, as further chondral damage is not expected. So trochleoplasty can be seen as a primary intervention for patellar instability because of trochlear dysplasia as the risk for cartilage damage is low.

  12. Effects of Triclosan on Neural Stem Cell Viability and Survival

    PubMed Central

    Park, Bo Kyung; Gonzales, Edson Luck T.; Yang, Sung Min; Bang, Minji; Choi, Chang Soon; Shin, Chan Young

    2016-01-01

    Triclosan is an antimicrobial or sanitizing agent used in personal care and household products such as toothpaste, soaps, mouthwashes and kitchen utensils. There are increasing evidence of the potentially harmful effects of triclosan in many systemic and cellular processes of the body. In this study, we investigated the effects of triclosan in the survivability of cultured rat neural stem cells (NSCs). Cortical cells from embryonic day 14 rat embryos were isolated and cultured in vitro. After stabilizing the culture, triclosan was introduced to the cells with concentrations ranging from 1 μM to 50 μM and in varied time periods. Thereafter, cell viability parameters were measured using MTT assay and PI staining. TCS decreased the cell viability of treated NSC in a concentration-dependent manner along with increased expressions of apoptotic markers, cleaved caspase-3 and Bax, while reduced expression of Bcl2. To explore the mechanisms underlying the effects of TCS in NSC, we measured the activation of MAPKs and intracellular ROS. TCS at 50 μM induced the activations of both p38 and JNK, which may adversely affect cell survival. In contrast, the activities of ERK, Akt and PI3K, which are positively correlated with cell survival, were inhibited. Moreover, TCS at this concentration augmented the ROS generation in treated NSC and depleted the glutathione activity. Taken together, these results suggest that TCS can induce neurodegenerative effects in developing rat brains through mechanisms involving ROS activation and apoptosis initiation. PMID:26759708

  13. Protein turnover and cellular stress in mildly and severely affected muscles from patients with limb girdle muscular dystrophy type 2I.

    PubMed

    Hauerslev, Simon; Sveen, Marie L; Vissing, John; Krag, Thomas O

    2013-01-01

    Patients with Limb girdle muscular dystrophy type 2I (LGMD2I) are characterized by progressive muscle weakness and wasting primarily in the proximal muscles, while distal muscles often are spared. Our aim was to investigate if wasting could be caused by impaired regeneration in the proximal compared to distal muscles. Biopsies were simultaneously obtained from proximal and distal muscles of the same patients with LGMD2I (n = 4) and healthy subjects (n = 4). The level of past muscle regeneration was evaluated by counting internally nucleated fibers and determining actively regenerating fibers by using the developmental markers embryonic myosin heavy chain (eMHC) and neural cell adhesion molecule (NCAM) and also assessing satellite cell activation status by myogenin positivity. Severe muscle histopathology was occasionally observed in the proximal muscles of patients with LGMD2I whereas distal muscles were always relatively spared. No difference was found in the regeneration markers internally nucleated fibers, actively regenerating fibers or activation status of satellite cells between proximal and distal muscles. Protein turnover, both synthesis and breakdown, as well as cellular stress were highly increased in severely affected muscles compared to mildly affected muscles. Our results indicate that alterations in the protein turnover and myostatin levels could progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy.

  14. Establishing lunar resource viability

    NASA Astrophysics Data System (ADS)

    Carpenter, J.; Fisackerly, R.; Houdou, B.

    2016-11-01

    Recent research has highlighted the potential of lunar resources as an important element of space exploration but their viability has not been demonstrated. Establishing whether or not they can be considered in future plans is a multidisciplinary effort, requiring scientific expertise and delivering scientific results. To this end various space agencies and private entities are looking to lunar resources, extracted and processed in situ, as a potentially game changing element in future space architectures, with the potential to increase scale and reduce cost. However, before any decisions can be made on the inclusion of resources in exploration roadmaps or future scenarios some big questions need to be answered about the viability of different resource deposits and the processes for extraction and utilisation. The missions and measurements that will be required to answer these questions, and which are being prepared by agencies and others, can only be performed through the engagement and support of the science community. In answering questions about resources, data and knowledge will be generated that is of fundamental scientific importance. In supporting resource prospecting missions the science community will de facto generate new scientific knowledge. Science enables exploration and exploration enables science.

  15. The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion.

    PubMed

    Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela

    2013-09-01

    Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs. Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection.

  16. The knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens.

    PubMed

    Finka, Andrija; Saidi, Younousse; Goloubinoff, Pierre; Neuhaus, Jean-Marc; Zrÿd, Jean-Pierre; Schaefer, Didier G

    2008-10-01

    The seven subunit Arp2/3 complex is a highly conserved nucleation factor of actin microfilaments. We have isolated the genomic sequence encoding a putative Arp3a protein of the moss Physcomitrella patens. The disruption of this ARP3A gene by allele replacement has generated loss-of-function mutants displaying a complex developmental phenotype. The loss-of function of ARP3A gene results in shortened, almost cubic chloronemal cells displaying affected tip growth and lacking differentiation to caulonemal cells. In moss arp3a mutants, buds differentiate directly from chloronemata to form stunted leafy shoots having differentiated leaves similar to wild type. Yet, rhizoids never differentiate from stem epidermal cells. To characterize the F-actin organization in the arp3a-mutated cells, we disrupted ARP3A gene in the previously described HGT1 strain expressing conditionally the GFP-talin marker. In vivo observation of the F-actin cytoskeleton during P. patens development demonstrated that loss-of-function of Arp3a is associated with the disappearance of specific F-actin cortical structures associated with the establishment of localized cellular growth domains. Finally, we show that constitutive expression of the P. patens Arp3a and its Arabidopsis thaliana orthologs efficiently complement the mutated phenotype indicating a high degree of evolutionary conservation of the Arp3 function in land plants.

  17. In vitro amphotericin B effects on growth, viability and dimorphism of Paracoccidioides brasiliensis: reversal of the treatment.

    PubMed

    Jesuino, R S; Soares, R de B; Salem-Izacc, S M; Pereira, M; Felipe, M S; Soares, C M

    1997-01-01

    The in vitro effects of amphotericin B deoxycholate suspension (fungizone) on Paracoccidioides brasiliensis growth, cell viability and transformation were investigated. We also analyzed the protein synthesis patterns of both cellular forms, yeast and mycelium in the presence of AmB. This drug, at 30 micrograms/ml, highly inhibited yeast growth, which could be recovered depending on treatment time, where the most effective reversion was observed after 6 hr of incubation. The yeast cell viability, that had been partially affected by the drug, could also be efficiently recovered after AmB was removed. The effect of AmB on the cellular dimorphism process showed a strong reduction in the mycelium to yeast transformation (80% inhibition compared to the control without the drug). On the other hand, the transformation from yeast to mycelium in the presence of AmB was 50% affected, relative to the control. In contrast to the growth and cell viability experiments, the reversion effects on dimorphism were partial when the drug was removed, even with only 6 hr treatment. The two-dimensional gels of 35S-labeled proteins revealed a strong reduction in the three species of 80, 71 and 56 kDa in yeast and mycelium when treated with AmB.

  18. The Bioavailability of Soluble Cigarette Smoke Extract Is Reduced through Interactions with Cells and Affects the Cellular Response to CSE Exposure

    PubMed Central

    Bourgeois, Jeffrey S.; Jacob, Jeeva; Garewal, Aram; Ndahayo, Renata; Paxson, Julia

    2016-01-01

    Cellular exposure to cigarette smoke leads to an array of complex responses including apoptosis, cellular senescence, telomere dysfunction, cellular aging, and neoplastic transformation. To study the cellular response to cigarette smoke, a common in vitro model exposes cultured cells to a nominal concentration (i.e. initial concentration) of soluble cigarette smoke extract (CSE). However, we report that use of the nominal concentration of CSE as the only measure of cellular exposure is inadequate. Instead, we demonstrate that cellular response to CSE exposure is dependent not only on the nominal concentration of CSE, but also on specific experimental variables, including the total cell number, and the volume of CSE solution used. As found in other similar xenobiotic assays, our work suggests that the effective dose of CSE is more accurately related to the amount of bioavailable chemicals per cell. In particular, interactions of CSE components both with cells and other physical factors limit CSE bioavailability, as demonstrated by a quantifiably reduced cellular response to CSE that is first modified by such interactions. This has broad implications for the nature of cellular response to CSE exposure, and for the design of in vitro assays using CSE. PMID:27649082

  19. Viability of stored seed: extension by cathodic protection.

    PubMed

    Pammenter, N W; Adamson, J H; Berjak, P

    1974-12-20

    Placing seeds on a negatively charged conductor extended their viability during artificial aging. Such cathodic protection may reduce free radical attack by providing a source of electrons. The results stupport the hypothesis of free radical damage to cellular components and are consistent with such damage being important in deteriorative senescence changes.

  20. Should human chondrocytes fly? The impact of electromagnetic irradiation on chondrocyte viability and implications for their use in tissue engineering.

    PubMed

    Koehler, C; Niederbichler, A D; Scholz, T; Bode, B; Roos, J; Jung, F J; Hoerstrup, S P; Hellermann, J P; Wedler, V

    2006-12-01

    A significant logistic factor as to the successful clinical application of the autologous tissue engineering concept is efficient transportation: the donor cells need to be delivered to tissue processing facilities which in most cases requires air transportation. This study was designed to evaluate how human chondrocytes react to X-ray exposure. Primary cell cultures were established, cultured, incubated and exposed to different doses and time periods of radiation. Subsequently, quantitative cell proliferation assays were done and qualitative evaluation of cellular protein production were performed. Our results show that after irradiation of chondrocytes with different doses, no significant differences in terms of cellular viability occurred compared with the control group. These results were obtained when chondrocytes were exposed to luggage transillumination doses as well as exposure to clinically used radiation doses. Any damage affecting cell growth or quality was not observed in our study. However, information about damage of cellular DNA remains incomplete.

  1. Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes

    PubMed Central

    Twigg, Stephen R.F.; Babbs, Christian; van den Elzen, Marijke E.P.; Goriely, Anne; Taylor, Stephen; McGowan, Simon J.; Giannoulatou, Eleni; Lonie, Lorne; Ragoussis, Jiannis; Akha, Elham Sadighi; Knight, Samantha J.L.; Zechi-Ceide, Roseli M.; Hoogeboom, Jeannette A.M.; Pober, Barbara R.; Toriello, Helga V.; Wall, Steven A.; Rita Passos-Bueno, M.; Brunner, Han G.; Mathijssen, Irene M.J.; Wilkie, Andrew O.M.

    2013-01-01

    Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries—a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5′ untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5′ UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females. PMID:23335590

  2. Cell viability test after laser guidance

    NASA Astrophysics Data System (ADS)

    Rosenbalm, Tabitha N.; Owens, Sarah; Bakken, Daniel; Gao, Bruce Z.

    2006-02-01

    To precisely control the position of multiple types of cells in a coculture for the study of cell-cell interactions, we have developed a laser micropatterning technique. The technique employs the optical forces generated by a weakly focused laser beam. In the beam's focal region, the optical force draws microparticles, such as cells, into the center of the beam, propels them along the beam axis, and guides them onto a target surface. Specific patterns are created through computercontrolled micromanipulation of the substrate relative to the laser beam. Preliminary data have demonstrated cell viability after laser guidance. This project was designed to systematically vary the controllable laser parameters, namely, intensity and exposure time of the laser on single cells, and thus determine the laser parameters that allow negligible cell damage with functional cellular position control. To accomplish this goal, embryonic day 7 (E7) chick forebrain neurons were cultured in 35 mm petri dishes. Control and test cells were selected one hour after cell placement to allow cell attachment. Test cells were subjected to the laser at the focal region. The experimental parameters were chosen as: wavelength - 800 nm, intensities - 100 mW, 200 mW, and 300 mW, and exposure times - 10 s and 60 s. Results were analyzed based on neurite outgrowth and the Live/Dead assay (Viability/Cytoxicity kit from Molecular Probes). No statistical difference (p >> 0.1, student t-test) in viability or function was found between the control neurons and those exposed to the laser. This confirms that laser guidance seems to be a promising method for cellular manipulation.

  3. Short Peptides Enhance Single Cell Adhesion and Viability on Microarrays

    PubMed Central

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

    2011-01-01

    Single cell patterning holds important implications for biology, biochemistry, biotechnology, medicine, and bioinformatics. The challenge for single cell patterning is to produce small islands hosting only single cells and retaining their viability for a prolonged period of time. This study demonstrated a surface engineering approach that uses a covalently-bound short peptide as a mediator to pattern cells with improved single cell adhesion and prolonged cellular viability on gold patterned SiO2 substrates. The underlying hypothesis is that cell adhesion is regulated by the type, availability and stability of effective cell adhesion peptides, and thus covalently bound short peptides would promote cell spreading and thus, single cell adhesion and viability. The effectiveness of this approach and the underlying mechanism for the increased probability of single cell adhesion and prolonged cell viability by short peptides were studied by comparing cellular behavior of human umbilical cord vein endothelial cells on three model surfaces whose gold electrodes were immobilized with fibronectin, physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently-bound Lys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and binding properties were characterized by reflectance Fourier transform infrared spectroscopy. Both short peptides were superior to fibronectin in producing adhesion of only single cells, while the covalently bound peptide also reduced apoptosis and necrosis of adhered cells. Controlling cell spreading by peptide binding domains to regulate apoptosis and viability represents a fundamental mechanism in cell-materials interaction and provides an effective strategy in engineering arrays of single cells. PMID:17371055

  4. Effect of heat challenge on peripheral blood mononuclear cell viability: comparison of a tropical and temperate pig breed.

    PubMed

    Bambou, Jean-Christophe; Gourdine, Jean-Luc; Grondin, Roxanne; Vachiery, Nathalie; Renaudeau, David

    2011-12-01

    We evaluated the effect of heat challenge on cell viability, concanavalin A-induced proliferation and heat shock protein (HSPs) mRNA expression in peripheral mononuclear blood cells (PBMC) isolated from Creole (CR) and Large White (LW) pigs. The PBMCs were cultured for 9 h at 37 °C before being subjected to heat challenge: (1) at 42 °C or 45 °C for 2, 4, 6 and 9 h to monitor cell viability;(2) at 45 °C for 2 and 9 h followed by stimulation for 24 h at 37 °C with concanavalin A to evaluate mitogen-induced proliferation; and (3) at 45 °C for 3, 6 and 9 h to measure induction of HSP70.2 and HSP90 mRNA. Cell viability was affected by breed and temperature (P < 0.01), and the viability decrease caused by heat challenge was greater for LW than CR pigs. For mitogen-stimulated PBMCs, incubation at 45 °C reduced lymphoblastogenesis equally in both breeds (P < 0.01). Although heat challenge for 3 and 6 h at 45°C induced expression of HSP70.2 and HSP90 mRNA, no breed difference was observed. In conclusion, differences in heat resistance between these two breeds at the whole organism level are reflected at the cellular level. Neither HSP70.2 nor HSP90 mRNA expression levels explain this effect.

  5. Analysis of cell viability in intervertebral disc: Effect of endplate permeability on cell population.

    PubMed

    Shirazi-Adl, A; Taheri, M; Urban, J P G

    2010-05-07

    Responsible for making and maintaining the extracellular matrix, the cells of intervertebral discs are supplied with essential nutrients by diffusion from the blood supply through mainly the cartilaginous endplates (CEPs) and disc tissue. Decrease in transport rate and increase in cellular activity may adversely disturb the intricate supply-demand balance leading ultimately to cell death and disc degeneration. The present numerical study aimed to introduce for the first time cell viability criteria into nonlinear coupled nutrition transport equations thereby evaluating the dynamic nutritional processes governing viable cell population and concentrations of oxygen, glucose and lactic acid in the disc as CEP exchange area dropped from a fully permeable condition to an almost impermeable one. A uniaxial model of an in vitro cell culture analogue of the disc is first employed to examine and validate cell viability criteria. An axisymmetric model of the disc with four distinct regions was subsequently used to investigate the survival of cells at different CEP exchange areas. In agreement with measurements, predictions of the diffusion chamber model demonstrated substantial cell death as essential nutrient concentrations fell to levels too low to support cells. Cells died away from the nutrient supply and at higher cell densities. In the disc model, the nucleus region being farthest away from supply sources was most affected; cell death initiated first as CEP exchange area dropped below approximately 40% and continued exponentially thereafter to depletion as CEP calcified further. In cases with loss of endplate permeability and/or disruptions therein, as well as changes in geometry and fall in diffusivity associated with fluid outflow, the nutrient concentrations could fall to levels inadequate to maintain cellular activity or viability, resulting in cell death and disc degeneration.

  6. Pneumocystis melanins confer enhanced organism viability.

    PubMed

    Icenhour, Crystal R; Kottom, Theodore J; Limper, Andrew H

    2006-06-01

    Pneumocystis continues to represent an important opportunistic fungal pathogen of those with compromised immunity. Thus, it is crucial to identify factors that affect its viability and pathogenicity. We previously reported the first identification of melanins in Pneumocystis. In the present study, we sought to further characterize these components and define the function for these melanins. Melanins extracted from Pneumocystis and melanized Pneumocystis cells were analyzed by electron spin resonance spectroscopy, revealing spectra consistent with melanins from other fungi. Immunofluorescence assays using anti-melanin monoclonal antibodies showed that melanins are widely present across Pneumocystis host species, including mouse-, ferret-, and human-derived Pneumocystis organisms, as well as Pneumocystis carinii derived from rat. Using immunoelectron microscopy, melanins were found to localize to the cell wall and cytoplasm of P. carinii cysts, as well as to intracystic bodies within mature cysts. Next, the role of melanins on the maintenance of Pneumocystis viability was determined by using quantitative reverse transcription-PCR measurement of the heat shock protein mRNA under adverse environmental conditions. Using a new method to promote the melanization of Pneumocystis, we observed that strongly melanized Pneumocystis retained viability to a greater degree when exposed to UV irradiation or desiccation compared to less-pigmented organisms. These studies support our previous identification of Pneumocystis melanins across the genus, further characterize these Pneumocystis components, and demonstrate that melanins protect Pneumocystis from environmental stressors.

  7. Endothelial Cellular Responses to Biodegradable Metal Zinc.

    PubMed

    Ma, Jun; Zhao, Nan; Zhu, Donghui

    Biodegradable zinc (Zn) metals, a new generation of biomaterials, have attracted much attention due to their excellent biodegradability, bioabsorbability, and adaptability to tissue regeneration. Compared with magnesium (Mg) and iron (Fe), Zn exhibits better corrosion and mechanical behaviors in orthopedic and stent applications. After implantation, Zn containing material will slowly degrade, and Zn ions (Zn(2+)) will be released to the surrounding tissue. For stent applications, the local Zn(2+)concentration near endothelial tissue/cells could be high. However, it is unclear how endothelia will respond to such high concentrations of Zn(2+), which is pivotal to vascular remodeling and regeneration. Here, we evaluated the short-term cellular behaviors of primary human coronary artery endothelial cells (HCECs) exposed to a concentration gradient (0-140 μM) of extracellular Zn(2+). Zn(2+) had an interesting biphasic effect on cell viability, proliferation, spreading, and migration. Generally, low concentrations of Zn(2+) promoted viability, proliferation, adhesion, and migration, while high concentrations of Zn(2+) had opposite effects. For gene expression profiles, the most affected functional genes were related to cell adhesion, cell injury, cell growth, angiogenesis, inflammation, vessel tone, and coagulation. These results provide helpful information and guidance for Zn-based alloy design as well as the controlled release of Zn(2+)in stent and other related medical applications.

  8. Probiotic fermented sausage: viability of probiotic microorganisms and sensory characteristics.

    PubMed

    Rouhi, M; Sohrabvandi, S; Mortazavian, A M

    2013-01-01

    Probiotics are from functional foods that bring health benefits for humans. Nowadays, a major development in functional foods is related to food containing probiotic cultures, mainly lactic acid bacteria or bifidobacteria. Probiotics must be alive and ingested in sufficient amounts to exert the positive effects on the health and the well-being of the host. Therefore, viability of probiotic products (the minimum viable probiotic cells in each gram or milliliter of product till the time of consumption) is their most important characteristic. However, these organisms often show poor viability in fermented products due to their detrimental conditions. Today, the variety of fermented meat products available around the world is nearly equal to that of cheese. With meat products, raw fermented sausages could constitute an appropriate vehicle for such microorganisms into the human gastrointestinal tract. In present article, the viability of probiotic microorganisms in fermented sausage, the main factors affect their viability, and the sensorial characteristics of final product are discussed.

  9. Viability and Virulence of Entomopathogenic Nematodes Exposed to Ultraviolet Radiation

    PubMed Central

    Shapiro-Ilan, David I.; Hazir, Selcuk; Lete, Luis

    2015-01-01

    is not a sufficient measure for potential impact on biocontrol efficacy as other characters such as virulence may be severely affected even when viability remains high. PMID:26527839

  10. Loss of Cellular Sialidases Does Not Affect the Sialylation Status of the Prion Protein but Increases the Amounts of Its Proteolytic Fragment C1

    PubMed Central

    Katorcha, Elizaveta; Klimova, Nina; Makarava, Natallia; Savtchenko, Regina; Pan, Xuefang; Annunziata, Ida; Takahashi, Kohta; Miyagi, Taeko; Pshezhetsky, Alexey V.; d’Azzo, Alessandra; Baskakov, Ilia V.

    2015-01-01

    The central molecular event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC), which is a sialoglycoprotein, into the disease-associated, transmissible form denoted PrPSc. Recent studies revealed a correlation between the sialylation status of PrPSc and incubation time to disease and introduced a new hypothesis that progression of prion diseases could be controlled or reversed by altering the sialylation level of PrPC. Of the four known mammalian sialidases, the enzymes that cleave off sialic acid residues, only NEU1, NEU3 and NEU4 are expressed in the brain. To test whether cellular sialidases control the steady-state sialylation level of PrPC and to identify the putative sialidase responsible for desialylating PrPC, we analyzed brain-derived PrPC from knockout mice deficient in Neu1, Neu3, Neu4, or from Neu3/Neu4 double knockouts. Surprisingly, no differences in the sialylation of PrPC or its proteolytic product C1 were noticed in any of the knockout mice tested as compared to the age-matched controls. However, significantly higher amounts of the C1 fragment relative to full-length PrPC were detected in the brains of Neu1 knockout mice as compared to WT mice or to the other knockout mice. Additional experiments revealed that in neuroblastoma cell line the sialylation pattern of C1 could be changed by an inhibitor of sialylatransferases. In summary, this study suggests that targeting cellular sialidases is apparently not the correct strategy for altering the sialylation levels of PrPC, whereas modulating the activity of sialylatransferases might offer a more promising approach. Our findings also suggest that catabolism of PrPC involves its α-cleavage followed by desialylation of the resulting C1 fragments by NEU1 and consequent fast degradation of the desialylated products. PMID:26569607

  11. A methanolic extract of Ganoderma lucidum fruiting body inhibits the growth of a gastric cancer cell line and affects cellular autophagy and cell cycle.

    PubMed

    Oliveira, Marta; Reis, Filipa S; Sousa, Diana; Tavares, Catarina; Lima, Raquel T; Ferreira, Isabel C F R; dos Santos, Tiago; Vasconcelos, M Helena

    2014-07-25

    Ganoderma lucidum is one of the most extensively studied mushrooms as a functional food and as a chemopreventive agent due to its recognized medicinal properties. Some G. lucidum extracts have shown promising antitumor potential. In this study, the bioactive properties of various extracts of G. lucidum, from both the fruiting body and the spores, were investigated. The most potent extract identified was the methanolic fruiting body extract, which inhibited the growth of a gastric cancer cell line (AGS) by interfering with cellular autophagy and cell cycle.

  12. Behaviour of Saccharomyces cerevisiae wine strains during adaptation to unfavourable conditions of fermentation on synthetic medium: cell lipid composition, membrane integrity, viability and fermentative activity.

    PubMed

    Mannazzu, Ilaria; Angelozzi, Daniele; Belviso, Simona; Budroni, Marilena; Farris, Giovanni Antonio; Goffrini, Paola; Lodi, Tiziana; Marzona, Mario; Bardi, Laura

    2008-01-15

    During must fermentation wine strains are exposed to a variety of biotic and abiotic stresses which, when prevailing over the cellular defence systems, can affect cell viability with negative consequences on the progression of the fermentative process. To investigate the ability of wine strains to survive and adapt to unfavourable conditions of fermentation, the lipid composition, membrane integrity, cell viability and fermentative activity of three strains of Saccharomyces cerevisiae were analysed during hypoxic growth in a sugar-rich medium lacking lipid nutrients. These are stressful conditions, not unusual during must fermentation, which, by affecting lipid biosynthesis may exert a negative effect on yeast viability. The results obtained showed that the three strains were able to modulate cell lipid composition during fermentation. However, only two of them, which showed highest viability and membrane integrity at the end of the fermentation process, reached a fatty acid composition which seemed to be optimal for a successful adaptation. In particular, C16/TFA and UFA/TFA ratios, more than total lipid and ergosterol contents, seem to be involved in yeast adaptation.

  13. Tetracapsuloides bryosalmonae infection affects the expression of genes involved in cellular signal transduction and iron metabolism in the kidney of the brown trout Salmo trutta.

    PubMed

    Kumar, Gokhlesh; Sarker, Subhodeep; Menanteau-Ledouble, Simon; El-Matbouli, Mansour

    2015-06-01

    Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease in various species of salmonids in Europe and North America. The life cycle of the European strain of T. bryosalmonae generally completes in an invertebrate host freshwater bryozoan and vertebrate host brown trout (Salmo trutta) Linnaeus, 1758. Little is known about the gene expression in the kidney of brown trout during the developmental stages of T. bryosalmonae. In the present study, quantitative real-time PCR was applied to quantify the target genes of interest in the kidney of brown trout at different time points of T. bryosalmonae development. PCR primers specific for target genes were designed and optimized, and their gene expression levels were quantified in the cDNA kidney samples using SYBR Green Supermix. Expression of Rab GDP dissociation inhibitor beta, integral membrane protein 2B, NADH dehydrogenase 1 beta subcomplex subunit 6, and 26S protease regulatory subunit S10B were upregulated significantly in infected brown trout, while the expression of the ferritin M middle subunit was downregulated significantly. These results suggest that host genes involved in cellular signal transduction, proteasomal activities, including membrane transporters and cellular iron storage, are differentially upregulated or downregulated in the kidney of brown trout during parasite development. The gene expression pattern of infected renal tissue may support the development of intraluminal sporogonic stages of T. bryosalmonae in the renal tubular lumen of brown trout which may facilitate the release of viable parasite spores to transmit to the invertebrate host bryozoan.

  14. NMDA-R inhibition affects cellular process formation in Tilapia melanocytes; a model for pigmented adrenergic neurons in process formation and retraction.

    PubMed

    Ogundele, Olalekan Michael; Okunnuga, Adetokunbo Adedotun; Fabiyi, Temitope Deborah; Olajide, Olayemi Joseph; Akinrinade, Ibukun Dorcas; Adeniyi, Philip Adeyemi; Ojo, Abiodun Ayodele

    2014-06-01

    Parkinson's disease has long been described to be a product of dopamine and (or) melanin loss in the substanstia nigra (SN). Although most studies have focused on dopaminergic neurons, it is important to consider the role of pigment cells in the etiology of the disease and to create an in vitro live cell model for studies involving pigmented adrenergic cells of the SN in Parkinsonism. The Melanocytes share specific features with the pigmented adrenergic neurons as both cells are pigmented, contain adrenergic receptors and have cellular processes. Although the melanocyte cellular processes are relatively short and observable only when stimulated appropriately by epinephrine and other factors or molecules. This study employs the manipulation of N-Methyl-D-Aspartate Receptor (NMDA-R), a major receptor in neuronal development, in the process formation pattern of the melanocyte in order to create a suitable model to depict cellular process elongation and shortening in pigmented adrenergic cells. NMDA-R is an important glutamate receptor implicated in neurogenesis, neuronal migration, maturation and cell death, thus we investigated the role of NMDA-R potentiation by glutamate/KCN and its inhibition by ketamine in the behavior of fish scale melanocytes in vitro. This is aimed at establishing the regulatory role of NMDA-R in this cell type (melanocytes isolated form Tilapia) in a similar manner to what is observable in the mammalian neurons. In vitro live cell culture was prepared in modified Ringer's solution following which the cells were treated as follows; Control, Glutamate, Ketamine, Glutamate + Ketamine, KCN + Ketamine and KCN. The culture was maintained for 10 min and the changes were captured in 3D-Time frame at 0, 5 and 10 min for the control and 5, 7 and 10 min for each of the treatment category. Glutamate treatment caused formation of short cellular processes localized directly on the cell body while ketamine treatment (inhibition of NMDA-R) facilitated

  15. The influence of bisphosphonates on viability, migration, and apoptosis of human oral keratinocytes--in vitro study.

    PubMed

    Pabst, Andreas M; Ziebart, Thomas; Koch, Felix P; Taylor, Katherine Y; Al-Nawas, Bilal; Walter, Christian

    2012-02-01

    Bisphosphonate-associated osteonecrosis of the jaw (BP-ONJ) is one of the most often seen side effects in patients treated with bisphosphonates, presenting clinically as a non-healing wound. One theory of BP-ONJ etiology describes a negative effect on soft tissues, especially on keratinocytes, which play an important role in oral wound healing and oral soft tissue regeneration. A high cell viability of keratinocytes, which can migrate to the affected location, is essential for wound healing. The aim of this in vitro study was to investigate the effect of differently potent bisphosphonates on human oral keratinocytes (HOK).Three nitrogen-containing bisphosphonates (ibandronate, pamidronate, and zoledronate) and one non-nitrogen-containing bisphosphonate (clodronate) were compared concerning their potency on cell viability (calcein assay and MTT assay), migration ability (Boyden chamber migration assay and scratch wound proliferation assay), and apoptosis (TUNEL assay) of HOK.The nitrogen-containing bisphosphonates, particularly highly potent pamidronate and zoledronate preparations, had a strong negative influence on cell viability, migration ability, and apoptosis of HOK. The non-nitrogen-containing clodronate even increased cell viability in higher concentrations.This study demonstrates that bisphosphonates have a strong influence on HOK on different cellular levels like cell viability, migration ability, and apoptosis rate. The results support the theory that BP-ONJ is a multifactorially caused disease.Furthermore, this in vitro study confirms the theory that perioperative interruption of bisphosphonate application during dental surgical procedures might be feasible to promote better tissue regeneration and wound healing.

  16. Bisphenol A Disrupts Transcription and Decreases Viability in Aging Vascular Endothelial Cells

    PubMed Central

    Ribeiro-Varandas, Edna; Pereira, H. Sofia; Monteiro, Sara; Neves, Elsa; Brito, Luísa; Boavida Ferreira, Ricardo; Viegas, Wanda; Delgado, Margarida

    2014-01-01

    Bisphenol A (BPA) is a widely utilized endocrine disruptor capable of mimicking endogenous hormones, employed in the manufacture of numerous consumer products, thereby interfering with physiological cellular functions. Recent research has shown that BPA alters epigenetic cellular mechanisms in mammals and may be correlated to enhanced cellular senescence. Here, the effects of BPA at 10 ng/mL and 1 µg/mL, concentrations found in human samples, were analyzed on HT29 human colon adenocarcinona cell line and Human Umbilical Vein Endothelial Cells (HUVEC). Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) transcriptional analysis of the Long Interspersed Element-1 (LINE-1) retroelement showed that BPA induces global transcription deregulation in both cell lines, although with more pronounced effects in HUVEC cells. Whereas there was an increase in global transcription in HT29 exclusively after 24 h of exposure, this chemical had prolonged effects on HUVEC. Immunoblotting revealed that this was not accompanied by alterations in the overall content of H3K9me2 and H3K4me3 epigenetic marks. Importantly, cell viability assays and transcriptional analysis indicated that prolonged BPA exposure affects aging processes in senescent HUVEC. To our knowledge this is the first report that BPA interferes with senescence in primary vascular endothelial cells, therefore, suggesting its association to the etiology of age-related human pathologies, such as atherosclerosis. PMID:25207595

  17. Bisphenol A disrupts transcription and decreases viability in aging vascular endothelial cells.

    PubMed

    Ribeiro-Varandas, Edna; Pereira, H Sofia; Monteiro, Sara; Neves, Elsa; Brito, Luísa; Ferreira, Ricardo Boavida; Viegas, Wanda; Delgado, Margarida

    2014-09-09

    Bisphenol A (BPA) is a widely utilized endocrine disruptor capable of mimicking endogenous hormones, employed in the manufacture of numerous consumer products, thereby interfering with physiological cellular functions. Recent research has shown that BPA alters epigenetic cellular mechanisms in mammals and may be correlated to enhanced cellular senescence. Here, the effects of BPA at 10 ng/mL and 1 µg/mL, concentrations found in human samples, were analyzed on HT29 human colon adenocarcinona cell line and Human Umbilical Vein Endothelial Cells (HUVEC). Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) transcriptional analysis of the Long Interspersed Element-1 (LINE-1) retroelement showed that BPA induces global transcription deregulation in both cell lines, although with more pronounced effects in HUVEC cells. Whereas there was an increase in global transcription in HT29 exclusively after 24 h of exposure, this chemical had prolonged effects on HUVEC. Immunoblotting revealed that this was not accompanied by alterations in the overall content of H3K9me2 and H3K4me3 epigenetic marks. Importantly, cell viability assays and transcriptional analysis indicated that prolonged BPA exposure affects aging processes in senescent HUVEC. To our knowledge this is the first report that BPA interferes with senescence in primary vascular endothelial cells, therefore, suggesting its association to the etiology of age-related human pathologies, such as atherosclerosis.

  18. An Arabidopsis senescence-associated protein SAG29 regulates cell viability under high salinity.

    PubMed

    Seo, Pil Joon; Park, Jung-Min; Kang, Seok Ki; Kim, Sang-Gyu; Park, Chung-Mo

    2011-01-01

    The plasma membrane is an important cellular organ that perceives incoming developmental and environmental signals and integrates these signals into cellular regulatory mechanisms. It also acts as a barrier against unfavorable extracellular factors to maintain cell viability. Despite its importance for cell viability, molecular components determining cell viability and underlying mechanisms are largely unknown. Here, we show that a plasma membrane-localized MtN3 protein SAG29 regulates cell viability under high salinity in Arabidopsis. The SAG29 gene is expressed primarily in senescing plant tissues. It is induced by osmotic stresses via an abscisic acid-dependent pathway. Whereas the SAG29-overexpressing transgenic plants (35S:SAG29) exhibited an accelerated senescence and were hypersensitive to salt stress, the SAG29-deficient mutants were less sensitive to high salinity. Consistent with this, the 35S:SAG29 transgenic plants showed reduced cell viability in the roots under normal growth condition. In contrast, cell viability in the SAG29-deficient mutant roots was indistinguishable from that in the roots of control plants. Notably, the mutant roots exhibited enhanced cell viability under high salinity. Our observations indicate that the senescence-associated SAG29 protein is associated with cell viability under high salinity and other osmotic stress conditions. We propose that the SAG29 protein may serve as a molecular link that integrates environmental stress responses into senescing process.

  19. Bog bilberry (Vaccinium uliginosum L.) extract reduces cultured Hep-G2, Caco-2, and 3T3-L1 cell viability, affects cell cycle progression, and has variable effects on membrane permeability.

    PubMed

    Liu, Jia; Zhang, Wei; Jing, Hao; Popovich, David G

    2010-04-01

    Bog bilberry (Vaccinium uliginosum L.) is a blue-pigmented edible berry related to bilberry (Vaccinium myrtillus L.) and the common blueberry (Vaccinium corymbosum). The objective of this study was to investigate the effect of a bog bilberry anthocyanin extract (BBAE) on cell growth, membrane permeability, and cell cycle of 2 malignant cancer cell lines, Caco-2 and Hep-G2, and a nonmalignant murine 3T3-L1 cell line. BBAE contained 3 identified anthocyanins. The most abundant anthocyanin was cyanidin-3-glucoside (140.9 +/- 2.6 microg/mg of dry weight), followed by malvidin-3-glucoside (10.3 +/- 0.3 microg/mg) and malvidin-3-galactoside (8.1 +/- 0.4 microg/mg). Hep-G2 LC50 was calculated to be 0.563 +/- 0.04 mg/mL, Caco-2 LC50 was 0.390 +/- 0.30 mg/mL and 0.214 +/- 0.02 mg/mL for 3T3-L1 cells. LDH release, a marker of membrane permeability, was significantly increased in Hep-G2 cells and Caco-2 cells after 48 and 72 h compared to 24 h. The increase was 21% at 48 h and 57% at 72 h in Caco-2 cells and 66% and 139% in Hep-G2 cells compared to 24 h. However, 3T3-L1 cells showed an unexpected significant lower LDH activity (P < or = 0.05) after 72 h of exposure corresponding to a 21% reduction in LDH release. BBAE treatment increased sub-G1 in all 3 cell lines without influencing cells in the G2/M phase. BBAE treatment reduced the growth and increased the accumulation of sub-G1 cells in 2 malignant and 1 nonmalignant cell line; however, the effect on membrane permeability differs considerably between the malignant and nonmalignant cells and may in part be due to differences in cellular membrane composition.

  20. Difference between Extra- and Intracellular T1 Values of Carboxylic Acids Affects the Quantitative Analysis of Cellular Kinetics by Hyperpolarized NMR.

    PubMed

    Karlsson, Magnus; Jensen, Pernille Rose; Ardenkjaer-Larsen, Jan Henrik; Lerche, Mathilde H

    2016-10-17

    Incomplete knowledge of the longitudinal relaxation time constant (T1 ) leads to incorrect assumptions in quantitative kinetic models of cellular systems, studied by hyperpolarized real-time NMR. Using an assay that measures the intracellular signal of small carboxylic acids in living cells, the intracellular T1 of the carboxylic acid moiety of acetate, keto-isocaproate, pyruvate, and butyrate was determined. The intracellular T1 is shown to be up to four-fold shorter than the extracellular T1 . Such a large difference in T1 values between the inside and the outside of the cell has significant influence on the quantification of intracellular metabolic activity. It is expected that the significantly shorter T1 value of the carboxylic moieties inside cells is a result of macromolecular crowding. An artificial cytosol has been prepared and applied to predict the T1 of other carboxylic acids. We demonstrate the value of this prediction tool.

  1. Previous chronic exogenous glucocorticoid administration in vivo does not affect functional characteristics and cellular lifespan of human skin fibroblasts in vitro.

    PubMed

    Pratsinis, Harris; Dimozi, Anastasia; Pilichos, Konstantinos; Tsagarakis, Stylianos; Yiacoumettis, Andreas M; Kletsas, Dimitris

    2011-06-01

    Excess of glucocorticoids (GCs) has been reported to lead to skin atrophy and impaired wound healing. The present study investigates whether human skin fibroblasts suffer permanent damages due to a long-term exposure to GC excess. Fibroblasts obtained from patients being under GC treatment for periods over one year were cultured under standard conditions in vitro, and studied regarding pivotal parameters involved in skin homeostasis and aging, i.e. collagen production, cell proliferation, and cellular replicative lifespan. No statistical differences were observed regarding these functions compared to those of normal human skin fibroblasts. Furthermore, no differences between normal and patient-derived cells were observed regarding their sensitivity to a supra-physiological cortisol concentration. In conclusion, the prolonged exposure of human skin fibroblasts in vivo to high concentrations of exogenously-administered GC does not lead to persistent adverse effects on their physiology.

  2. The Dark Recovery Rate in the Photocycle of the Bacterial Photoreceptor YtvA Is Affected by the Cellular Environment and by Hydration

    PubMed Central

    Losi, Aba; Mandalari, Carmen; Bedotti, Roberta; Viappiani, Cristiano; Zanacchi, Francesca Cella; Diaspro, Alberto; Gärtner, Wolfgang

    2014-01-01

    We report thermal recovery kinetics of the lit state into the parental dark state, measured for the blue light-sensing photoreceptor YtvA inside overexpressing E. coli and B. subtilis bacterial cells, performed for the wild type and several mutated proteins. Recovery was followed as a recovery of the fluorescence, as this property is only found for the parental but not for the photochemically generated lit state. When cells were deposited onto a microscope glass plate, the observed thermal recovery rate in the photocycle was found ca. ten times faster in comparison to purified YtvA in solution. When the E. coli or B. subtilis colonies were soaked in an isotonic buffer, the dark relaxation became again much slower and was very similar to that observed for YtvA in solution. The observed effects show that rate constants can be tuned by the cellular environment through factors such as hydration. PMID:25211155

  3. Expression of R132H mutational IDH1 in human U87 glioblastoma cells affects the SREBP1a pathway and induces cellular proliferation.

    PubMed

    Zhu, Jian; Cui, Gang; Chen, Ming; Xu, Qinian; Wang, Xiuyun; Zhou, Dai; Lv, Shengxiang; Fu, Linshan; Wang, Zhong; Zuo, Jianling

    2013-05-01

    Sterol regulatory element-binding protein-1a (SREBP1a) is a member of the SREBP family of transcription factors, which mainly controls homeostasis of lipids. SREBP1a can also activate the transcription of isocitrate dehydrogenase 1 (IDH1) by binding to its promoter region. IDH1 mutations, especially R132H mutation of IDH1, are a common feature of a major subset of human gliomas. There are few data available on the relationship between mutational IDH1 expression and SREBP1a pathway. In this study, we investigated cellular effects and SREBP1a pathway alterations caused by R132H mutational IDH1 expression in U87 cells. Two glioma cell lines, stably expressing mutational (U87/R132H) or wild type (U87/wt) IDH1, were established. A cell line, stably transfected with pcDNA3.1(+) (U87/vector), was generated as a control. Click-iT EdU assay, sulforhodamine B assay, and wound healing assay respectively showed that the expression of R132H induced cellular proliferation, cell growth, and cell migration. Western blot revealed that SREBP1 was increased in U87/R132H compared with that in U87/wt. Elevated SREBP1a and several its target genes, but not SREBP1c, were detected by real-time polymerase chain reaction in U87/R132H. All these findings indicated that R132H mutational IDH1 is involved in the regulation of proliferation, growth, and migration of glioma cells. These effects may partially be mediated by SREBP1a pathway.

  4. Kinetic viability assays using DRAQ7 probe.

    PubMed

    Wlodkowic, Donald; Akagi, Jin; Dobrucki, Jurek; Errington, Rachel; Smith, Paul J; Takeda, Kazuo; Darzynkiewicz, Zbigniew

    2013-07-01

    Cell death within cell populations is a stochastic process where cell-to-cell variation in temporal progression through the various stages of cell death arises from asynchrony of subtle fluctuations in the signaling pathways. Most cell death assays rely on detection of the specific marker of cell demise at the end-point of cell culturing. Such an approach cannot account for the asynchrony and the stochastic nature of cell response to the death-inducing signal. There is a need therefore for rapid and high-throughput bioassays capable of continuously tracking viability of individual cells from the time of encountering a stress signal up to final stages of their demise. In this context, a new anthracycline derivative, DRAQ7, is gaining increasing interest as an easy-to-use marker capable of long-term monitoring of cell death in real-time. This novel probe neither penetrates the plasma membrane of living cells nor does it affect the cells' susceptibility to the death-inducing agents. However, when the membrane integrity is compromised, DRAQ7 enters cells undergoing demise and binds readily to nuclear DNA to report cell death. Here, we provide three sets of protocols for viability assays using DRAQ7 probe. The first protocol describes the innovative use of single-color DRAQ7 real-time assay to dynamically track cell viability. The second protocol outlines a simplified end-point DRAQ7 staining approach. The final protocol highlights the real-time and multiparametric apoptosis assay utilizing DRAQ7 dye concurrently with tetramethylrhodamine methyl ester (TMRM), the mitochondrial trans-membrane electrochemical potential (ΔΨm) sensing probe.

  5. Bioluminescence assay for cell viability.

    PubMed

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N

    2015-06-01

    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  6. Comparison of reintroduction and enhancement effects on metapopulation viability

    USGS Publications Warehouse

    Halsey, Samniqueka J; Bell, Timothy J.; McEachern, Kathryn; Pavlovic, Noel B.

    2015-01-01

    Metapopulation viability depends upon a balance of extinction and colonization of local habitats by a species. Mechanisms that can affect this balance include physical characteristics related to natural processes (e.g. succession) as well as anthropogenic actions. Plant restorations can help to produce favorable metapopulation dynamics and consequently increase viability; however, to date no studies confirm this is true. Population viability analysis (PVA) allows for the use of empirical data to generate theoretical future projections in the form of median time to extinction and probability of extinction. In turn, PVAs can inform and aid the development of conservation, recovery, and management plans. Pitcher's thistle (Cirsium pitcheri) is a dune endemic that exhibited metapopulation dynamics. We projected viability of three natural and two restored populations with demographic data spanning 15–23 years to determine the degree the addition of reintroduced population affects metapopulation viability. The models were validated by comparing observed and projected abundances and adjusting parameters associated with demographic and environmental stochasticity to improve model performance. Our chosen model correctly predicted yearly population abundance for 60% of the population-years. Using that model, 50-year projections showed that the addition of reintroductions increases metapopulation viability. The reintroduction that simulated population performance in early-successional habitats had the maximum benefit. In situ enhancements of existing populations proved to be equally effective. This study shows that restorations can facilitate and improve metapopulation viability of species dependent on metapopulation dynamics for survival with long-term persistence of C. pitcheri in Indiana likely to depend on continued active management.

  7. Genetic Dominance & Cellular Processes

    ERIC Educational Resources Information Center

    Seager, Robert D.

    2014-01-01

    In learning genetics, many students misunderstand and misinterpret what "dominance" means. Understanding is easier if students realize that dominance is not a mechanism, but rather a consequence of underlying cellular processes. For example, metabolic pathways are often little affected by changes in enzyme concentration. This means that…

  8. The cellular proteome is affected by a gelsolin (BbGEL1) during morphological transitions in aerobic surface versus liquid growth in the entomopathogenic fungus Beauveria bassiana.

    PubMed

    He, Pu-Hong; Dong, Wei-Xia; Chu, Xin-Ling; Feng, Ming-Guang; Ying, Sheng-Hua

    2016-11-01

    The gelsolin superfamily includes seven protein members: gelsolin, villin, adseverin, CapG, advillin, supervillin and flightless I. The gelsolin proteins are actin-binding proteins that contain three or six gelsolin-like domains, and they play important roles in remodelling actin dynamics and cellular processes in eukaryotes. The entomopathogenic fungus Beauveria bassiana expresses a unique CapG protein (BbGEL1) that contains three gelsolin-like domains. BbGEL1p is associated with actin during mycelial growth and plays an important role in fungal morphological transitions under both aerobic and submerged conditions. The ΔBbGEL1 mutant displays abnormal spore-producing structures that reduce the conidial and blastospore yields by approximately 70% and 90% respectively. The virulence of the ΔBbGEL1 mutant is notably reduced as indicated by topical and intrahemocoel injection assays. Two comparative proteomics analyses indicated that BbGEL1 has significantly different roles in the development of conidia and blastospores, and the results revealed the potential targets of BbGEL1 in the corresponding developmental processes. Additionally, as an overlapping downstream protein of BbGEL1, the hydrophobin-like protein gene BbHyd3 is required for conidiation but has a negative role in blastospore formation. Our findings indicate that in addition to its function as an actin-interacting protein, BbGEL1 contributes to fungal morphological transitions via broad genetic pathways.

  9. Cellular Prion Protein Combined with Galectin-3 and -6 Affects the Infectivity Titer of an Endogenous Retrovirus Assayed in Hippocampal Neuronal Cells

    PubMed Central

    Shin, Hae-Young; Goto, Joy J.; Carp, Richard I.; Choi, Eun-Kyoung; Kim, Yong-Sun

    2016-01-01

    Prion diseases are infectious and fatal neurodegenerative diseases which require the cellular prion protein, PrPC, for development of diseases. The current study shows that the PrPC augments infectivity and plaque formation of a mouse endogenous retrovirus, MuLV. We have established four neuronal cell lines expressing mouse PrPC, PrP+/+; two express wild type PrPC (MoPrPwild) and the other two express mutant PrPC (MoPrPmut). Infection of neuronal cells from various PrP+/+ and PrP-/- (MoPrPKO) lines with MuLV yielded at least three times as many plaques in PrP+/+ than in PrP-/-. Furthermore, among the four PrP+/+ lines, one mutant line, P101L, had at least 2.5 times as many plaques as the other three PrP+/+ lines. Plaques in P101L were four times larger than those in other PrP+/+ lines. Colocalization of PrP and CAgag was seen in MuLV-infected PrP+/+ cells. In the PrP-MuLV interaction, the involvement of galectin-3 and -6 was observed by immunoprecipitation with antibody to PrPC. These results suggest that PrPC combined with galectin-3 and -6 can act as a receptor for MuLV. P101L, the disease form of mutant PrPC results suggest the genetic mutant form of PrPC may be more susceptible to viral infection. PMID:27936017

  10. Genetic improvement of Saccharomyces cerevisiae wine strains for enhancing cell viability after desiccation stress.

    PubMed

    López-Martínez, Gema; Pietrafesa, Rocchina; Romano, Patrizia; Cordero-Otero, Ricardo; Capece, Angela

    2013-08-01

    In the last few decades spontaneous grape must fermentations have been replaced by inoculated fermentation with Saccharomyces cerevisiae strains as active dry yeast (ADY). Among the essential genes previously characterized to overcome the cell-drying/rehydration process, six belong to the group of very hydrophilic proteins known as hydrophilins. Among them, only SIP18 has shown early transcriptional response during dehydration stress. In fact, the overexpression in S. cerevisiae of gene SIP18 increases cell viability after the dehydration process. The purpose of this study was to characterize dehydration stress tolerance of three wild and one commercial S. cerevisiae strains of wine origin. The four strains were submitted to transformation by insertion of the gene SIP18. Selected transformants were submitted to the cell-drying-rehydration process and yeast viability was evaluated by both viable cell count and flow cytometry. The antioxidant capacity of SIP18p was illustrated by ROS accumulation reduction after H2 O2 attack. Growth data as cellular duplication times and lag times were calculated to estimate cell vitality after the cell rehydration process. The overexpressing SIP18 strains showed significantly longer time of lag phase despite less time needed to stop the leakage of intracellular compounds during the rehydration process. Subsequently, the transformants were tested in inoculated grape must fermentation at laboratory scale in comparison to untransformed strains. Chemical analyses of the resultant wines indicated that no significant change for the content of secondary compounds was detected. The obtained data showed that the transformation enhances the viability of ADY without affecting fermentation efficiency and metabolic behaviour.

  11. Influence of novel resin monomer on viability of L-929 mouse fibroblasts in vitro.

    PubMed

    Jinno, Satoshi; Kawai, Tatsushi; Ishikawa, Atsuko; Suzuki, Tomoo; Hattori, Nobuaki; Okeya, Hiroyuki; Hayashi, Tatsuhide; Maeda, Hatsuhiko; Ohno, Yuzo; Ito, Masamitsu; Noguchi, Toshihide

    2006-12-01

    We have previously synthesized a novel acrylic resin monomer, methacryloyloxyethyl methyl succinate (TA). The aim of this in vitro study, therefore, was to examine its influence on cell viability using L-929 mouse fibroblasts and then compare the results with MMA, EMA, and LMA. Medium containing each monomer was changed every 15 minutes as some monomers were volatile. After one hour of exposure, these mediums were replaced with a normal medium and cells were further incubated for 72 hours. IC50 value for each monomer was determined, and chronological cell viability and cytomorphologic observation were evaluated. Viability was impaired in a dose-dependent manner. All monomers, except TA, tended to correlate between molecular weight and cell viability. On the other hand, TA showed excellent viability and did not impair growth abruptly. These results thus demonstrated that cellular damage by TA was much lower than that by other monomers.

  12. Spatial and Temporal Measurements of Temperature and Cell Viability in Response to Nanoparticle Mediated Photothermal Therapy

    SciTech Connect

    Whitney, Jon R; Rodgers, Amanda; Harvie, Erica; Carswell, William; Torti, Suzy; Puretzky, Alexander A; Rouleau, Christopher M; Geohegan, David B; Rylander, Christopher; Rylander, Nichole M

    2012-01-01

    Aim: Nanoparticle enhanced photothermal therapy is a promising alternative to tumor resection. However, quantitative measurements of cellular response to these treatments are limited. This paper introduces a Bimodal Enhanced Analysis of Spatiotemporal Temperature (BEAST) algorithm to rapidly determine the viability of cancer cells in vitro following photothermal therapy alone or in combination with nanoparticles. Materials & Methods: To illustrate the capability of the BEAST viability algorithm, single wall carbon nanohorns were added to renal cancer (RENCA) cells in vitro and time-dependent spatial temperature maps measured with an infrared camera during laser therapy were correlated with post-treatment cell viability distribution maps obtained by cell-staining fluorescent microscopy. Conclusion: The BEAST viability algorithm accurately and rapidly determined the cell viability as function of time, space, and temperature.

  13. 3D finite element analysis of nutrient distributions and cell viability in the intervertebral disc: effects of deformation and degeneration.

    PubMed

    Jackson, Alicia R; Huang, Chun-Yuh C; Brown, Mark D; Gu, Wei Yong

    2011-09-01

    The intervertebral disc (IVD) receives important nutrients, such as glucose, from surrounding blood vessels. Poor nutritional supply is believed to play a key role in disc degeneration. Several investigators have presented finite element models of the IVD to investigate disc nutrition; however, none has predicted nutrient levels and cell viability in the disc with a realistic 3D geometry and tissue properties coupled to mechanical deformation. Understanding how degeneration and loading affect nutrition and cell viability is necessary for elucidating the mechanisms of disc degeneration and low back pain. The objective of this study was to analyze the effects of disc degeneration and static deformation on glucose distributions and cell viability in the IVD using finite element analysis. A realistic 3D finite element model of the IVD was developed based on mechano-electrochemical mixture theory. In the model, the cellular metabolic activities and viability were related to nutrient concentrations, and transport properties of nutrients were dependent on tissue deformation. The effects of disc degeneration and mechanical compression on glucose concentrations and cell density distributions in the IVD were investigated. To examine effects of disc degeneration, tissue properties were altered to reflect those of degenerated tissue, including reduced water content, fixed charge density, height, and endplate permeability. Two mechanical loading conditions were also investigated: a reference (undeformed) case and a 10% static deformation case. In general, nutrient levels decreased moving away from the nutritional supply at the disc periphery. Minimum glucose levels were at the interface between the nucleus and annulus regions of the disc. Deformation caused a 6.2% decrease in the minimum glucose concentration in the normal IVD, while degeneration resulted in an 80% decrease. Although cell density was not affected in the undeformed normal disc, there was a decrease in cell

  14. Carbon nanotubes affect the toxicity of CuO nanoparticles to denitrification in marine sediments by altering cellular internalization of nanoparticle

    NASA Astrophysics Data System (ADS)

    Zheng, Xiong; Su, Yinglong; Chen, Yinguang; Wan, Rui; Li, Mu; Huang, Haining; Li, Xu

    2016-06-01

    Denitrification is an important pathway for nitrate transformation in marine sediments, and this process has been observed to be negatively affected by engineered nanomaterials. However, previous studies only focused on the potential effect of a certain type of nanomaterial on microbial denitrification. Here we show that the toxicity of CuO nanoparticles (NPs) to denitrification in marine sediments is highly affected by the presence of carbon nanotubes (CNTs). It was found that the removal efficiency of total NOX‑-N (NO3‑-N and NO2‑-N) in the presence of CuO NPs was only 62.3%, but it increased to 81.1% when CNTs appeared in this circumstance. Our data revealed that CuO NPs were more easily attached to CNTs rather than cell surface because of the lower energy barrier (3.5 versus 36.2 kT). Further studies confirmed that the presence of CNTs caused the formation of large, incompact, non-uniform dispersed, and more negatively charged CuO-CNTs heteroaggregates, and thus reduced the nanoparticle internalization by cells, leading to less toxicity to metabolism of carbon source, generation of reduction equivalent, and activities of nitrate reductase and nitrite reductase. These results indicate that assessing nanomaterial-induced risks in real circumstances needs to consider the “mixed” effects of nanomaterials.

  15. Carbon nanotubes affect the toxicity of CuO nanoparticles to denitrification in marine sediments by altering cellular internalization of nanoparticle

    PubMed Central

    Zheng, Xiong; Su, Yinglong; Chen, Yinguang; Wan, Rui; Li, Mu; Huang, Haining; Li, Xu

    2016-01-01

    Denitrification is an important pathway for nitrate transformation in marine sediments, and this process has been observed to be negatively affected by engineered nanomaterials. However, previous studies only focused on the potential effect of a certain type of nanomaterial on microbial denitrification. Here we show that the toxicity of CuO nanoparticles (NPs) to denitrification in marine sediments is highly affected by the presence of carbon nanotubes (CNTs). It was found that the removal efficiency of total NOX−-N (NO3−-N and NO2−-N) in the presence of CuO NPs was only 62.3%, but it increased to 81.1% when CNTs appeared in this circumstance. Our data revealed that CuO NPs were more easily attached to CNTs rather than cell surface because of the lower energy barrier (3.5 versus 36.2 kT). Further studies confirmed that the presence of CNTs caused the formation of large, incompact, non-uniform dispersed, and more negatively charged CuO-CNTs heteroaggregates, and thus reduced the nanoparticle internalization by cells, leading to less toxicity to metabolism of carbon source, generation of reduction equivalent, and activities of nitrate reductase and nitrite reductase. These results indicate that assessing nanomaterial-induced risks in real circumstances needs to consider the “mixed” effects of nanomaterials. PMID:27279546

  16. Micro-organism and cell viability on antimicrobially modified titanium.

    PubMed

    Omori, S; Shibata, Y; Arimoto, T; Igarashi, T; Baba, K; Miyazaki, T

    2009-10-01

    When titanium is anodized by discharge in NaCl solution, both antimicrobial activity and osteoconductivity are conferred. The viability of adherent micro-organisms and cells on antimicrobial titanium remains uncertain. We hypothesized that a thin peroxidation barrier would efficiently destroy adherent bacteria, whereas adherent osteoblastic cells would be viable, since these cells adhere to the surface indirectly though serum proteins. The efficacy of antimicrobial titanium appears to be based on peroxidation, since peroxidation products were detected in parallel with the destruction of bacterial cell-surface structures. The peroxidation effect of antimicrobial titanium was confined to the surface within narrow limits. The viability of osteoblastic cells on the surface was strongly dependent on the presence of serum protein, whereas that of adherent Streptococcus mutans was not affected by the presence of serum proteins. Therefore, differences in the adherent systems used by bacteria and osteoblastic cells are important determinants of their viability on antimicrobial titanium.

  17. Exploring Cellular Interactions of Liposomes Using Protein Corona Fingerprints and Physicochemical Properties.

    PubMed

    Bigdeli, Arafeh; Palchetti, Sara; Pozzi, Daniela; Hormozi-Nezhad, Mohammad Reza; Baldelli Bombelli, Francesca; Caracciolo, Giulio; Mahmoudi, Morteza

    2016-03-22

    To control liposomes fate and transport upon contact with biofluids, it is essential to consider several parameters affecting the synthetic and biological identity of liposomes, as well as liposome-protein corona (PC) aspects. As a powerful tool in this data mining adventure, quantitative structure-activity relationship (QSAR) approach is used to correlate physicochemical properties of liposomes and their PC fingerprints to multiple quantified biological responses. In the present study, the relationship between cellular interactions of a set of structurally diverse liposomal formulations and their physicochemical and PC properties has been investigated via linear and nonlinear QSAR models. Significant parameters affecting cellular uptake and cell viability of liposomes in two important cancer cell lines (PC3 and HeLa) have been identified. The developed QSARs have the capacity to be implemented in advanced targeted delivery of liposomal drugs.

  18. Hydrogen Supplementation of Preservation Solution Improves Viability of Osteochondral Grafts

    PubMed Central

    Yamada, Takuya; Onuma, Kenji; Kuzuno, Jun; Ujihira, Masanobu; Kurokawa, Ryosuke; Sakai, Rina; Takaso, Masashi

    2014-01-01

    Allogenic osteochondral tissue (OCT) is used for the treatment of large cartilage defects. Typically, OCTs collected during the disease-screening period are preserved at 4°C; however, the gradual reduction in cell viability during cold preservation adversely affects transplantation outcomes. Therefore, improved storage methods that maintain the cell viability of OCTs are needed to increase the availability of high-quality OCTs and improve treatment outcomes. Here, we evaluated whether long-term hydrogen delivery to preservation solution improved the viability of rat OCTs during cold preservation. Hydrogen-supplemented Dulbecco's Modified Eagles Medium (DMEM) and University of Wisconsin (UW) solution both significantly improved the cell viability of OCTs during preservation at 4°C for 21 days compared to nonsupplemented media. However, the long-term cold preservation of OCTs in DMEM containing hydrogen was associated with the most optimal maintenance of chondrocytes with respect to viability and morphology. Our findings demonstrate that OCTs preserved in DMEM supplemented with hydrogen are a promising material for the repair of large cartilage defects in the clinical setting. PMID:25506061

  19. Predicting embryo presence and viability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pregnancy establishment, followed by birth of live offspring, is essential to all mammals. The biological processes leading up to pregnancy establishment, maintenance, and birth are complex and dependent on the coordinated timing of a series of events at the molecular, cellular, and physiological le...

  20. Non-disruptive measurement system of cell viability in bioreactors

    NASA Astrophysics Data System (ADS)

    Rudek, F.; Nelsen, B. L.; Baselt, T.; Berger, T.; Wiele, M.; Prade, I.; Hartmann, P.

    2016-04-01

    Nutrient and oxygen transport, as well as the removal of metabolic waste are essential processes to support and maintain viable tissue. Current bioreactor technology used to grow tissue cultures in vitro has a fundamental limit to the thickness of tissues. Based on the low diffusion limit of oxygen a maximum tissue thickness of 200 μm is possible. The efficiency of those systems is currently under investigation. During the cultivation process of the artificial tissue in bioreactors, which lasts 28 days or longer, there are no possibilities to investigate the viability of cells. This work is designed to determine the influence of a non-disruptive cell viability measuring system on cellular activity. The measuring system uses a natural cellular marker produced during normal metabolic activity. Nicotinamide adenine dinucleotide (NADH) is a coenzyme naturally consumed and produced during cellular metabolic processes and has thoroughly been studied to determine the metabolic state of a cell. Measuring the fluorescence of NADH within the cell represents a non-disruptive marker for cell viability. Since the measurement process is optical in nature, NADH fluorescence also provides a pathway for sampling at different measurement depths within a given tissue sample. The measurement system we are using utilizes a special UV light source, to excite the NADH fluorescence state. However, the high energy potentially alters or harms the cells. To investigate the influence of the excitation signal, the cells were irradiated with a laser operating at a wavelength of 355 nm and examined for cytotoxic effects. The aim of this study was to develop a non-cytotoxic system that is applicable for large-scale operations during drug-tissue interaction testing.

  1. Perturbations of amino acid metabolism associated with glyphosate-dependent inhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundance of proteins involved in photosynthesis and photorespiration.

    PubMed

    Vivancos, Pedro Diaz; Driscoll, Simon P; Bulman, Christopher A; Ying, Liu; Emami, Kaveh; Treumann, Achim; Mauve, Caroline; Noctor, Graham; Foyer, Christine H

    2011-09-01

    The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway.

  2. Perturbations of Amino Acid Metabolism Associated with Glyphosate-Dependent Inhibition of Shikimic Acid Metabolism Affect Cellular Redox Homeostasis and Alter the Abundance of Proteins Involved in Photosynthesis and Photorespiration1[W][OA

    PubMed Central

    Vivancos, Pedro Diaz; Driscoll, Simon P.; Bulman, Christopher A.; Ying, Liu; Emami, Kaveh; Treumann, Achim; Mauve, Caroline; Noctor, Graham; Foyer, Christine H.

    2011-01-01

    The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway. PMID:21757634

  3. Multianalyte microphysiometry reveals changes in cellular bioenergetics upon exposure to fluorescent dyes.

    PubMed

    Shinawi, Tesniem F; Kimmel, Danielle W; Cliffel, David E

    2013-12-17

    Fluorescent dyes have been designed for internal cellular component specificity and have been used extensively in the scientific community as a means to monitor cell growth, location, morphology, and viability. However, it is possible that the introduction of these dyes influences the basal function of the cell and, in turn, the results of these studies. Electrochemistry provides a noninvasive method for probing the unintended cellular affects of these dyes. The multianalyte microphysiometer (MAMP) is capable of simultaneous electrochemical measurement of extracellular metabolites in real-time. In this study, analytes central to cellular metabolism, glucose, lactate, oxygen, as well as extracellular acidification were monitored to determine the immediate metabolic effects of nuclear stains, including SYTO, DAPI dilactate, Hoechst 33342, and FITC dyes upon the pheochromocytoma PC-12 cells and RAW 264.7 macrophages. The experimental results revealed that the SYTO dye 13 significantly decreased glucose and oxygen consumption and increased extracellular acidification and lactate production in both cell lines, indicating a shift to anaerobic respiration. No other dyes caused significantly definitive changes in cellular metabolism upon exposure. This study shows that fluorescent dyes can have unintended effects on cellular metabolism and care should be taken when using these probes to investigate cellular function and morphology.

  4. Regionally Aligned Forces: Concept Viability and Implementation

    DTIC Science & Technology

    2015-03-01

    Carlisle Compendia of Collaborative Research United States Army War College Student Publications Regionally Aligned Forces: Concept Viability and...Concept Viability and Implementation 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER...Carlisle Compendia of Collaborative Research United States Army War College Student Publications Regionally Aligned Forces: Concept Viability and

  5. Economic viability of anaerobic digestion

    SciTech Connect

    Wellinger, A.

    1996-01-01

    The industrial application of anaerobic digestion is a relatively new, yet proven waste treatment technology. Anaerobic digestion reduces and upgrades organic waste, and is a good way to control air pollution as it reduces methane and nitrous gas emissions. For environmental and energy considerations, anaerobic digestion is a nearly perfect waste treatment process. However, its economic viability is still in question. A number of parameters - type of waste (solid or liquid), digester system, facility size, product quality and end use, environmental requirements, cost of alternative treatments (including labor), and interest rates - define the investment and operating costs of an anaerobic digestion facility. Therefore, identical facilities that treat the same amount and type of waste may, depending on location, legislation, and end product characteristics, reveal radically different costs. A good approach for evaluating the economics of anaerobic digestion is to compare it to treatment techniques such as aeration or conventional sewage treatment (for industrial wastewater), or composting and incineration (for solid organic waste). For example, the cost (per ton of waste) of in-vessel composting with biofilters is somewhat higher than that of anaerobic digestion, but the investment costs 1 1/2 to 2 times more than either composting or anaerobic digestion. Two distinct advantages of anaerobic digestion are: (1) it requires less land than either composting or incinerating, which translates into lower costs and milder environmental and community impacts (especially in densely populated areas); and (2) it produces net energy, which can be used to operate the facility or sold to nearby industries.

  6. Evaluation of skin viability effect on ethosome and liposome-mediated psoralen delivery via cell uptake.

    PubMed

    Zhang, Yong-Tai; Shen, Li-Na; Wu, Zhong-Hua; Zhao, Ji-Hui; Feng, Nian-Ping

    2014-10-01

    This study investigated the effect of skin viability on its permeability to psoralen delivered by ethosomes, as compared with liposomes. With decreasing skin viability, the amount of liposome-delivered psoralen that penetrated through the skin increased, whereas skin deposition of psoralen from both ethosomes and liposomes reduced. Psoralen delivery to human-immortalized epidermal cells was more effective using liposomes, whereas delivery to human embryonic skin fibroblast cells was more effective when ethosomes were used. These findings agreed with those of in vivo studies showing that skin psoralen deposition from ethosomes and liposomes first increased and then plateaued overtime, which may indicate gradual saturation of intracellular drug delivery. It also suggested that the reduced deposition of ethosome- or liposome-delivered psoralen in skin with reduced viability may relate to reduced cellular uptake. This work indicated that the effects of skin viability should be taken into account when evaluating nanocarrier-mediated drug skin permeation.

  7. Role of cytoskeleton and elastic moduli in cellular response to nanosecond pulsed electric fields

    NASA Astrophysics Data System (ADS)

    Thompson, Gary L.; Roth, Caleb; Tolstykh, Gleb; Kuipers, Marjorie; Ibey, Bennett L.

    2013-02-01

    Nanosecond pulsed electric fields (nsPEFs) are known to increase cell membrane permeability to small molecules in accordance with dosages. As previous work has focused on nsPEF exposures in whole cells, electrodeformation may contribute to this induced-permeabilization in addition to other biological mechanisms. Here, we hypothesize that cellular elasticity, based upon the cytoskeleton, affects nsPEF-induced decrease in cellular viability. Young's moduli of various types of cells have been calculated from atomic force microscopy (AFM) force curve data, showing that CHO cells are stiffer than non-adherent U937 and Jurkat cells, which are more susceptible to nsPEF exposure. To distinguish any cytoskeletal foundation for these observations, various cytoskeletal reagents were applied. Inhibiting actin polymerization significantly decreased membrane integrity, as determined by relative propidium uptake and phosphatidylserine externalization, upon exposure at 150 kV/cm with 100 pulses of 10 ns pulse width. Exposure in the presence of other drugs resulted in insignificant changes in membrane integrity and 24-hour viability. However, Jurkat cells showed greater lethality than latrunculin-treated CHO cells of comparable elasticity. From these results, it is postulated that cellular elasticity rooted in actin-membrane interaction is only a minor contributor to the differing responses of adherent and non-adherent cells to nsPEF insults.

  8. Viability and functional integrity of washed platelets

    SciTech Connect

    Pineda, A.A.; Zylstra, V.W.; Clare, D.E.; Dewanjee, M.K.; Forstrom, L.A.

    1989-07-01

    The viability and functional integrity of saline- and ACD-saline-washed platelets were compared with those of unwashed platelets. After template bleeding time (TBT) was measured, 15 healthy volunteers underwent plateletpheresis and ingested 600 mg of aspirin. Autologous /sup 111/In-labeled platelets were transfused: unwashed (n = 5), washed with 0.9 percent saline solution (SS) (n = 5), and washed with a buffered 12.6 percent solution of ACD-A in 0.9 percent saline solution (n = 5). After transfusion, we measured TBT at 1, 4, and 24 hours; platelet survival at 10 minutes and 1, 4, and 24 hours and daily for 6 days; and the percentage of uptake in liver and spleen by quantitative whole-body radionuclide scintigraphy at 24 and 190 hours. We found that saline washing affected platelet recovery, 23.47 +/- 12 percent (p less than 0.001) as compared to 52.43 +/- 17 percent (p less than 0.002) for ACD-saline and 73.17 +/- 8 percent for control; that saline washing resulted in a greater liver uptake than control and ACD-saline-washed platelets (31.9 +/- 8% (p less than 0.001) vs 17.7 +/- 4.1 and 19.3 +/- 2.1% (p greater than 0.1), respectively); that, unlike control and ACD-saline-washed platelets, saline-washed platelets did not shorten bleeding time; and that neither type of washing affected survival. Although ACD-saline washing affects recovery, it also results in intact function, normal survival, higher recovery than SS platelets, and no significant liver uptake.

  9. Experiments with the Viability of Chicken Eggs

    ERIC Educational Resources Information Center

    Garigliano, Leonard J.

    1975-01-01

    Presents the results of an experiment designed to test two hypotheses: (1) a delay of two weeks at room temperature will have no effect on the viability of fertile chicken eggs and (2) refrigeration will have no effect on the viability of fertile chicken eggs. Experimenters were the author and two ninth-grade students. (PEB)

  10. Physicochemical, morphological and cellular uptake properties of lutein nanodispersions prepared by using surfactants with different stabilizing mechanisms.

    PubMed

    Tan, Tai Boon; Chu, Wern Cui; Yussof, Nor Shariffa; Abas, Faridah; Mirhosseini, Hamed; Cheah, Yoke Kqueen; Nehdi, Imededdine Arbi; Tan, Chin Ping

    2016-04-01

    In this study, we prepared a series of lutein nanodispersions via the solvent displacement method, by using surfactants with different stabilizing mechanisms. The surfactants used include Tween 80 (steric stabilization), sodium dodecyl sulfate (SDS; electrostatic stabilization), sodium caseinate (electrosteric stabilization) and SDS-Tween 80 (electrostatic-steric stabilization). We then characterized the resulting lutein nanodispersions in terms of their particle size, particle size distribution, zeta potential, lutein content, flow behavior, apparent viscosity, transmittance, color, morphological properties and their effects on cell viability and cellular uptake. The type of surfactant used significantly (p < 0.05) affected the physical properties of the nanodispersions, but the chemical properties (lutein content) remained unaffected. Transmission electron microscopy (TEM) images obtained from this study demonstrated that the solvent displacement method was capable of producing lutein nanodispersions containing spherical particles with sizes ranging from 66.20-125.25 nm, depending on the type of surfactant used. SDS and SDS-Tween 80 surfactants negatively affected the viability of the HT-29 cells used in this study. Thus, for the cellular uptake determination, only Tween 80 and sodium caseinate surfactants were used. The cellular uptake of the lutein nanodispersion stabilized by sodium caseinate was higher than that which was stabilized by Tween 80. All things considered, the type of surfactant with different stabilizing mechanisms did produce lutein nanodispersions with different characteristics. These findings would aid in future selection of surfactants in order to produce nanodispersions with desirable properties.

  11. Recovery and viability of Dirofilaria immitis microfilariae.

    PubMed

    Grieve, R B; Mika-Grieve, M; Lok, J B; Marchell, T F; Cupp, E W

    1984-09-01

    The viability of Dirofilaria immitis microfilariae recovered from canine blood by different methods was determined. Microfilaria recovery techniques included saponin lysis, saponin lysis with a trypsin treatment, dextran sedimentation and phytohemagglutinin treatment. Criteria for evaluating viability were microfilarial motility in vitro at 37 degrees C, microfilarial development in mosquitoes and the ability of microfilariae to circulate in mice. Although each method produced motile microfilariae, differences among groups of microfilariae recovered by different techniques were apparent by each of the criteria for viability. Saponin lysis gave superior yields of viable microfilariae.

  12. Fluorescein diacetate for determination of cell viability in 3D fibroblast-collagen-GAG constructs.

    PubMed

    Powell, Heather M; Armour, Alexis D; Boyce, Steven T

    2011-01-01

    Quantification of cell viability and distribution within engineered tissues currently relies on representative histology, phenotypic assays, and destructive assays of viability. To evaluate uniformity of cell density throughout 3D collagen scaffolds prior to in vivo use, a nondestructive, field assessment of cell viability is advantageous. Here, we describe a field measure of cell viability in lyophilized collagen-glycosaminoglycan (C-GAG) scaffolds in vitro using fluorescein diacetate (FdA). Fibroblast-C-GAG constructs are stained 1 day after cellular inoculation using 0.04 mg/ml FdA followed by exposure to 366 nm UV light. Construct fluorescence quantified using Metamorph image analysis is correlated with inoculation density, MTT values, and histology of corresponding biopsies. Construct fluorescence correlates significantly with inoculation density (p  <  0.001) and MTT values (p  <  0.001) of biopsies collected immediately after FdA staining. No toxicity is detected in the constructs, as measured by MTT assay before and after the FdA assay at different time points; normal in vitro histology is demonstrated for the FdA-exposed constructs. In conclusion, measurement of intracellular fluorescence with FdA allows for the early, comprehensive measurement of cellular distributions and viability in engineered tissue.

  13. Controlling Cellular Endocytosis at the Nanoscale

    NASA Astrophysics Data System (ADS)

    Battaglia, Giuseppe

    2011-03-01

    , amphiphilic molecules, and hydrophilic molecules without affecting the viability of cells or even triggering inflammatory pathways. Finally we show how size, surface chemistry and surface topology of the vesicles affect their interaction with the cell membrane and hence their cellular uptake. References: C. Lo Presti, M. Massignani, T. Smart, H. Lomas, and G. Battaglia J. Mater. Chem. (2009) 19, 3576-3590 H. Lomas, I. Canton, S. MacNeil, J. Du, S.P. Armes, A.J. Ryan, A.L. Lewis and G. Battaglia Adv. Mater. (2007). 19, 4238-4243 M. Massignani, I. Canton, N. Patikarnmonthon, N. J. Warren, S. P. Armes, A. L. Lewis and G. Battaglia, Nature Prec., 2010, http://hdl.handle.net/10101/npre.2010.4427.1 M. Massignani, C. LoPresti, A. Blanazs, J. Madsen, S. P. Armes, A. L. Lewis and G. Battaglia Small, 2009, 5, 2424-2432. M. Massignani, T. Sun, A. Blanazs, V. Hearnden, I. Canton, P. Desphande, S. Armes, S. MacNeil, A. Lewis and G. Battaglia PLoS One, 2010, 5, e10459.

  14. Viability and DNA fragmentation in differently sorted boar spermatozoa.

    PubMed

    De Ambrogi, M; Spinaci, M; Galeati, G; Tamanini, C

    2006-11-01

    Sperm cell defense against DNA damage relies on two factors: the tight packaging of chromatin, based on condensation and substitution of histones with protamines, and the antioxidant agents present in seminal plasma. These defenses are extremely important as mature sperm is unable to repair DNA damage and even if a successful fertilization occurs, embryo undergoes apoptosis at the time of genomic activation. Sex-sorting exposes spermatozoa to stress sources such as high pressure, laser beam and electrical charge. The aim of this work was to determine how sorting procedures affect viability and DNA integrity in boar spermatozoa, by using the newly developed Sperm-Sus-Halomax. Four sperm populations were considered: CONTROL (no treatment), REAL (sex-sorted semen), BULK (semen sorted without sex separation) and NO LASER (semen only exposed to the high pressure, but including also cells normally discarded from sex-sorting). A significantly (P=0.019) lower viability in NO LASER (64.71%) than in CONTROL (78.6%) and REAL (80.5%) groups was found; this was accompanied by a significantly (P=0.001) higher DNA fragmentation index (DFI) in NO LASER group (6.86%) respect to CONTROL (3.30%) and REAL (3.42%) groups. BULK group did not show any difference in viability or DFI as compared to the other groups. In conclusion, we may believe that sex-sorting procedure as a whole does not affect either viability or DFI and that shear mechanical forces are a relevant source of DNA damage for sorted semen.

  15. A cellular uptake and cytotoxicity properties study of gallic acid-loaded mesoporous silica nanoparticles on Caco-2 cells

    NASA Astrophysics Data System (ADS)

    Rashidi, Ladan; Vasheghani-Farahani, Ebrahim; Soleimani, Masoud; Atashi, Amir; Rostami, Khosrow; Gangi, Fariba; Fallahpour, Masoud; Tahouri, Mohammad Taher

    2014-03-01

    In this study, the effects of intracellular delivery of various concentrations of gallic acid (GA) as a semistable antioxidant, gallic acid-loaded mesoporous silica nanoparticles (MSNs-GA), and cellular uptake of nanoparticles into Caco-2 cells were investigated. MSNs were synthesized and loaded with GA, then characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, N2 adsorption isotherms, X-ray diffraction, and thermal gravimetric analysis. The cytotoxicity of MSNs and MSNs-GA at low and high concentrations were studied by means of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry. MSNs did not show significant toxicity in various concentrations (0-500 μg/ml) on Caco-2 cells. For MSNs-GA, cell viability was reduced as a function of incubation time and different concentrations of nanoparticles. The in vitro GA release from MSNs-GA exhibited the same antitumor properties as free GA on Caco-2 cells. Flow cytometry results confirmed those obtained using MTT assay. TEM and fluorescent microscopy confirmed the internalization of MSNs by Caco-2 cells through nonspecific cellular uptake. MSNs can easily internalize into Caco-2 cells without deleterious effects on cell viability. The cell viability of Caco-2 cells was affected during MSNs-GA uptake. MSNs could be designed as suitable nanocarriers for antioxidants delivery.

  16. Sperm storage and viability in Photinus fireflies.

    PubMed

    Demary, Kristian C

    2005-07-01

    In many species females mate with and store sperm from multiple males, and some female insects have evolved multiple compartments for sperm storage. Sperm storage and sperm viability were investigated in two firefly species, Photinus greeni and P. ignitus, which differ in the morphology of the female reproductive tract. Although the primary spermatheca is similar in both species, P. greeni females have an additional, conspicuous outpocketing within the bursa copulatrix whose potential role in sperm storage was investigated in this study. An assay that distinguishes between live and dead sperm was used to examine sperm viability in male seminal vesicles and sperm storage sites within the female reproductive tract. For both Photinus species, sperm from male seminal vesicles showed significantly higher viability compared to sperm from the primary spermatheca of single mated females. In single mated P. greeni females, sperm taken from the channel outpocketing (secondary spermatheca) showed significantly higher viability compared to sperm from the primary spermatheca. This sperm viability difference was not evident in double mated females. There were no significant differences between P. greeni and P. ignitus females in the viability of sperm from the primary spermatheca. These studies contribute to our understanding of post-mating processes that may influence paternity success, and suggest that sexual conflict over control of fertilizations may occur in multiply mated firefly females.

  17. [Influence of microcystin-LR on cell viability and surface characteristics of Pseudomonas putida].

    PubMed

    Deng, Ting-jin; Ye, Jin-shao; Peng, Hui; Liu, Zhi-chen; Liu, Ze-hua; Yin, Hua; Chen, Shuo-na

    2015-01-01

    In microcystin-LR (MC-LR) degradation system, the change in surface characteristics and cell viability of Pseudomonas putida was studied. The purpose of this study was to reveal the influence of MC-LR on P. putida and elucidate the toxicity of MC-LR on microorganisms. The result demonstrated that MC-LR enhanced the cytoplasmic membrane permeability, as well as affected the ion metabolism and protein release of P. putida. The soluble sugar and Na+, Cl-release increased with the rising concentration of MC-LR ranging from 0 mg x L(-1) to 2.0 mg x L(-1). Flow Cytometry Method(FCM) analysis revealed that MC-LR accelerated the death of P. putida, and the death rate increased with the ascending concentration of MC-LR. Compared with the control, the death rate on day 5 increased by nearly 30% when 2.5 mg x L(-1) MC-LR was added. Scanning electron microscopy (SEM) analysis showed that the cells were deformed under the toxicity of MC-LR. After 5-day exposure to 2.5 mg x L(-1) MC-LR, the majority of the cells were ruptured and the intracellular materials flew out. The cellular structure was severely damaged under this condition.

  18. Quantification of cell viability and rapid screening anti-cancer drug utilizing nanomechanical fluctuation.

    PubMed

    Wu, Shangquan; Liu, Xiaoli; Zhou, Xiarong; Liang, Xin M; Gao, Dayong; Liu, Hong; Zhao, Gang; Zhang, Qingchuan; Wu, Xiaoping

    2016-03-15

    Cancer is a serious threat to human health. Although numerous anti-cancer drugs are available clinically, many have shown toxic side effects due to poor tumor-selectivity, and reduced effectiveness due to cancers rapid development of resistance to treatment. The development of new highly efficient and practical methods to quantify cell viability and its change under drug treatment is thus of significant importance in both understanding of anti-cancer mechanism and anti-cancer drug screening. Here, we present an approach of utilizing a nanomechanical fluctuation based highly sensitive microcantilever sensor, which is capable of characterizing the viability of cells and quantitatively screening (within tens of minutes) their responses to a drug with the obvious advantages of a rapid, label-free, quantitative, noninvasive, real-time and in-situ assay. The microcantilever sensor operated in fluctuation mode was used in evaluating the paclitaxel effectiveness on breast cancer cell line MCF-7. This study demonstrated that the nanomechanical fluctuations of the microcantilever sensor are sensitive enough to detect the dynamic variation in cellular force which is provided by the cytoskeleton, using cell metabolism as its energy source, and the dynamic instability of microtubules plays an important role in the generation of the force. We propose that cell viability consists of two parts: biological viability and mechanical viability. Our experimental results suggest that paclitaxel has little effect on biological viability, but has a significant effect on mechanical viability. This new method provides a new concept and strategy for the evaluation of cell viability and the screening of anti-cancer drugs.

  19. Fungal Spores Viability on the International Space Station

    NASA Astrophysics Data System (ADS)

    Gomoiu, I.; Chatzitheodoridis, E.; Vadrucci, S.; Walther, I.; Cojoc, R.

    2016-11-01

    long term experiment lost the outer layer of their coat without affecting the viability since they were still protected by the middle and the inner layer of the coating. This research highlights a new protocol to perform spaceflight experiments inside the ISS with fungal spores in microgravity conditions, under the additional effect of possible cosmic radiation. According to this protocol the results are expressed in terms of viability, microscopic and morphological changes.

  20. Fungal Spores Viability on the International Space Station.

    PubMed

    Gomoiu, I; Chatzitheodoridis, E; Vadrucci, S; Walther, I; Cojoc, R

    2016-11-01

    long term experiment lost the outer layer of their coat without affecting the viability since they were still protected by the middle and the inner layer of the coating. This research highlights a new protocol to perform spaceflight experiments inside the ISS with fungal spores in microgravity conditions, under the additional effect of possible cosmic radiation. According to this protocol the results are expressed in terms of viability, microscopic and morphological changes.

  1. Metronidazole decreases viability of DLD-1 colorectal cancer cell line.

    PubMed

    Sadowska, Anna; Krętowski, Rafał; Szynaka, Beata; Cechowska-Pasko, Marzanna; Car, Halina

    2013-10-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test.

  2. DNA ligase I is not essential for mammalian cell viability.

    PubMed

    Han, Li; Masani, Shahnaz; Hsieh, Chih-lin; Yu, Kefei

    2014-04-24

    Of the three DNA ligases present in all vertebrates, DNA ligase I (Lig1) has been considered essential for ligating Okazaki fragments during DNA replication and thereby essential for cell viability. Here, we report the striking finding that a Lig1-null murine B cell line is viable. Surprisingly, the Lig1-null cells exhibit normal proliferation and normal immunoglobulin heavy chain class switch recombination and are not hypersensitive to a wide variety of DNA damaging agents. These findings demonstrate that Lig1 is not absolutely required for cellular DNA replication and repair and that either Lig3 or Lig4 can substitute for the role of Lig1 in joining Okazaki fragments. The establishment of a Lig1-null cell line will greatly facilitate the characterization of DNA ligase function in mammalian cells, but the finding alone profoundly reprioritizes the role of ligase I in DNA replication, repair, and recombination.

  3. Hydrostatic Compress Force Enhances the Viability and Decreases the Apoptosis of Condylar Chondrocytes through Integrin-FAK-ERK/PI3K Pathway

    PubMed Central

    Ma, Dandan; Kou, Xiaoxing; Jin, Jing; Xu, Taotao; Wu, Mengjie; Deng, Liquan; Fu, Lusi; Liu, Yi; Wu, Gang; Lu, Haiping

    2016-01-01

    Reduced mechanical stimuli in many pathological cases, such as hemimastication and limited masticatory movements, can significantly affect the metabolic activity of mandibular condylar chondrocytes and the growth of mandibles. However, the molecular mechanisms for these phenomena remain unclear. In this study, we hypothesized that integrin-focal adhesion kinase (FAK)-ERK (extracellular signal–regulated kinase)/PI3K (phosphatidylinositol-3-kinase) signaling pathway mediated the cellular response of condylar chondrocytes to mechanical loading. Primary condylar chondrocytes were exposed to hydrostatic compressive forces (HCFs) of different magnitudes (0, 50, 100, 150, 200, and 250 kPa) for 2 h. We measured the viability, morphology, and apoptosis of the chondrocytes with different treatments as well as the gene, protein expression, and phosphorylation of mechanosensitivity-related molecules, such as integrin α2, integrin α5, integrin β1, FAK, ERK, and PI3K. HCFs could significantly increase the viability and surface area of condylar chondrocytes and decrease their apoptosis in a dose-dependent manner. HCF of 250 kPa resulted in a 1.51 ± 0.02-fold increase of cell viability and reduced the ratio of apoptotic cells from 18.10% ± 0.56% to 7.30% ± 1.43%. HCFs could significantly enhance the mRNA and protein expression of integrin α2, integrin α5, and integrin β1 in a dose-dependent manner, but not ERK1, ERK2, or PI3K. Instead, HCF could significantly increase phosphorylation levels of FAK, ERK1/2, and PI3K in a dose-dependent manner. Cilengitide, the potent integrin inhibitor, could dose-dependently block such effects of HCFs. HCFs enhances the viability and decreases the apoptosis of condylar chondrocytes through the integrin-FAK-ERK/PI3K pathway. PMID:27827993

  4. A New Methodology for Evaluation of Nematode Viability

    PubMed Central

    Ferreira, Sebastião Rodrigo; Mendes, Tiago Antônio Oliveira; Bueno, Lilian Lacerda; de Araújo, Jackson Victor; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio

    2015-01-01

    Nematodes infections are responsible for debilitating conditions and economic losses in domestic animals as well as livestock and are considered an important public health problem due to the high prevalence in humans. The nematode resistance for drugs has been reported for livestock, highlighting the importance for development of new anthelmintic compounds. The aim of the current study was to apply and compare fluorimetric techniques using Sytox and propidium iodide for evaluating the viability of C. elegans larvae after treatment with anthelmintic drugs. These fluorescent markers were efficient to stain larvae treated with ivermectin and albendazole sulfoxide. We observed that densitometric values were proportional to the concentration of dead larvae stained with both markers. Furthermore, data on motility test presented an inverse correlation with fluorimetric data when ivermectin was used. Our results showed that lower concentrations of drugs were effective to interfere in the processes of cellular transport while higher drugs concentrations were necessary in order to result in any damage to cell integrity. The methodology described in this work might be useful for studies that aim to evaluate the viability of nematodes, particularly for testing of new anthelminthic compounds using an easy, economic, reproducible, and no time-consuming technique. PMID:25866820

  5. Effects of Fluid Shear Stress on Cancer Stem Cell Viability

    NASA Astrophysics Data System (ADS)

    Sunday, Brittney; Triantafillu, Ursula; Domier, Ria; Kim, Yonghyun

    2014-11-01

    Cancer stem cells (CSCs), which are believed to be the source of tumor formation, are exposed to fluid shear stress as a result of blood flow within the blood vessels. It was theorized that CSCs would be less susceptible to cell death than non-CSCs after both types of cell were exposed to a fluid shear stress, and that higher levels of fluid shear stress would result in lower levels of cell viability for both cell types. To test this hypothesis, U87 glioblastoma cells were cultured adherently (containing smaller populations of CSCs) and spherically (containing larger populations of CSCs). They were exposed to fluid shear stress in a simulated blood flow through a 125-micrometer diameter polyetheretherketone (PEEK) tubing using a syringe pump. After exposure, cell viability data was collected using a BioRad TC20 Automated Cell Counter. Each cell type was tested at three physiological shear stress values: 5, 20, and 60 dynes per centimeter squared. In general, it was found that the CSC-enriched U87 sphere cells had higher cell viability than the CSC-depleted U87 adherent cancer cells. Interestingly, it was also observed that the cell viability was not negatively affected by the higher fluid shear stress values in the tested range. In future follow-up studies, higher shear stresses will be tested. Furthermore, CSCs from different tumor origins (e.g. breast tumor, prostate tumor) will be tested to determine cell-specific shear sensitivity. National Science Foundation Grant #1358991 supported the first author as an REU student.

  6. Effect of silica nanoparticles with variable size and surface functionalization on human endothelial cell viability and angiogenic activity

    NASA Astrophysics Data System (ADS)

    Guarnieri, Daniela; Malvindi, Maria Ada; Belli, Valentina; Pompa, Pier Paolo; Netti, Paolo

    2014-02-01

    Silica nanoparticles could be promising delivery vehicles for drug targeting or gene therapy. However, few studies have been undertaken to determine the biological behavior effects of silica nanoparticles on primary endothelial cells. Here we investigated uptake, cytotoxicity and angiogenic properties of silica nanoparticle with positive and negative surface charge and sizes ranging from 25 to 115 nm in primary human umbilical vein endothelial cells. Dynamic light scattering measurements and nanoparticle tracking analysis were used to estimate the dispersion status of nanoparticles in cell culture media, which was a key aspect to understand the results of the in vitro cellular uptake experiments. Nanoparticles were taken up by primary endothelial cells in a size-dependent manner according to their degree of agglomeration occurring after transfer in cell culture media. Functionalization of the particle surface with positively charged groups enhanced the in vitro cellular uptake, compared to negatively charged nanoparticles. However, this effect was contrasted by the tendency of particles to form agglomerates, leading to lower internalization efficiency. Silica nanoparticle uptake did not affect cell viability and cell membrane integrity. More interestingly, positively and negatively charged 25 nm nanoparticles did not influence capillary-like tube formation and angiogenic sprouting, compared to controls. Considering the increasing interest in nanomaterials for several biomedical applications, a careful study of nanoparticle-endothelial cells interactions is of high relevance to assess possible risks associated to silica nanoparticle exposure and their possible applications in nanomedicine as safe and effective nanocarriers for vascular transport of therapeutic agents.

  7. Viability and Resilience of Languages in Competition

    PubMed Central

    Chapel, Laetitia; Castelló, Xavier; Bernard, Claire; Deffuant, Guillaume; Eguíluz, Víctor M.; Martin, Sophie; Miguel, Maxi San

    2010-01-01

    We study the viability and resilience of languages, using a simple dynamical model of two languages in competition. Assuming that public action can modify the prestige of a language in order to avoid language extinction, we analyze two cases: (i) the prestige can only take two values, (ii) it can take any value but its change at each time step is bounded. In both cases, we determine the viability kernel, that is, the set of states for which there exists an action policy maintaining the coexistence of the two languages, and we define such policies. We also study the resilience of the languages and identify configurations from where the system can return to the viability kernel (finite resilience), or where one of the languages is lead to disappear (zero resilience). Within our current framework, the maintenance of a bilingual society is shown to be possible by introducing the prestige of a language as a control variable. PMID:20126655

  8. Poxvirus viability and signatures in historical relics.

    PubMed

    McCollum, Andrea M; Li, Yu; Wilkins, Kimberly; Karem, Kevin L; Davidson, Whitni B; Paddock, Christopher D; Reynolds, Mary G; Damon, Inger K

    2014-02-01

    Although it has been >30 years since the eradication of smallpox, the unearthing of well-preserved tissue material in which the virus may reside has called into question the viability of variola virus decades or centuries after its original occurrence. Experimental data to address the long-term stability and viability of the virus are limited. There are several instances of well-preserved corpses and tissues that have been examined for poxvirus viability and viral DNA. These historical specimens cause concern for potential exposures, and each situation should be approached cautiously and independently with the available information. Nevertheless, these specimens provide information on the history of a major disease and vaccination against it.

  9. Enhanced cellular uptake of aminosilane-coated superparamagnetic iron oxide nanoparticles in mammalian cell lines

    PubMed Central

    Zhu, Xiao-Ming; Wang, Yi-Xiang J; Leung, Ken Cham-Fai; Lee, Siu-Fung; Zhao, Feng; Wang, Da-Wei; Lai, Josie MY; Wan, Chao; Cheng, Christopher HK; Ahuja, Anil T

    2012-01-01

    Purpose To compare the cellular uptake efficiency and cytotoxicity of aminosilane (SiO2-NH2)-coated superparamagnetic iron oxide (SPIO@SiO2-NH2) nanoparticles with three other types of SPIO nanoparticles coated with SiO2 (SPIO@SiO2), dextran (SPIO@dextran), or bare SPIO in mammalian cell lines. Materials and methods Four types of monodispersed SPIO nanoparticles with a SPIO core size of 7 nm and an overall size in a range of 7–15 nm were synthesized. The mammalian cell lines of MCF-7, MDA-MB-231, HT-29, RAW264.7, L929, HepG2, PC-3, U-87 MG, and mouse mesenchymal stem cells (MSCs) were incubated with four types of SPIO nanoparticles for 24 hours in the serum-free culture medium Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 μg/mL iron concentration. The cellular uptake efficiencies of SPIO nanoparticles were compared by Prussian blue staining and intracellular iron quantification. In vitro magnetic resonance imaging of MSC pellets after SPIO labeling was performed at 3 T. The effect of each SPIO nanoparticle on the cell viability of RAW 264.7 (mouse monocyte/macrophage) cells was also evaluated. Results Transmission electron microscopy demonstrated surface coating with SiO2-NH2, SiO2, and dextran prevented SPIO nanoparticle aggregation in DMEM culture medium. MCF-7, MDA-MB-231, and HT-29 cells failed to show notable iron uptake. For all the remaining six cell lines, Prussian blue staining and intracellular iron quantification demonstrated that SPIO@ SiO2-NH2 nanoparticles had the highest cellular uptake efficiency. SPIO@SiO2-NH2, bare SPIO, and SPIO@dextran nanoparticles did not affect RAW 264.7 cell viability up to 200 μg Fe/mL, while SPIO@SiO2 reduced RAW 264.7 cell viability from 10 to 200 μg Fe/mL in a dose-dependent manner. Conclusion Cellular uptake efficiency of SPIO nanoparticles depends on both the cell type and SPIO surface characteristics. Aminosilane surface coating enhanced the cellular uptake efficiency without inducing cytotoxicity in a

  10. Viability assessment of primo-node slices from organ surface primo-vascular tissues in rats.

    PubMed

    Han, Tae Hee; Lim, Chae Jeong; Choi, Jae-Hong; Lee, So Yeong; Ryu, Pan Dong

    2010-12-01

    The primo-vascular system is a novel thread-like structure which is recently rediscovered, but its cellular properties are largely unknown. In this study, a slice preparation for primo-nodes was developed to facilitate study of the cellular properties of primo-node cells in vitro. Slices (4-8 slices; 200 μm thick) were sectioned from single primo-nodes collected from the abdominal organ surface of rats and incubated in oxygenated Krebs solution at 25°C or 31°C for up to 7 hours. Trypan blue staining and whole-cell patch-clamp recordings were performed to estimate the viability of cells in the slices. Viability was largely maintained during the first 3 hours, but subsequently decreased (from 80% to 21%, p < 0.001). In addition, the viability of slices incubated at 31°C was higher than those incubated at 25°C (80%vs. 47%, p < 0.001). In whole-cell patch-clamp experiments, high resistance seals readily formed and primo-node cells showed a mean resting membrane potential (-38 mV) comparable to that recorded with sharp electrodes and outwardly-rectifying current-voltage relationships. The results show that the primo-node slices developed in this study maintained viability for up to 4 hours in vitro.

  11. Predicting cell viability within tissue scaffolds under equiaxial strain: multi-scale finite element model of collagen-cardiomyocytes constructs.

    PubMed

    Elsaadany, Mostafa; Yan, Karen Chang; Yildirim-Ayan, Eda

    2017-01-16

    Successful tissue engineering and regenerative therapy necessitate having extensive knowledge about mechanical milieu in engineered tissues and the resident cells. In this study, we have merged two powerful analysis tools, namely finite element analysis and stochastic analysis, to understand the mechanical strain within the tissue scaffold and residing cells and to predict the cell viability upon applying mechanical strains. A continuum-based multi-length scale finite element model (FEM) was created to simulate the physiologically relevant equiaxial strain exposure on cell-embedded tissue scaffold and to calculate strain transferred to the tissue scaffold (macro-scale) and residing cells (micro-scale) upon various equiaxial strains. The data from FEM were used to predict cell viability under various equiaxial strain magnitudes using stochastic damage criterion analysis. The model validation was conducted through mechanically straining the cardiomyocyte-encapsulated collagen constructs using a custom-built mechanical loading platform (EQUicycler). FEM quantified the strain gradients over the radial and longitudinal direction of the scaffolds and the cells residing in different areas of interest. With the use of the experimental viability data, stochastic damage criterion, and the average cellular strains obtained from multi-length scale models, cellular viability was predicted and successfully validated. This methodology can provide a great tool to characterize the mechanical stimulation of bioreactors used in tissue engineering applications in providing quantification of mechanical strain and predicting cellular viability variations due to applied mechanical strain.

  12. Influence of electric current on bacterial viability in wastewater treatment.

    PubMed

    Wei, V; Elektorowicz, M; Oleszkiewicz, J A

    2011-10-15

    Minimizing the influence of electric current on bacterial viability in the electro-technologies such as electrophoresis and electrocoagulation is crucial in designing and operating the electric hybrid wastewater treatment system. In this study the biomass from a membrane bioreactor (MBR) was subjected to constant direct current and the bacterial viability was monitored against electrical intensity, duration as well as the spatial vicinity related to the electrodes. It was found that the bacterial viability was not significantly affected (less than 10% of death percentage) when the applied electric current density (CD) was less than 6.2 A/m2 after 4 h. The percentage of live cell dropped by 15% and 29% at CD of 12.3 A/m2 and 24.7 A/m2, respectively. The pH of electrolytic biomass fluid has shifted to alkaline (from nearly neutral to around pH 10) at CD above 12.3 A/m2, which could have been the contributing factor for the bacterial inactivation. The temperature change in the electrolytic media at all current densities during 4 h of experiment was less than 2 °C, thus temperature effects were negligible. Bacteria experienced different micro-environments in the electrochemical reactor. Bacterial cells on the cathode surface exhibited highest death rate, whereas bacteria outside the space between electrodes were the least affected. It was concluded that in an electro-technology integrated wastewater treatment process, sufficient mixing should be used to avoid localized inactivation of bacterial cells.

  13. Viability of nonminimally coupled f (R) gravity

    NASA Astrophysics Data System (ADS)

    Bertolami, Orfeu; Páramos, Jorge

    2016-03-01

    In this work we explore the viability of nonminimally coupled matter-curvature gravity theories, namely the conditions required for the absence of tachyon instabilities and ghost degrees of freedom. We contrast our finds with recent claims of a pathological behaviour of this class of models, which resorted to, in our view, an incorrect analogy with k-essence.

  14. Observability in strategic models of viability selection.

    PubMed

    Gámez, M; Carreño, R; Kósa, A; Varga, Z

    2003-10-01

    Strategic models of frequency-dependent viability selection, in terms of mathematical systems theory, are considered as a dynamic observation system. Using a general sufficient condition for observability of nonlinear systems with invariant manifold, it is studied whether, observing certain phenotypic characteristics of the population, the development of its genetic state can be recovered, at least near equilibrium.

  15. Viability of mesenchymal stem cells during electrospinning

    PubMed Central

    Zanatta, G.; Steffens, D.; Braghirolli, D.I.; Fernandes, R.A.; Netto, C.A.; Pranke, P.

    2011-01-01

    Tissue engineering is a technique by which a live tissue can be re-constructed and one of its main goals is to associate cells with biomaterials. Electrospinning is a technique that facilitates the production of nanofibers and is commonly used to develop fibrous scaffolds to be used in tissue engineering. In the present study, a different approach for cell incorporation into fibrous scaffolds was tested. Mesenchymal stem cells were extracted from the wall of the umbilical cord and mononuclear cells from umbilical cord blood. Cells were re-suspended in a 10% polyvinyl alcohol solution and subjected to electrospinning for 30 min under a voltage of 21 kV. Cell viability was assessed before and after the procedure by exclusion of dead cells using trypan blue staining. Fiber diameter was observed by scanning electron microscopy and the presence of cells within the scaffolds was analyzed by confocal laser scanning microscopy. After electrospinning, the viability of mesenchymal stem cells was reduced from 88 to 19.6% and the viability of mononuclear cells from 99 to 8.38%. The loss of viability was possibly due to the high viscosity of the polymer solution, which reduced the access to nutrients associated with electric and mechanical stress during electrospinning. These results suggest that the incorporation of cells during fiber formation by electrospinning is a viable process that needs more investigation in order to find ways to protect cells from damage. PMID:22183245

  16. Fetal pain, abortion, viability, and the Constitution.

    PubMed

    Cohen, I Glenn; Sayeed, Sadath

    2011-01-01

    In early 2010, the Nebraska state legislature passed a new abortion restricting law asserting a new, compelling state interest in preventing fetal pain. In this article, we review existing constitutional abortion doctrine and note difficulties presented by persistent legal attention to a socially derived viability construct. We then offer a substantive biological, ethical, and legal critique of the new fetal pain rationale.

  17. Cellular interactions of surface modified nanoporous silicon particles

    NASA Astrophysics Data System (ADS)

    Bimbo, Luis M.; Sarparanta, Mirkka; Mäkilä, Ermei; Laaksonen, Timo; Laaksonen, Päivi; Salonen, Jarno; Linder, Markus B.; Hirvonen, Jouni; Airaksinen, Anu J.; Santos, Hélder A.

    2012-05-01

    In this study, the self-assembly of hydrophobin class II (HFBII) on the surface of thermally hydrocarbonized porous silicon (THCPSi) nanoparticles was investigated. The HFBII-coating converted the hydrophobic particles into more hydrophilic ones, improved the particles' cell viability in both HT-29 and Caco-2 cell lines compared to uncoated particles, and enhanced the particles' cellular association. The amount of HFBII adsorbed onto the particles was also successfully quantified by both the BCA assay and a HPLC method. Importantly, the permeation of a poorly water-soluble drug, indomethacin, loaded into THCPSi particles across Caco-2 monolayers was not affected by the protein coating. In addition, 125I-radiolabelled HFBII did not extensively permeate the Caco-2 monolayer and was found to be stably adsorbed onto the THCPSi nanoparticles incubated in pH 7.4, which renders the particles the possibility for further track-imaging applications. The results highlight the potential of HFBII coating for improving wettability, increasing biocompatibility and possible intestinal association of PSi nanoparticulates for drug delivery applications.In this study, the self-assembly of hydrophobin class II (HFBII) on the surface of thermally hydrocarbonized porous silicon (THCPSi) nanoparticles was investigated. The HFBII-coating converted the hydrophobic particles into more hydrophilic ones, improved the particles' cell viability in both HT-29 and Caco-2 cell lines compared to uncoated particles, and enhanced the particles' cellular association. The amount of HFBII adsorbed onto the particles was also successfully quantified by both the BCA assay and a HPLC method. Importantly, the permeation of a poorly water-soluble drug, indomethacin, loaded into THCPSi particles across Caco-2 monolayers was not affected by the protein coating. In addition, 125I-radiolabelled HFBII did not extensively permeate the Caco-2 monolayer and was found to be stably adsorbed onto the THCPSi

  18. Linking Conservation Actions with Population Viability Models: Reducing Uncertainty to Better Predict Management Effects on Viability

    DTIC Science & Technology

    2011-05-01

    editors. Population viability analysis. University of Chicago Press, Chicago , Illinois, USA. Goodman, L. A. 1971. On the sensitivity of the...stochastic simulation of the extinction process. Version 9.71. Chicago Zoological Society, Brookfield, Illinois. Ladd, C., and L. Gass. 1999...Pages 511-520 in S.R. Beissinger and D.R. McCullough, editors. Population viability analysis. University of Chicago Press, Chicago , Illinois, USA

  19. Hierarchical cellular materials

    SciTech Connect

    Gibson, L.J.

    1991-12-31

    In this paper a method for estimating the contributions of both the composite and the cellular microstructures to the overall material properties and the mechanical efficiency of natural cellular solids will be described. The method will be demonstrated by focusing on the Young`s modulus; similar techniques can be used for other material properties. The results suggest efficient microstructures for engineered cellular materials.

  20. Hierarchical cellular materials

    SciTech Connect

    Gibson, L.J.

    1991-01-01

    In this paper a method for estimating the contributions of both the composite and the cellular microstructures to the overall material properties and the mechanical efficiency of natural cellular solids will be described. The method will be demonstrated by focusing on the Young's modulus; similar techniques can be used for other material properties. The results suggest efficient microstructures for engineered cellular materials.

  1. Air quality biomonitoring through pollen viability of Fabaceae.

    PubMed

    Duro, Anna; Piccione, Vincenzo; Zampino, Daniela

    2013-05-01

    In this study, pollen viability and germination of three plant species, Cercis siliquastrum L., Robinia pseudoacacia L., and Spartium junceum L., belonging to the Fabaceae family, was evaluated in sites with different intensity of road traffic, constantly monitored with continuous analysers for air pollutants (carbon monoxide (CO), sulphur dioxide (SO(2)), and nitrogen dioxide (NO(2))) by the Municipality of Catania. Two sites, in which road traffic was absent, were selected, too. The percentages of viable pollen by 2,3,5-trypheniltetrazolium chloride (TTC) test ranged from 59.0 to 90.2 % in C. siliquastrum, from 61.5 to 83.5 % in S. junceum and from 67.5 to 84.3 % in R. pseudoacacia. The percentages of germination varied from 41.0 to 72.7 % in C. siliquastrum, from 42.0 to 64.7 % in S. junceum and from 38.3 to 66.3 % in R. pseudoacacia. The highest percentages of viable pollens were found in no-road traffic stations by either TTC or germination tests, while the lowest values were detected in a site characterised by heavy road traffic. In the monitored period (2007-2009), pollen viability, germinability and tube length of C. siliquastrum resulted in a significant negative correlation to CO, SO(2) and NO(2), whereas data from TTC and germination tests on S. junceum and R. pseudoacacia pollens were not well correlated to air pollutants. The results showed that pollen viability, germination and tube growth in C. siliquastrum were affected by air pollution. S. junceum and R. pseudoacacia were not very influenced by air pollutants, suggesting a different pollen sensitivity of these species.

  2. The cybernetics of viability: an overview

    NASA Astrophysics Data System (ADS)

    Nechansky, Helmut

    2011-10-01

    A three-level approach to viability is developed, considering (1) living systems, (2) a niche, understood as the area within the reach of their actions, and (3) an environment. A systematic analysis of the interrelations between these levels shows that living systems emerge with matter/energy processing systems. These can add controller structures when producing excess energy. A three-sensor controller structure enables a living system to deal with unfavourable and scarce environments. Further evolution of these controller structures offers improved ways to act on niches. Maintaining niches in scarce environments can require technology or economy. So social systems emerge, which are understood as aggregates of living systems. Basic patterns of interactions within social systems are analysed. So the introduction of the notion of the niche into the discussion of viability allows us to explain phenomena ranging from properties of single living systems to societal organization.

  3. Hospital Viability during a Pandemic Influenza Outbreak

    DTIC Science & Technology

    2009-06-01

    provide protection from the next pandemic. 15. SUBJECT TERMS Hospital Security, Pandemic Influenza , Viability Checklist, 1918 Spanish Flu, 2003 SARS...pandemic influenza : 1918 Spanish Flu, 1957 Asian Flu, 1968 Hong Kong Flu, 2003 SARS epidemic, and 2005 Hurricane Katrina. Understanding the...emergency management events: 1918 , Spanish flu (H1N1); 2003, SARS outbreak; 2005, Hurricane Katrina, and the 2009, Swine flu (H1N1) outbreak, for the

  4. Membrane transporters and protein traffic networks differentially affecting metal tolerance: a genomic phenotyping study in yeast

    PubMed Central

    Ruotolo, Roberta; Marchini, Gessica; Ottonello, Simone

    2008-01-01

    Background The cellular mechanisms that underlie metal toxicity and detoxification are rather variegated and incompletely understood. Genomic phenotyping was used to assess the roles played by all nonessential Saccharomyces cerevisiae proteins in modulating cell viability after exposure to cadmium, nickel, and other metals. Results A number of novel genes and pathways that affect multimetal as well as metal-specific tolerance were discovered. Although the vacuole emerged as a major hot spot for metal detoxification, we also identified a number of pathways that play a more general, less direct role in promoting cell survival under stress conditions (for example, mRNA decay, nucleocytoplasmic transport, and iron acquisition) as well as proteins that are more proximally related to metal damage prevention or repair. Most prominent among the latter are various nutrient transporters previously not associated with metal toxicity. A strikingly differential effect was observed for a large set of deletions, the majority of which centered on the ESCRT (endosomal sorting complexes required for transport) and retromer complexes, which - by affecting transporter downregulation and intracellular protein traffic - cause cadmium sensitivity but nickel resistance. Conclusion The data show that a previously underestimated variety of pathways are involved in cadmium and nickel tolerance in eukaryotic cells. As revealed by comparison with five additional metals, there is a good correlation between the chemical properties and the cellular toxicity signatures of various metals. However, many conserved pathways centered on membrane transporters and protein traffic affect cell viability with a surprisingly high degree of metal specificity. PMID:18394190

  5. Effect of various commercial buffers on sperm viability and capacitation.

    PubMed

    Andrisani, Alessandra; Donà, Gabriella; Ambrosini, Guido; Bonanni, Guglielmo; Bragadin, Marcantonio; Cosmi, Erich; Clari, Giulio; Armanini, Decio; Bordin, Luciana

    2014-08-01

    A wide variety of sperm preparation protocols are currently available for assisted conception. They include density gradient separation and washing methods. Both aim at isolating and capacitating as much motile sperm as possible for subsequent oocyte fertilization. The aim of this study was to examine the effects of four commercial sperm washing buffers on sperm viability and capacitation. Semen samples from 48 healthy donors (normal values of sperm count, motility, morphology, and volume) were analyzed. After separation (density gradient 40/80%), sperm were incubated in various buffers then analysed for reactive oxygen species (ROS) production, viability, tyrosine phosphorylation (Tyr-P), cholera toxin B subunit (CTB) labeling, and the acrosome reaction (AR). The buffers affected ROS generation in various ways resulting either in rapid cell degeneration (when the amount of ROS was too high for cell survival) or the inability of the cells to maintain correct functioning (when ROS were too few). Only when the correct ROS generation curve was maintained, suitable membrane reorganization, evidenced by CTB labeling was achieved, leading to the highest percentages of both Tyr-P- and acrosome-reacted-cells. Distinguishing each particular pathological state of the sperm sample would be helpful to select the preferred buffer treatment since both ROS production and membrane reorganization can be significantly altered by commercial buffers.

  6. Cell Viability and Functionality of Probiotic Bacteria in Dairy Products

    PubMed Central

    Vinderola, Gabriel; Binetti, Ana; Burns, Patricia; Reinheimer, Jorge

    2011-01-01

    Probiotic bacteria, according to the definition adopted by the World Health Organization in 2002, are live microorganisms, which when administered in adequate amounts confer a health benefit to the host. Recent studies show that the same probiotic strain produced and/or preserved under different storage conditions, may present different responses regarding their susceptibility to the adverse conditions of the gastrointestinal tract, its capacity to adhere to the intestinal epithelium, or its immunomodulating capacity, the functionality being affected without changes in cell viability. This could imply that the control of cell viability is not always enough to guarantee the functionality (probiotic capacity) of a strain. Therefore, a new challenge arises for food technologists and microbiologists when it comes to designing and monitoring probiotic food: to be able to monitor the functionality of a probiotic microorganism throughout all the stages the strain goes through from the moment it is produced and included in the food vehicle, until the moment of consumption. Conventional methodological tools or others still to be developed must be used. The application of cell membrane functionality markers, the use of tests of resistance to intestinal barriers, the study of surface properties and the application of in vivo models come together as complementary tools to assess the actual capacity of a probiotic organism in a specific food, to exert functional effects regardless of the number of viable cells present at the moment of consumption. PMID:21833320

  7. Cell viability and functionality of probiotic bacteria in dairy products.

    PubMed

    Vinderola, Gabriel; Binetti, Ana; Burns, Patricia; Reinheimer, Jorge

    2011-01-01

    Probiotic bacteria, according to the definition adopted by the World Health Organization in 2002, are live microorganisms, which when administered in adequate amounts confer a health benefit to the host. Recent studies show that the same probiotic strain produced and/or preserved under different storage conditions, may present different responses regarding their susceptibility to the adverse conditions of the gastrointestinal tract, its capacity to adhere to the intestinal epithelium, or its immunomodulating capacity, the functionality being affected without changes in cell viability. This could imply that the control of cell viability is not always enough to guarantee the functionality (probiotic capacity) of a strain. Therefore, a new challenge arises for food technologists and microbiologists when it comes to designing and monitoring probiotic food: to be able to monitor the functionality of a probiotic microorganism throughout all the stages the strain goes through from the moment it is produced and included in the food vehicle, until the moment of consumption. Conventional methodological tools or others still to be developed must be used. The application of cell membrane functionality markers, the use of tests of resistance to intestinal barriers, the study of surface properties and the application of in vivo models come together as complementary tools to assess the actual capacity of a probiotic organism in a specific food, to exert functional effects regardless of the number of viable cells present at the moment of consumption.

  8. The cellular magnetic response and biocompatibility of biogenic zinc- and cobalt-doped magnetite nanoparticles

    PubMed Central

    Moise, Sandhya; Céspedes, Eva; Soukup, Dalibor; Byrne, James M.; El Haj, Alicia J.; Telling, Neil D.

    2017-01-01

    The magnetic moment and anisotropy of magnetite nanoparticles can be optimised by doping with transition metal cations, enabling their properties to be tuned for different biomedical applications. In this study, we assessed the suitability of bacterially synthesized zinc- and cobalt-doped magnetite nanoparticles for biomedical applications. To do this we measured cellular viability and activity in primary human bone marrow-derived mesenchymal stem cells and human osteosarcoma-derived cells. Using AC susceptibility we studied doping induced changes in the magnetic response of the nanoparticles both as stable aqueous suspensions and when associated with cells. Our findings show that the magnetic response of the particles was altered after cellular interaction with a reduction in their mobility. In particular, the strongest AC susceptibility signal measured in vitro was from cells containing high-moment zinc-doped particles, whilst no signal was observed in cells containing the high-anisotropy cobalt-doped particles. For both particle types we found that the moderate dopant levels required for optimum magnetic properties did not alter their cytotoxicity or affect osteogenic differentiation of the stem cells. Thus, despite the known cytotoxicity of cobalt and zinc ions, these results suggest that iron oxide nanoparticles can be doped to sufficiently tailor their magnetic properties without compromising cellular biocompatibility. PMID:28045082

  9. The cellular magnetic response and biocompatibility of biogenic zinc- and cobalt-doped magnetite nanoparticles

    NASA Astrophysics Data System (ADS)

    Moise, Sandhya; Céspedes, Eva; Soukup, Dalibor; Byrne, James M.; El Haj, Alicia J.; Telling, Neil D.

    2017-01-01

    The magnetic moment and anisotropy of magnetite nanoparticles can be optimised by doping with transition metal cations, enabling their properties to be tuned for different biomedical applications. In this study, we assessed the suitability of bacterially synthesized zinc- and cobalt-doped magnetite nanoparticles for biomedical applications. To do this we measured cellular viability and activity in primary human bone marrow-derived mesenchymal stem cells and human osteosarcoma-derived cells. Using AC susceptibility we studied doping induced changes in the magnetic response of the nanoparticles both as stable aqueous suspensions and when associated with cells. Our findings show that the magnetic response of the particles was altered after cellular interaction with a reduction in their mobility. In particular, the strongest AC susceptibility signal measured in vitro was from cells containing high-moment zinc-doped particles, whilst no signal was observed in cells containing the high-anisotropy cobalt-doped particles. For both particle types we found that the moderate dopant levels required for optimum magnetic properties did not alter their cytotoxicity or affect osteogenic differentiation of the stem cells. Thus, despite the known cytotoxicity of cobalt and zinc ions, these results suggest that iron oxide nanoparticles can be doped to sufficiently tailor their magnetic properties without compromising cellular biocompatibility.

  10. Relevant aspects in the surface properties in titanium dental implants for the cellular viability.

    PubMed

    Velasco-Ortega, E; Alfonso-Rodríguez, C A; Monsalve-Guil, L; España-López, A; Jiménez-Guerra, A; Garzón, I; Alaminos, M; Gil, F J

    2016-07-01

    Roughness and topographical features are the most relevant of the surface properties for a dental implant for its osseointegration. For that reason, we studied the four surfaces more used in titanium dental implants: machined, sandblasted, acid etching and sandblasted plus acid etching. The roughness and wettability (contact angle and surface free energy) was studied by means 3D-interferometric microscope and sessile drop method. Normal human gingival fibroblasts (HGF) were obtained from small oral mucosa biopsies and were used for cell cultures. To analyze cell integrity, we first quantified the total amount of DNA and LDH released from dead cells to the culture medium. Then, LIVE/DEAD assay was used as a combined method assessing cell integrity and metabolism. All experiments were carried out on each cell type cultured on each Ti material for 24h, 48h and 72h. To evaluate the in vivo cell adhesion capability of each Ti surface, the four types of discs were grafted subcutaneously in 5 Wistar rats. Sandblasted surfaces were significantly rougher than acid etching and machined. Wettability and surface free energy decrease when the roughness increases in sand blasted samples. This fact favors the protein adsorption. The DNA released by cells cultured on the four Ti surfaces did not differ from that of positive control cells (p>0.05). The number of cells per area was significantly lower (p<0.05) in the sand-blasted surface than in the machined and surface for both cell types (7±2 cells for HGF and 10±5 cells for SAOS-2). The surface of the machined-type discs grafted in vivo had a very small area occupied by cells and/or connective tissue (3.5%), whereas 36.6% of the sandblasted plus acid etching surface, 75.9% of sandblasted discs and 59.6% of acid etching discs was covered with cells and connective tissue. Cells cultured on rougher surfaces tended to exhibit attributes of more differentiated osteoblasts than cells cultured on smoother surfaces. These surface properties justify that the sandblasted implants is able to significantly increase bone contact and bone growth with very good osseointegration results in vivo.

  11. Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

    PubMed Central

    Dennis, SG.; Trusk, T.; Richards, D.; Jia, J.; Tan, Y.; Mei, Y.; Fann, S.; Markwald, R.; Yost, M.

    2016-01-01

    Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters. PMID:26436877

  12. Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer.

    PubMed

    Dennis, Sarah Grace; Trusk, Thomas; Richards, Dylan; Jia, Jia; Tan, Yu; Mei, Ying; Fann, Stephen; Markwald, Roger; Yost, Michael

    2015-09-22

    Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

  13. Analysis of cellular senescence induced by lipopolysaccharide in pulmonary alveolar epithelial cells.

    PubMed

    Kim, Chang Oh; Huh, Ae Jung; Han, Sang Hoon; Kim, June Myung

    2012-01-01

    In this work, it was examined the possibility of lipopolysaccharide (LPS) causing cellular senescence in lung alveolar epithelial cells. Then, it was clarified how this cellular senescence phenomenon is associated with oxidative stress effect induced by LPS and whether antioxidants could inhibit reduced cellular viability by oxidant stress effect of LPS. In cell viability using cell counting kit-8, exposure to LPS decreased cellular viability and induced growth arrest in a concentration-dependent manner. The pre-apoptotic concentration of LPS was determined by caspase activation using a Caspase-Glo 3/7 luminescence assay kit. This concentration of LPS caused morphologic characteristics shown in senescent cells and elevated senescence-associated β-galactosidase activity. In addition, lysosomal content associated with senescence was increased by LPS at the pre-apoptotic concentration. However, this concentration of LPS did not shorten the telomere length. Exposure to LPS resulted in the formation of hydrogen peroxide in a concentration-dependent manner. The ability of LPS to reduce cellular viability was inhibited by the presence of glutathione. This study revealed that LPS could induce cellular senescence in lung alveloar epithelial cells, and these phenomena were closely associated with hydrogen peroxide production by LPS. Taken together, it is suggested that LPS-induced cellular senescence may play an important role in limiting the tissue repair response after sepsis.

  14. Imaging self-assembly dependent spatial distribution of small molecules in a cellular environment.

    PubMed

    Gao, Yuan; Kuang, Yi; Du, Xuewen; Zhou, Jie; Chandran, Preethi; Horkay, Ferenc; Xu, Bing

    2013-12-10

    Self-assembly of small molecules, as a more common phenomenon than one previously thought, can be either beneficial or detrimental to cells. Despite its profound biological implications, how the self-assembly of small molecules behave in a cellular environment is largely unknown and barely explored. This work studies four fluorescent molecules that consist of the same peptidic backbone (e.g., Phe-Phe-Lys) and enzyme trigger (e.g., a phosphotyrosine residue), but bear different fluorophores on the side chain of the lysine residue of the peptidic motif. These molecules, however, exhibit a different ability of self-assembly before and after enzymatic transformation (e.g., dephosphorylation). Fluorescent imaging reveals that self-assembly directly affects the distribution of these small molecules in a cellular environment. Moreover, cell viability tests suggest that the states and the locations of the molecular assemblies in the cellular environment control the phenotypes of the cells. For example, the molecular nanofibers of one of the small molecules apparently stabilize actin filaments and alleviate the insult of an F-actin toxin (e.g., latrunculin A). Combining fluorescent imaging and enzyme-instructed self-assembly of small peptidic molecules, this work demonstrates self-assembly as a key factor for dictating the spatial distribution of small molecules in a cellular environment. In addition, it illustrates a useful approach, based on enzyme-instructed self-assembly of small molecules, to modulate spatiotemporal profiles of small molecules in a cellular environment, which allows the use of the emergent properties of small molecules to control the fate of cells.

  15. Impact of release dynamics of laser-irradiated polymer micropallets on the viability of selected adherent cells

    PubMed Central

    Ma, Huan; Mismar, Wael; Wang, Yuli; Small, Donald W.; Ras, Mat; Allbritton, Nancy L.; Sims, Christopher E.; Venugopalan, Vasan

    2012-01-01

    We use time-resolved interferometry, fluorescence assays and computational fluid dynamics (CFD) simulations to examine the viability of confluent adherent cell monolayers to selection via laser microbeam release of photoresist polymer micropallets. We demonstrate the importance of laser microbeam pulse energy and focal volume position relative to the glass–pallet interface in governing the threshold energies for pallet release as well as the pallet release dynamics. Measurements using time-resolved interferometry show that increases in laser pulse energy result in increasing pallet release velocities that can approach 10 m s−1 through aqueous media. CFD simulations reveal that the pallet motion results in cellular exposure to transient hydrodynamic shear stress amplitudes that can exceed 100 kPa on microsecond timescales, and which produces reduced cell viability. Moreover, CFD simulation results show that the maximum shear stress on the pallet surface varies spatially, with the largest shear stresses occurring on the pallet periphery. Cell viability of confluent cell monolayers on the pallet surface confirms that the use of larger pulse energies results in increased rates of necrosis for those cells situated away from the pallet centre, while cells situated at the pallet centre remain viable. Nevertheless, experiments that examine the viability of these cell monolayers following pallet release show that proper choices for laser microbeam pulse energy and focal volume position lead to the routine achievement of cell viability in excess of 90 per cent. These laser microbeam parameters result in maximum pallet release velocities below 6 m s−1 and cellular exposure of transient hydrodynamic shear stresses below 20 kPa. Collectively, these results provide a mechanistic understanding that relates pallet release dynamics and associated transient shear stresses with subsequent cellular viability. This provides a quantitative, mechanistic basis for determining

  16. Evaluation of an automated instrument for viability and concentration measurements of cryopreserved hematopoietic cells.

    PubMed

    Szabo, S E; Monroe, S L; Fiorino, S; Bitzan, J; Loper, K

    2004-01-01

    Two important parameters for determination of deleterious effects of cellular processing on hematopoietic progenitor cells are cell viability and concentration. The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Hospital evaluated the Beckman Coulter Vi-Cell automated instrument for the measurement of these two parameters. Using 33 thawed hematopoietic progenitor cell samples, automated Vi-Cell viability results were compared to those obtained using the standard trypan blue manual method. In addition, cell concentrations from these samples were compared with results from the Model Z2 Coulter Counter. Chinese Hamster Ovary cells were used for the evaluation of Vi-Cell linearity at the Beckman Coulter Cellular Analysis Development Center. Significant correlation was obtained when the two methods were compared for both cell concentration and percentage viability (P < .0001). The results of the linearity study indicated that the Vi-Cell is linear from approximately 5 x 10(4) to greater than 1 x 10(7) cells/mL. The Vi-Cell uses sample volumes as low as 0.5 mL; cell diameters may be 2 to 70 microns. The Vi-Cell automated instrument offers many significant advantages for cell analyses in today's busy laboratory environment.

  17. The comparison of the viability of crushed, morselized and diced cartilage grafts: a confocal microscopic study.

    PubMed

    Kayabasoglu, Gurkan; Ozbek, Elvan; Yanar, Sevinc; Sahin, Fikrettin; Keles, Osman Nuri; Yilmaz, Mahmut Sinan; Guven, Mehmet

    2015-05-01

    To compare the cellular viability of diced, crushed, and morselized cartilage used in nasal surgeries. In this study, cartilage was extracted from the ears of seven New Zealand rabbits and was subsequently either diced, crushed or morselized to an amorphous state, or left unmodified. The four types of grafts were then implanted in the back regions of the rabbits. After 3 months, the cellular viability from four groups was compared to a control group using confocal microscopy. Analysis of the data obtained from the enumeration of live cells showed no statistically significant difference between the unmodified graft group and the control group. The diced, crushed, and morselized cartilage groups did show a statistically significant difference in terms of live cell count with the highest number of live cells in diced cartilage group. A statistically significant decrease in live cell count was detected in crushed cartilage group. Our study shows that the viability of cells in diced cartilage grafts is greater than those in crushed or morselized cartilage grafts.

  18. The metastatic microenvironment: lung-derived factors control the viability of neuroblastoma lung metastasis.

    PubMed

    Maman, Shelly; Edry-Botzer, Liat; Sagi-Assif, Orit; Meshel, Tsipi; Yuan, Weirong; Lu, Wuyuan; Witz, Isaac P

    2013-11-15

    Recent data suggest that the mechanisms determining whether a tumor cell reaching a secondary organ will enter a dormant state, progress toward metastasis, or go through apoptosis are regulated by the microenvironment of the distant organ. In neuroblastoma, 60-70% of children with high-risk disease will ultimately experience relapse due to the presence of micrometastases. The main goal of this study is to evaluate the role of the lung microenvironment in determining the fate of neuroblastoma lung metastases and micrometastases. Utilizing an orthotopic mouse model for human neuroblastoma metastasis, we were able to generate two neuroblastoma cell populations-lung micrometastatic (MicroNB) cells and lung macrometastatic (MacroNB) cells. These two types of cells share the same genetic background, invade the same distant organ, but differ in their ability to create metastasis in the lungs. We hypothesize that factors present in the lung microenvironment inhibit the propagation of MicroNB cells preventing them from forming overt lung metastasis. This study indeed shows that lung-derived factors significantly reduce the viability of MicroNB cells by up regulating the expression of pro-apoptotic genes, inducing cell cycle arrest and decreasing ERK and FAK phosphorylation. Lung-derived factors affected various additional progression-linked cellular characteristics of neuroblastoma cells, such as the expression of stem-cell markers, morphology, and migratory capacity. An insight into the microenvironmental effects governing neuroblastoma recurrence and progression would be of pivotal importance as they could have a therapeutic potential for the treatment of neuroblastoma residual disease.

  19. Cell viability after osteotomy and bone harvesting: comparison of piezoelectric surgery and conventional bur.

    PubMed

    Mouraret, S; Houschyar, K S; Hunter, D J; Smith, A A; Jew, O S; Girod, S; Helms, J A

    2014-08-01

    The aim of this study was to evaluate and compare the influence of a piezoelectric device versus a conventional bur on osteocyte viability and osteoblast and osteoclast activity using an in vivo mouse model. Osteotomies were created and bone grafts were harvested using either a conventional bur or a piezoelectric device; the resulting injuries and bone grafts were evaluated over an extended time-course using molecular and cellular assays for cell death (TUNEL assay), cell viability (4',6-diamidino-2-phenylindole (DAPI) staining), the onset of mineralization (alkaline phosphatase activity), and bone remodelling (tartrate-resistant acid phosphatase activity). Osteotomies created with a piezoelectric device showed greater osteocyte viability and reduced cell death. Bone grafts harvested with a piezoelectric device exhibited greater short-term cell viability than those harvested with a bur, and exhibited slightly more new bone deposition and bone remodelling. The difference in response of osteocytes, osteoblasts, and osteoclasts to bone cutting via a bur and via a piezoelectric device is negligible in vivo. Given the improved visibility and the margin of safety afforded by a piezoelectric device, they are the instrument of choice when cutting or harvesting bone to preserve soft tissue.

  20. In Vitro Electrochemical Corrosion and Cell Viability Studies on Nickel-Free Stainless Steel Orthopedic Implants

    PubMed Central

    Salahinejad, Erfan; Hadianfard, Mohammad Jafar; Macdonald, Digby Donald; Sharifi-Asl, Samin; Mozafari, Masoud; Walker, Kenneth J.; Rad, Armin Tahmasbi; Madihally, Sundararajan V.; Tayebi, Lobat

    2013-01-01

    The corrosion and cell viability behaviors of nanostructured, nickel-free stainless steel implants were studied and compared with AISI 316L. The electrochemical studies were conducted by potentiodynamic polarization and electrochemical impedance spectroscopic measurements in a simulated body fluid. Cytocompatibility was also evaluated by the adhesion behavior of adult human stem cells on the surface of the samples. According to the results, the electrochemical behavior is affected by a compromise among the specimen's structural characteristics, comprising composition, density, and grain size. The cell viability is interpreted by considering the results of the electrochemical impedance spectroscopic experiments. PMID:23630603

  1. The cellular memory disc of reprogrammed cells.

    PubMed

    Anjamrooz, Seyed Hadi

    2013-04-01

    The crucial facts underlying the low efficiency of cellular reprogramming are poorly understood. Cellular reprogramming occurs in nuclear transfer, induced pluripotent stem cell (iPSC) formation, cell fusion, and lineage-switching experiments. Despite these advances, there are three fundamental problems to be addressed: (1) the majority of cells cannot be reprogrammed, (2) the efficiency of reprogramming cells is usually low, and (3) the reprogrammed cells developed from a patient's own cells activate immune responses. These shortcomings present major obstacles for using reprogramming approaches in customised cell therapy. In this Perspective, the author synthesises past and present observations in the field of cellular reprogramming to propose a theoretical picture of the cellular memory disc. The current hypothesis is that all cells undergo an endogenous and exogenous holographic memorisation such that parts of the cellular memory dramatically decrease the efficiency of reprogramming cells, act like a barrier against reprogramming in the majority of cells, and activate immune responses. Accordingly, the focus of this review is mainly to describe the cellular memory disc (CMD). Based on the present theory, cellular memory includes three parts: a reprogramming-resistance memory (RRM), a switch-promoting memory (SPM) and a culture-induced memory (CIM). The cellular memory arises genetically, epigenetically and non-genetically and affects cellular behaviours. [corrected].

  2. A Cellular Biophysics Textbook

    NASA Astrophysics Data System (ADS)

    Wilder, Alan Joseph

    2011-12-01

    In the past two decades, great advances have been made in understanding of the biophysical mechanisms of the protein machines that carry out the fundamental processes of the cell. It is now known that all major eukaryotic cellular processes require a complicated assemblage of proteins acting via a series of concerted motions. In order to grasp current understanding of cellular mechanisms, the new generation of cell biologists needs to be trained in the general characteristics of these cellular properties and the methods with which to study them. This cellular biophysics textbook, to be used in conjunction with the cellular biophysics course (MCB143) at UC-Davis, provides a great tool in the instruction of the new generation of cellular biologists. It provides a hierarchical view of the cell, from atoms to protein machines and explains in depth the mechanisms of cytoskeletal force generators as an example of these principles.

  3. [Nicotinamide influence on pancreatic cells viability].

    PubMed

    Kuchmerovs'ka, T M; Donchenko, H V; Tykhonenko, T M; Huzyk, M M; Stavniĭchuk, R V; Ianits'ka, L V; Stepanenko, S P; Klymenko, A P

    2012-01-01

    The study was undertaken to investigate the modulating effect of nicotinamide (NAm) in different concentrations and under different glucose concentrations on the viability and oxidative stress induced by streptozotocin (STZ, 5 mmol/l) and hydrogen peroxide (H2O2, 100 micromol/l) on isolated rat pancreatic cells of the Langerhans islets in vitro. Cell viability did not depend on the concentration of glucose in the range of 5-20 mmol/l, and in subsequent studies we used glucose in concentration of 10 mmol/l to protect cells against its hypo- and hyperglycemic action. Cytoprotective effect of NAm in concentrations from 5 to 20 mmol/l on cells survival was the same. It was found that the destructive action of STZ and H2O2 during 24 hours on isolated cells of the pancreas resulted in the significant cell death. It was revealed that NAm in concentration of 5 mmol/l not only had cytoprotective effects against STZ and H2O2 but also partially reduced the level of oxidative stress in the investigated cells induced by these compounds. High concentration of NAm, 35 mmol/l, causes cytotoxic effect on the viability of pancreatic islet cells and increase of oxidative stress induced by STZ and H2O2. Most likely these effects could be associated with direct modulatory action of NAm on important effector mechanisms involved in cell death, including PARP-dependent processes, or/and indirectly, through metabolic and antioxidant effects of the compound.

  4. Echinococcus metacestode: in search of viability markers

    PubMed Central

    Gottstein, Bruno; Wang, Junhua; Blagosklonov, Oleg; Grenouillet, Frédéric; Millon, Laurence; Vuitton, Dominique A.; Müller, Norbert

    2014-01-01

    Epidemiological studies have demonstrated that most humans infected with Echinococcus spp. exhibit resistance to disease. When infection leads to disease, the parasite is partially controlled by host immunity: in case of immunocompetence, the normal alveolar echinococcosis (AE) or cystic echinococcosis (CE) situation, the metacestode grows slowly, and first clinical signs appear years after infection; in case of impaired immunity (AIDS; other immunodeficiencies), uncontrolled proliferation of the metacestode leads to rapidly progressing disease. Assessing Echinococcus multilocularis viability in vivo following therapeutic interventions in AE patients may be of tremendous benefit when compared with the invasive procedures used to perform biopsies. Current options are F18-fluorodeoxyglucose-positron emission tomography (FDG-PET), which visualizes periparasitic inflammation due to the metabolic activity of the metacestode, and measurement of antibodies against recEm18, a viability-associated protein, that rapidly regresses upon metacestode inactivation. For Echinococcus granulosus, similar prognosis-associated follow-up parameters are still lacking but a few candidates may be listed. Other possible markers include functional and diffusion-weighted Magnetic Resonance Imaging (MRI), and measurement of products from the parasite (circulating antigens or DNA), and from the host (inflammation markers, cytokines, or chemokines). Even though some of them have been promising in pilot studies, none has been properly validated in an appropriate number of patients until now to be recommended for further use in clinical settings. There is therefore still a need to develop reliable tools for improved viability assessment to provide the sufficient information needed to reliably withdraw anti-parasite benzimidazole chemotherapy, and a basis for the development of new alternative therapeutic tools. PMID:25429386

  5. Rapid onsite assessment of spore viability.

    SciTech Connect

    Branda, Steven; Lane, Todd W.; VanderNoot, Victoria A.; Gaucher, Sara P.; Jokerst, Amanda S.

    2005-12-01

    This one year LDRD addresses problems of threat assessment and restoration of facilities following a bioterror incident like the incident that closed down mail facilities in late 2001. Facilities that are contaminated with pathogenic spores such as B. anthracis spores must be shut down while they are treated with a sporicidal agent and the effectiveness of the treatment is ascertained. This process involves measuring the viability of spore test strips, laid out in a grid throughout the facility; the CDC accepted methodologies require transporting the samples to a laboratory and carrying out a 48 hr outgrowth experiment. We proposed developing a technique that will ultimately lead to a fieldable microfluidic device that can rapidly assess (ideally less than 30 min) spore viability and effectiveness of sporicidal treatment, returning facilities to use in hours not days. The proposed method will determine viability of spores by detecting early protein synthesis after chemical germination. During this year, we established the feasibility of this approach and gathered preliminary results that should fuel a future more comprehensive effort. Such a proposal is currently under review with the NIH. Proteomic signatures of Bacillus spores and vegetative cells were assessed by both slab gel electrophoresis as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection. The conditions for germination using a number of chemical germinants were evaluated and optimized and the time course of protein synthesis was ascertained. Microseparations were carried out using both viable spores and spores inactivated by two different methods. A select number of the early synthesis proteins were digested into peptides for analysis by mass spectrometry.

  6. Quintessence reconstructed: New constraints and tracker viability

    SciTech Connect

    Sahlen, Martin; Liddle, Andrew R.; Parkinson, David

    2007-01-15

    We update and extend our previous work reconstructing the potential of a quintessence field from current observational data. We extend the cosmological data set to include new supernova data, plus information from the cosmic microwave background and from baryon acoustic oscillations. We extend the modeling by considering Pade approximant expansions as well as Taylor series, and by using observations to assess the viability of the tracker hypothesis. We find that parameter constraints have improved by a factor of 2, with a strengthening of the preference of the cosmological constant over evolving quintessence models. Present data show some signs, though inconclusive, of favoring tracker models over nontracker models under our assumptions.

  7. Mps1 kinase regulates tumor cell viability via its novel role in mitochondria

    PubMed Central

    Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

    2016-01-01

    Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells. PMID:27383047

  8. Use of flow cytometry to assess chondrocyte viability after laser reshaping of cartilage

    NASA Astrophysics Data System (ADS)

    Rasouli, Alexandre; Kim, Charlton C.; Basu, Reshmi; Wong, Brian J.

    2000-05-01

    Lasers have been shown to cause permanent shape change in cartilage via photothermally induced mechanical stress relaxation. While the biophysical properties of cartilage during laser irradiation have been studied, tissue viability following laser irradiation has not been fully characterized. In this study, cell viability staining and flow cytometry were used to determine chondrocyte viability following photothermal stress relaxation. Porcine septal cartilage slabs (10 X 25 X 1.5 mm) were irradiated with light from a Nd:YAG laser ((lambda) equals 1.32 micrometer, 25 W/cm2) while surface temperature, stress relaxation, and diffuse reflectance were recorded. Each slab received one, two, or three laser exposures (respective exposure times of 6.7, 7.2, 10 s), determined from measurements of diffuse reflectance, which correlate with mechanical stress relaxation. Irradiated samples were then divided into two groups analyzed immediately and at five days following laser exposure (the latter group was maintained in culture). Chondrocytes were isolated following serial enzymatic digestion with hyaluronidase, protease, and collagenase II for a total of 17 hours. Chondrocytes were then stained using SYTOR/DEAD RedTM (Molecular Probes; Eugene, OR) wherein live cells stained green (530 nm) and dead cells stained red (630 nm) when excited at 488 nm. A flow cytometer (FACScan, Becton Dickinson, Franklin Lakes, NJ) was then used to detect differential cell fluorescence; size; granularity; and the number of live cells, dead cells, and post irradiation debris in each treatment population. Nearly 60% of chondrocytes from reshaped cartilage samples isolated shortly after irradiation, were viable as determined using flow cytometry while non- irradiated controls were 100 percent viable. Specimens irradiated two or three times with the laser demonstrated increasing amounts of cellular debris along with a reduction in chondrocyte viability: 31 percent following two laser exposures, and 16

  9. Artificial evolution by viability rather than competition.

    PubMed

    Maesani, Andrea; Fernando, Pradeep Ruben; Floreano, Dario

    2014-01-01

    Evolutionary algorithms are widespread heuristic methods inspired by natural evolution to solve difficult problems for which analytical approaches are not suitable. In many domains experimenters are not only interested in discovering optimal solutions, but also in finding the largest number of different solutions satisfying minimal requirements. However, the formulation of an effective performance measure describing these requirements, also known as fitness function, represents a major challenge. The difficulty of combining and weighting multiple problem objectives and constraints of possibly varying nature and scale into a single fitness function often leads to unsatisfactory solutions. Furthermore, selective reproduction of the fittest solutions, which is inspired by competition-based selection in nature, leads to loss of diversity within the evolving population and premature convergence of the algorithm, hindering the discovery of many different solutions. Here we present an alternative abstraction of artificial evolution, which does not require the formulation of a composite fitness function. Inspired from viability theory in dynamical systems, natural evolution and ethology, the proposed method puts emphasis on the elimination of individuals that do not meet a set of changing criteria, which are defined on the problem objectives and constraints. Experimental results show that the proposed method maintains higher diversity in the evolving population and generates more unique solutions when compared to classical competition-based evolutionary algorithms. Our findings suggest that incorporating viability principles into evolutionary algorithms can significantly improve the applicability and effectiveness of evolutionary methods to numerous complex problems of science and engineering, ranging from protein structure prediction to aircraft wing design.

  10. Population Viability Analysis of Riverine Fishes

    SciTech Connect

    Bates, P.; Chandler, J.; Jager, H.I.; Lepla, K.; Van Winkle, W.

    1999-04-12

    Many utilities face conflkts between two goals: cost-efficient hydropower generation and protecting riverine fishes. Research to develop ecological simulation tools that can evaluate alternative mitigation strategies in terms of their benefits to fish populations is vital to informed decision-making. In this paper, we describe our approach to population viability analysis of riverine fishes in general and Snake River white sturgeon in particular. We are finding that the individual-based modeling approach used in previous in-stream flow applications is well suited to addressing questions about the viability of species of concern for several reasons. Chief among these are: (1) the abiIity to represent the effects of individual variation in life history characteristics on predicted population viabili~, (2) the flexibili~ needed to quanti~ the ecological benefits of alternative flow management options by representing spatial and temporal variation in flow and temperaturty and (3) the flexibility needed to quantifi the ecological benefits of non-flow related manipulations (i.e., passage, screening and hatchery supplementation).

  11. Protein Extraction by Means of Electroporation from E. coli with Preserved Viability.

    PubMed

    Haberl Meglic, Sasa; Marolt, Tilen; Miklavcic, Damijan

    2015-10-01

    Extracting proteins by means of electroporation from different microorganisms is gaining on its importance, as electroporation is a quick, chemical-free, and cost-effective method. Since complete cell destruction (to obtain proteins) necessitates additional work, and cost of purifying the end-product is high, pulses have to be adjusted in order to prevent total disintegration. Namely, total disintegration of the cell releases bacterial membrane contaminants in the final sample. Therefore, our goal was to study different electric pulse parameters in order to extract as much proteins as possible from E. coli bacteria, while preserving bacterial viability. Our results show that by increasing electric field strength the concentration of extracted proteins increases and viability reduces. The correlation is reasonable, since high electric field destroys bacterial envelope, releasing all intracellular components into surrounding media. The strong correlation was also found with pulse duration. However, at longer pulses we obtained more proteins, while bacterial viability was not as much affected. Pulse number and/or pulse repetition frequency at our conditions have no or little effect on concentration of extracted proteins and/or bacterial viability. We can conclude that the most promising pulse protocol for protein extraction by means of electroporation based on our experience would be longer pulses with lower pulse amplitude assuring high protein yield and low effect on bacterial viability.

  12. Effects of mutagenized X chromosomes of Drosophila melanogaster on viability and fitness in males

    SciTech Connect

    Simmons, M.J.; Raymond, J.D.; Anklesaria, F.X.; Hawkins, E.T.; Johnson, N.A.; Cooper, J.S.; Cox, N.M.

    1984-01-01

    Drosophila melanogaster males were treated with different doses of X-rays or ethyl methanesulfonate (EMS) and mated so that mutagenized X chromosomes could be recovered and tested for lethal mutations affecting viability and other aspects of fitness. The Poisson-corrected frequencies of the lethal mutations increased linearly with dose for both X-rays and EMS. Based on the data, l Krad X-rays given acutely induces the same number of lethals as 0.55 mM EMS administered by feeding. For some of the X-ray and EMS doses, the mutagenized chromosomes that were nonlethal reduced the viability of their carriers by a small amount, but there was no discernable dose-effect relationship. However in every case where a viability effect was seen, the percentage reduction was less than the corresponding frequency of lethals. All the groups of mutagenized nonlethal chromosomes reduced overall fitness by a significant percentage. Wherever a meaningful comparison was possible, this reduction was 2-3 times the reduction in viability, but as in the viability data, no dose-effect relationship was discernable.

  13. Efficient intracellular delivery of molecules with high cell viability using nanosecond-pulsed laser-activated carbon nanoparticles.

    PubMed

    Sengupta, Aritra; Kelly, Sean C; Dwivedi, Nishant; Thadhani, Naresh; Prausnitz, Mark R

    2014-03-25

    Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5-9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability.

  14. Efficient Intracellular Delivery of Molecules with High Cell Viability Using Nanosecond-Pulsed Laser-Activated Carbon Nanoparticles

    PubMed Central

    2015-01-01

    Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5–9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability. PMID:24547946

  15. A multicellular spheroid array to realize spheroid formation, culture, and viability assay on a chip.

    PubMed

    Torisawa, Yu-suke; Takagi, Airi; Nashimoto, Yuji; Yasukawa, Tomoyuki; Shiku, Hitoshi; Matsue, Tomokazu

    2007-01-01

    We describe a novel multicellular spheroid culture system that facilitates the easy preparation and culture of a spheroid microarray for the long-term monitoring of cellular activity. A spheroid culture device with an array of pyramid-like microholes was constructed in a silicon chip that was equipped with elastomeric microchannels. A cell suspension was introduced via the microfluidic channel into the microstructure that comprised silicon microholes and elastomeric microwells. A single spheroid can be formed and localized precisely within each microstructure. Since the culture medium could be replaced via the microchannels, a long-term culture (of approximately 2 weeks) is available on the chip. Measurement of albumin production in the hepatoma cell line (HepG2) showed that the liver-specific functions were maintained for 2 weeks. Based on the cellular respiratory activity, the cellular viability of the spheroid array on the chip was evaluated using scanning electrochemical microscopy. Responses to four different chemical stimulations were simultaneously detected on the same chip, thus demonstrating that each channel could be evaluated independently under various stimulation conditions. Our spheroid culture system facilitated the understanding of spheroid formation, culture, and viability assay on a single chip, thus functioning as a useful drug-screening device for cancer and liver cells.

  16. Low level laser therapy modulates viability, alkaline phosphatase and matrix metalloproteinase-2 activities of osteoblasts.

    PubMed

    Oliveira, Flávia Amadeu de; Matos, Adriana Arruda; Matsuda, Sandra Satiko; Buzalaf, Marília Afonso Rabelo; Bagnato, Vanderley Salvador; Machado, Maria Aparecida de Andrade Moreira; Damante, Carla Andreotti; Oliveira, Rodrigo Cardoso de; Peres-Buzalaf, Camila

    2017-04-01

    Low level laser therapy (LLLT) has been shown to stimulate bone cell metabolism but their impact on the matrix metalloproteinase (MMP) expression and activity is little explored. This study evaluated the influence of LLLT at two different wavelengths, red and infrared, on MC3T3-E1 preosteoblast viability, alkaline phosphatase (ALP) and MMP-2 and -9 activities. To accomplish this, MC3T3-E1 cells were irradiated with a punctual application of either red (660nm; InGaAIP active medium) or infrared (780nm; GaAlAs active medium) lasers both at a potency of 20mW, energy dose of 0.08 or 0.16J, and energy density of 1.9J/cm(2) or 3.8J/cm(2), respectively. The control group received no irradiation. Cellular viability, ALP and MMP-2 and -9 activities were assessed by MTT assay, enzymatic activity and zymography, respectively, at 24, 48 and 72h. The treatment of cells with both red and infrared lasers significantly increased the cellular viability compared to the non-irradiated control group at 24 and 48h. The ALP activity was also up modulated in infrared groups at 24 and 72h, depending on the energy densities. In addition, the irradiation with red laser at the energy density of 1.9J/cm(2) promoted an enhancement of MMP-2 activity at 48 and 72h. However, no differences were observed for the MMP-9 activity. In conclusion, when used at these specific parameters, LLL modulates both preosteoblast viability and differentiation highlighted by the increased ALP and MMP-2 activities induced by irradiation.

  17. Coculture with mesenchymal stem cells results in improved viability and function of human hepatocytes.

    PubMed

    Fitzpatrick, Emer; Wu, Yue; Dhadda, Paramjeet; Hughes, Robin D; Mitry, Ragai R; Qin, Hong; Lehec, Sharon C; Heaton, Nigel D; Dhawan, Anil

    2015-01-01

    Hepatocyte transplantation is becoming an accepted therapy for acute liver failure, either as a bridge to liver regeneration or to organ transplantation. Hepatocytes provide liver function in place of the failing organ. The maintenance of sufficient viability and function of the transplanted hepatocytes is a concern. There is a lot of recent interest in mesenchymal stem cells (MSCs) for the provision of structural and trophic support to hepatocytes, but few studies currently use primary human hepatocytes. The aim of this study was to investigate if coculture of human MSCs with cryopreserved human hepatocytes may improve their function and viability, thus with potential for cellular therapy of liver disease. MSCs were isolated from human umbilical cord or adipose tissue. Hepatocytes were isolated from donor organs unsuitable for transplantation. MSCs and hepatocytes were cocultured in both direct and indirect contact. Conditioned medium (CM) from cocultured MSCs and hepatocytes was also used on hepatocytes. Viability and liver-specific function were compared between test and controls. Human hepatocytes that were cocultured directly with MSCs demonstrated improved production of albumin from day 5 to day 25 of culture. This effect was most prominent at day 15. Likewise, urea production was improved in coculture from day 5 to 25. Indirect coculture demonstrated improved albumin production by day 4 (1,107 ng/ml) versus hepatocyte monoculture (940 ng/ml). Hepatocytes in CM demonstrated a nonsignificant improvement in function. The viability of cocultured hepatocytes was superior to that of monocultured cells with up to a 16% improvement. Thus, coculture of human hepatocytes with MSCs demonstrates both improved function and viability. The effect is seen mainly with direct coculture but can also be seen in indirect culture and with CM. Such coculture conditions may convey major advantages in hepatocyte survival and function for cell transplantation.

  18. Novel Sensor-Enabled Ex Vivo Bioreactor: A New Approach towards Physiological Parameters and Porcine Artery Viability

    PubMed Central

    Mundargi, Raghavendra; Venkataraman, Divya; Kumar, Saranya; Mogal, Vishal; Ortiz, Raphael; Loo, Joachim; Venkatraman, Subbu; Steele, Terry

    2015-01-01

    The aim of the present work is to design and construct an ex vivo bioreactor system to assess the real time viability of vascular tissue. Porcine carotid artery as a model tissue was used in the ex vivo bioreactor setup to monitor its viability under physiological conditions such as oxygen, pressure, temperature, and flow. The real time tissue viability was evaluated by monitoring tissue metabolism through a fluorescent indicator “resorufin.” Our ex vivo bioreactor allows real time monitoring of tissue responses along with physiological conditions. These ex vivo parameters were vital in determining the tissue viability in sensor-enabled bioreactor and our initial investigations suggest that, porcine tissue viability is considerably affected by high shear forces and low oxygen levels. Histological evaluations with hematoxylin and eosin and Masson's trichrome staining show intact endothelium with fresh porcine tissue whereas tissues after incubation in ex vivo bioreactor studies indicate denuded endothelium supporting the viability results from real time measurements. Hence, this novel viability sensor-enabled ex vivo bioreactor acts as model to mimic in vivo system and record vascular responses to biopharmaceutical molecules and biomedical devices. PMID:26609536

  19. Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms.

    PubMed

    Skogman, Malena Elise; Vuorela, Pia Maarit; Fallarero, Adyary

    2012-09-01

    Despite that three types of assays (measuring biofilm viability, biomass, or matrix) are described to assess anti-biofilm activity, they are rarely used together. As infections can easily reappear if the matrix is not affected after antibiotic treatments, our goal was to explore the simultaneous effects of antibiotics on the viability, biomass and matrix of Staphylococcus aureus biofilms (ATCC 25923). Viability and biomass were quantified using resazurin and crystal violet staining sequentially in the same plate, while matrix staining was conducted with a wheat germ agglutinin-Alexa Fluor 488 fluorescent conjugate. Establishment of the detection limits and linearity ranges allowed concluding that all three methods were able to estimate biofilm formation in a similar fashion. In a susceptibility study with 18-h biofilms, two model compounds (penicillin G and ciprofloxacin) caused a reduction on the viability and biomass accompanied by an increase or not changed levels of the matrix, respectively. This response pattern was also proven for S. aureus Newman, S. epidermidis and E. coli biofilms. A classification of antibiotics based on five categories according to their effects on viability and matrix has been proposed earlier. Our data suggests a sixth group, represented by penicillin, causing decrease in bacterial viability but showing stimulatory effects on the matrix. Further, if effects on the matrix are not taken into account, the long-term chemotherapeutic effect of antibiotics can be jeopardized in spite of the positive effects on biofilms viability and biomass. Thus, measuring all these three endpoints simultaneously provide a more complete and accurate picture.

  20. Viability of Selected Microorganisms in Hydrocarbon Fuels.

    PubMed

    Hedrick, H G; Carroll, M T; Owen, H P; Pritchard, D J

    1963-11-01

    A laboratory study of the viability of selected microorganisms in a hydrocarbon fuel medium was carried out on 19 species of microorganisms, representative of the types found as natural contaminants in aircraft fuels. More species remained viable when inoculated in pure cultures than when inoculated in mixed (composite) cultures. Of the 19 species selected, 10 were still viable after 3 months and 5 were viable after 4 months in the pure culture inoculants. In the complete composite culture inoculant, the bacterial species which were viable at the end of 4 months were the same as those found in the pure culture inoculant. No fungi remained viable in the complete composite cultures after a 3-week period. The microorganisms which remain viable in a hydrocarbon fuel medium are considered indicative of a satisfactory inoculum to be used as a test culture in laboratory analysis of mechanical control techniques.

  1. VIABILITY OF COLIFORM BACTERIA IN ANTARCTIC SOIL.

    PubMed

    BOYD, W L; BOYD, J W

    1963-05-01

    Boyd, William L. (Ohio State University, Columbus) and Josephine W. Boyd. Viability of coliform bacteria in antarctic soil. J. Bacteriol. 85:1121-1123. 1963.-The distribution of coliform bacteria in soils of Ross Island and the nearby mainland was studied. None was found in almost all of the samples collected, including some from the Adelie penguin rookeries at Cape Royds and Cape Crozier and in soil at the McMurdo Base which had been recently contaminated by human sewage. Samples of pony manure left from previous expeditions were also negative, with one exception where Escherichia coli were present. Studies carried out with two freshly isolated human strains of E. coli and the isolate from pony manure showed that the death rate was extremely rapid, although the animal strain was much more resistant to the various factors of the environment causing death.

  2. Incorporating evolutionary processes into population viability models.

    PubMed

    Pierson, Jennifer C; Beissinger, Steven R; Bragg, Jason G; Coates, David J; Oostermeijer, J Gerard B; Sunnucks, Paul; Schumaker, Nathan H; Trotter, Meredith V; Young, Andrew G

    2015-06-01

    We examined how ecological and evolutionary (eco-evo) processes in population dynamics could be better integrated into population viability analysis (PVA). Complementary advances in computation and population genomics can be combined into an eco-evo PVA to offer powerful new approaches to understand the influence of evolutionary processes on population persistence. We developed the mechanistic basis of an eco-evo PVA using individual-based models with individual-level genotype tracking and dynamic genotype-phenotype mapping to model emergent population-level effects, such as local adaptation and genetic rescue. We then outline how genomics can allow or improve parameter estimation for PVA models by providing genotypic information at large numbers of loci for neutral and functional genome regions. As climate change and other threatening processes increase in rate and scale, eco-evo PVAs will become essential research tools to evaluate the effects of adaptive potential, evolutionary rescue, and locally adapted traits on persistence.

  3. Artificial Evolution by Viability Rather than Competition

    PubMed Central

    Maesani, Andrea; Fernando, Pradeep Ruben; Floreano, Dario

    2014-01-01

    Evolutionary algorithms are widespread heuristic methods inspired by natural evolution to solve difficult problems for which analytical approaches are not suitable. In many domains experimenters are not only interested in discovering optimal solutions, but also in finding the largest number of different solutions satisfying minimal requirements. However, the formulation of an effective performance measure describing these requirements, also known as fitness function, represents a major challenge. The difficulty of combining and weighting multiple problem objectives and constraints of possibly varying nature and scale into a single fitness function often leads to unsatisfactory solutions. Furthermore, selective reproduction of the fittest solutions, which is inspired by competition-based selection in nature, leads to loss of diversity within the evolving population and premature convergence of the algorithm, hindering the discovery of many different solutions. Here we present an alternative abstraction of artificial evolution, which does not require the formulation of a composite fitness function. Inspired from viability theory in dynamical systems, natural evolution and ethology, the proposed method puts emphasis on the elimination of individuals that do not meet a set of changing criteria, which are defined on the problem objectives and constraints. Experimental results show that the proposed method maintains higher diversity in the evolving population and generates more unique solutions when compared to classical competition-based evolutionary algorithms. Our findings suggest that incorporating viability principles into evolutionary algorithms can significantly improve the applicability and effectiveness of evolutionary methods to numerous complex problems of science and engineering, ranging from protein structure prediction to aircraft wing design. PMID:24489790

  4. Plasmonic Nanostructured Cellular Automata

    NASA Astrophysics Data System (ADS)

    Alkhazraji, Emad; Ghalib, A.; Manzoor, K.; Alsunaidi, M. A.

    2017-03-01

    In this work, we have investigated the scattering plasmonic resonance characteristics of silver nanospheres with a geometrical distribution that is modelled by Cellular Automata using time-domain numerical analysis. Cellular Automata are discrete mathematical structures that model different natural phenomena. Two binary one-dimensional Cellular Automata rules are considered to model the nanostructure, namely rule 30 and rule 33. The analysis produces three-dimensional scattering profiles of the entire plasmonic nanostructure. For the Cellular Automaton rule 33, the introduction of more Cellular Automata generations resulted only in slight red and blue shifts in the plasmonic modes with respect to the first generation. On the other hand, while rule 30 introduced significant red shifts in the resonance peaks at early generations, at later generations however, a peculiar effect is witnessed in the scattering profile as new peaks emerge as a feature of the overall Cellular Automata structure rather than the sum of the smaller parts that compose it. We strongly believe that these features that emerge as a result adopting the different 256 Cellular Automata rules as configuration models of nanostructures in different applications and systems might possess a great potential in enhancing their capability, sensitivity, efficiency, and power utilization.

  5. Effect of chlorine, blanching, freezing, and microwave heating on Cryptosporidium parvum viability inoculated on green peppers.

    PubMed

    Duhain, G L M C; Minnaar, A; Buys, E M

    2012-05-01

    Cryptosporidium parvum oocysts have been found on the surface of vegetables in both developed and developing countries. C. parvum can contaminate vegetables via various routes, including irrigation water. This study investigated the effect of individual treatments of chlorine, blanching, blast freezing, and microwave heating, as well as combined treatments of chlorine and freezing, and chlorine and microwave heating on the viability of C. parvum oocysts inoculated on green peppers. The viability of the oocysts after the treatments was assessed using propidium iodide and a flow cytometer. Based on the propidium iodide staining, the chlorine treatments did not affect the viability of the oocysts. Blast freezing significantly inactivated 20% of the oocysts. Microwave heating and blanching significantly inactivated 93% of oocysts. Treatment with chlorine followed by blast freezing did not affect the viability of the oocysts significantly. Treatment with chlorine and microwave heating was significantly more effective than microwave heating alone and inactivated 98% of the oocysts. The study indicates that C. parvum oocysts are sensitive to heat and, to some extent, to blast freezing, but are resistant to chlorine. Therefore, the use of chlorine during vegetable processing is not a critical control point for C. parvum oocysts, and the consumption of raw or minimally processed vegetables may constitute a health risk as C. parvum oocysts can still be found viable on ready-to-eat, minimally processed vegetables.

  6. Moxonidine modulates cytokine signalling and effects on cardiac cell viability.

    PubMed

    Aceros, Henry; Farah, Georges; Noiseux, Nicolas; Mukaddam-Daher, Suhayla

    2014-10-05

    Regression of left ventricular hypertrophy and improved cardiac function in SHR by the centrally acting imidazoline I1-receptor agonist, moxonidine, are associated with differential actions on circulating and cardiac cytokines. Herein, we investigated cell-type specific I1-receptor (also known as nischarin) signalling and the mechanisms through which moxonidine may interfere with cytokines to affect cardiac cell viability. Studies were performed on neonatal rat cardiomyocytes and fibroblasts incubated with interleukin (IL)-1β (5 ng/ml), tumor necrosis factor (TNF)-α (10 ng/ml), and moxonidine (10(-7) and 10(-5) M), separately and in combination, for 15 min, and 24 and 48 h for the measurement of MAPKs (ERK1/2, JNK, and p38) and Akt activation and inducible NOS (iNOS) expression, by Western blotting, and cardiac cell viability/proliferation and apoptosis by flow cytometry, MTT assay, and Live/Dead assay. Participation of imidazoline I1-receptors and the signalling proteins in the detected effects was identified using imidazoline I1-receptor antagonist and signalling protein inhibitors. The results show that IL-1β, and to a lower extent, TNF-α, causes cell death and that moxonidine protects against starvation- as well as IL-1β -induced mortality, mainly by maintaining membrane integrity, and in part, by improving mitochondrial activity. The protection involves activation of Akt, ERK1/2, p38, JNK, and iNOS. In contrast, moxonidine stimulates basal and IL-1β-induced fibroblast mortality by mechanisms that include inhibition of JNK and iNOS. Thus, apart from their actions on the central nervous system, imidazoline I1-receptors are directly involved in cardiac cell growth and death, and may play an important role in cardiovascular diseases associated with inflammation.

  7. Cellular Reflectarray Antenna

    NASA Technical Reports Server (NTRS)

    Romanofsky, Robert R.

    2010-01-01

    The cellular reflectarray antenna is intended to replace conventional parabolic reflectors that must be physically aligned with a particular satellite in geostationary orbit. These arrays are designed for specified geographical locations, defined by latitude and longitude, each called a "cell." A particular cell occupies nominally 1,500 square miles (3,885 sq. km), but this varies according to latitude and longitude. The cellular reflectarray antenna designed for a particular cell is simply positioned to align with magnetic North, and the antenna surface is level (parallel to the ground). A given cellular reflectarray antenna will not operate in any other cell.

  8. Different N-terminal isoforms of Oct-1 control expression of distinct sets of genes and their high levels in Namalwa Burkitt's lymphoma cells affect a wide range of cellular processes

    PubMed Central

    Pankratova, Elizaveta V.; Stepchenko, Alexander G.; Portseva, Tatiana; Mogila, Vladic A.; Georgieva, Sofia G.

    2016-01-01

    Oct-1 transcription factor has various functions in gene regulation. Its expression level is increased in several types of cancer and is associated with poor survival prognosis. Here we identified distinct Oct-1 protein isoforms in human cells and compared gene expression patterns and functions for Oct-1A, Oct-1L, and Oct-1X isoforms that differ by their N-terminal sequences. The longest isoform, Oct-1A, is abundantly expressed and is the main Oct-1 isoform in most of human tissues. The Oct-1L and the weakly expressed Oct-1X regulate the majority of Oct-1A targets as well as additional sets of genes. Oct-1X controls genes involved in DNA replication, DNA repair, RNA processing, and cellular response to stress. The high level of Oct-1 isoforms upregulates genes related to cell cycle progression and activates proliferation both in Namalwa Burkitt's lymphoma cells and primary human fibroblasts. It downregulates expression of genes related to antigen processing and presentation, cytokine-cytokine receptor interaction, oxidative metabolism, and cell adhesion, thus facilitating pro-oncogenic processes. PMID:27407111

  9. Cellular Localization of Dieldrin and Structure–Activity Relationship of Dieldrin Analogues in Dopaminergic Cells

    PubMed Central

    Allen, Erin M. G.; Florang, Virginia R.; Davenport, Laurie L.; Jinsmaa, Yunden; Doorn, Jonathan A.

    2015-01-01

    The incidence of Parkinson’s disease (PD) correlates with environmental exposure to pesticides, such as the organochlorine insecticide, dieldrin. Previous studies found an increased concentration of the pesticide in the striatal region of the brains of PD patients and also that dieldrin adversely affects cellular processes associated with PD. These processes include mitochondrial function and reactive oxygen species production. However, the mechanism and specific cellular targets responsible for dieldrin-mediated cellular dysfunction and the structural components of dieldrin contributing to its toxicity (toxicophore) have not been fully defined. In order to identify the toxicophore of dieldrin, a structure–activity approach was used, with the toxicity profiles of numerous analogues of dieldrin (including aldrin, endrin, and cis-aldrin diol) assessed in PC6-3 cells. The MTT and lactate dehydrogenase (LDH) assays were used to monitor cell viability and membrane permeability after treatment with each compound. Cellular assays monitoring ROS production and extracellular dopamine metabolite levels were also used. Structure and stereochemistry for dieldrin were found to be very important for toxicity and other end points measured. Small changes in structure for dieldrin (e.g., comparison to the stereoisomer endrin) yielded significant differences in toxicity. Interestingly, the cis-diol metabolite of dieldrin was found to be significantly more toxic than the parent compound. Disruption of dopamine catabolism yielded elevated levels of the neurotoxin, 3,4-dihydroxyphenylacetaldehyde, for many organochlorines. Comparisons of the toxicity profiles for each dieldrin analogue indicated a structure-specific effect important for elucidating the mechanisms of dieldrin neurotoxicity. PMID:23763672

  10. Evidence for substantial maintenance of membrane integrity and cell viability in normally developing grape (Vitis vinifera L.) berries throughout development.

    PubMed

    Krasnow, Mark; Matthews, Mark; Shackel, Ken

    2008-01-01

    Fluorescein diacetate (FDA) was used as a vital stain to assay membrane integrity (cell viability) in mesocarp tissue of the developing grape (Vitis vinifera L.) berry in order to test the hypothesis that there is a substantial loss of compartmentation in these cells during ripening. This technique was also used to determine whether loss of viability was associated with symptoms of a ripening disorder known as berry shrivel. FDA fluorescence of berry cells was rapid, bright, and stable for over 1 h at room temperature. Confocal microscopy detected FDA staining through two to three intact surface cell layers (300-400 mum) of bisected berries, and showed that the fluorescence was confined to the cytoplasm, indicating the maintenance of integrity in both cytoplasmic as well as vacuolar membranes, and the presence of active cytoplasmic esterases. FDA clearly discriminated between living cells and freeze-killed cells, and exhibited little, if any, non-specific staining. Propidium iodide and DAPI, both widely used to assess cell viability, were unable to discriminate between living and freeze-killed cells, and did not specifically stain the nuclei of dead cells. For normally developing berries under field conditions there was no evidence of viability loss until about 40 d after veraison, and the majority (80%) of mesocarp cells remained viable past commercial harvest (26 degrees Brix). These results are inconsistent with current models of grape berry development which hypothesize that veraison is associated with a general loss of compartmentation in mesocarp cells. The observed viability loss was primarily in the locule area around the seeds, suggesting that a localized loss of viability and compartmentation may occur as part of normal fruit development. The cell viability of berry shrivel-affected berries was similar to that of normally developing berries until the onset of visible symptoms (i.e. shrivelling), at which time viability declined in visibly shrivelled

  11. Glycosylation regulates prestin cellular activity.

    PubMed

    Rajagopalan, Lavanya; Organ-Darling, Louise E; Liu, Haiying; Davidson, Amy L; Raphael, Robert M; Brownell, William E; Pereira, Fred A

    2010-03-01

    Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin(NN163/166AA) is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin(NN163/166AA) is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin(NN163/166AA) exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin(NN163/166AA) participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin(NN163/166AA) shows a significant cholesterol-dependent decrease in cell-surface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin(NN163/166AA). These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by

  12. Cellular aging and cancer

    PubMed Central

    Hornsby, Peter J.

    2010-01-01

    Aging is manifest in a variety of changes over time, including changes at the cellular level. Cellular aging acts primarily as a tumor suppressor mechanism, but also may enhance cancer development under certain circumstances. One important process of cellular aging is oncogene-induced senescence, which acts as an important anti-cancer mechanism. Cellular senescence resulting from damage caused by activated oncogenes prevents the growth or potentially neoplastic cells. Moreover, cells that have entered senescence appear to be targets for elimination by the innnate immune system. In another aspect of cellular aging, the absence of telomerase activity in normal tissues results in such cells lacking a telomere maintenance mechanism. One consequence is that in aging there is an increase in cells with shortened telomeres. In the presence of active oncogenes that cause expansion of a neoplastic clone, shortening of telomeres leading to telomere dysfunction prevents the indefinite expansion of the clone because the cells enter crisis. Crisis results from fusions and other defects caused by dysfunctional telomeres and is a terminal state of the neoplastic clone. In this way the absence of telomerase in human cells, while one cause of cellular aging, also acts as an anti-cancer mechanism. PMID:20705476

  13. Fault Detection and Isolation using Viability Theory and Interval Observers

    NASA Astrophysics Data System (ADS)

    Ghaniee Zarch, Majid; Puig, Vicenç; Poshtan, Javad

    2017-01-01

    This paper proposes the use of interval observers and viability theory in fault detection and isolation (FDI). Viability theory develops mathematical and algorithmic methods for investigating the adaptation to viability constraints of evolutions governed by complex systems under uncertainty. These methods can be used for checking the consistency between observed and predicted behavior by using simple sets that approximate the exact set of possible behavior (in the parameter or state space). In this paper, fault detection is based on checking for an inconsistency between the measured and predicted behaviors using viability theory concepts and sets. Finally, an example is provided in order to show the usefulness of the proposed approach.

  14. Nutritional deprivation and LPS exposure as feasible methods for induction of cellular - A methodology to validate for vitro photobiomodulation studies.

    PubMed

    Basso, F G; Turrioni, A P S; Almeida, L F; Soares, D G; Oliveira, C F; Hebling, J; de Souza Costa, C A

    2016-06-01

    Previous studies have demonstrated that high biostimulation takes place when cells under stress are subjected to phototherapy by laser or light-emitting-diode (LED) devices. Several studies selected nutritional deprivation by reducing the concentration of fetal bovine serum (FBS) in the culture medium or the exposure of cultured cells to lipopolysaccharide (LPS) as an in vitro cellular stress condition. However, there are no data certifying that these stimuli cause stressful conditions for cultured cells. This investigation assessed the induction of cellular stress by decreasing the concentration of FBS or adding LPS to culture medium. Odontoblast-like cells (MDPC-23) were cultured in complete culture medium (DMEM) containing 10% FBS. After a 12-hour incubation period, the DMEM was replaced by fresh medium containing 10% FBS (control), low concentrations of FBS (0, 0.2, 0.5, 2, or 5%) or LPS from Escherichia coli (10μg/ml). After an additional 12-hour incubation, cell viability, total cell-counting, total protein production, and gene expression of heat shock protein 70 (HSP70) were assessed. Data were statistically analyzed by ANOVA complemented by the Tukey test, with 5% considered significant. Cell viability was negatively affected only for 0% FBS, while reduced viable cell numbers and total protein production were detected for FBS concentrations lower than 2%. Higher HSP70 gene expression was also observed for FBS concentrations lower than 2% and for cells exposed to LPS. The nutritional deprivation model with culture medium lower than 2% of FBS can be safely used to induce cellular stress for in vitro photobiomodulation studies.

  15. Cellular stress and RNA splicing.

    PubMed

    Biamonti, Giuseppe; Caceres, Javier F

    2009-03-01

    In response to physical and chemical stresses that affect protein folding and, thus, the execution of normal metabolic processes, cells activate gene-expression strategies aimed at increasing their chance of survival. One target of several stressing agents is pre-mRNA splicing, which is inhibited upon heat shock. Recently, the molecular basis of this splicing inhibition has begun to emerge. Interestingly, different mechanisms seem to be in place to block constitutive pre-mRNA splicing and to affect alternative splicing regulation. This could be important to modulate gene expression during recovery from stress. Thus, pre-mRNA splicing emerges as a central mechanism to integrate cellular and metabolic stresses into gene-expression profiles.

  16. Storage Viability and Optimization Web Service

    SciTech Connect

    Stadler, Michael; Marnay, Christ; Lai, Judy; Siddiqui, Afzal; Limpaitoon, Tanachai; Phan, Trucy; Megel, Olivier; Chang, Jessica; DeForest, Nicholas

    2010-10-11

    Non-residential sectors offer many promising applications for electrical storage (batteries) and photovoltaics (PVs). However, choosing and operating storage under complex tariff structures poses a daunting technical and economic problem that may discourage potential customers and result in lost carbon and economic savings. Equipment vendors are unlikely to provide adequate environmental analysis or unbiased economic results to potential clients, and are even less likely to completely describe the robustness of choices in the face of changing fuel prices and tariffs. Given these considerations, researchers at Lawrence Berkeley National Laboratory (LBNL) have designed the Storage Viability and Optimization Web Service (SVOW): a tool that helps building owners, operators and managers to decide if storage technologies and PVs merit deeper analysis. SVOW is an open access, web-based energy storage and PV analysis calculator, accessible by secure remote login. Upon first login, the user sees an overview of the parameters: load profile, tariff, technologies, and solar radiation location. Each parameter has a pull-down list of possible predefined inputs and users may upload their own as necessary. Since the non-residential sectors encompass a broad range of facilities with fundamentally different characteristics, the tool starts by asking the users to select a load profile from a limited cohort group of example facilities. The example facilities are categorized according to their North American Industry Classification System (NAICS) code. After the load profile selection, users select a predefined tariff or use the widget to create their own. The technologies and solar radiation menus operate in a similar fashion. After these four parameters have been inputted, the users have to select an optimization setting as well as an optimization objective. The analytic engine of SVOW is LBNL?s Distributed Energy Resources Customer Adoption Model (DER-CAM), which is a mixed

  17. Measurement Techniques for Cellular Biomechanics In Vitro

    PubMed Central

    Addae-Mensah, Kweku A; Wikswo, John P

    2014-01-01

    Living cells and tissues experience mechanical forces in their physiological environments that are known to affect many cellular processes. Also of importance are the mechanical properties of cells, as well as the microforces generated by cellular processes themselves in their microenvironments. The difficulty associated with studying these phenomena in vivo has led to alternatives such as using in vitro models. The need for experimental techniques for investigating cellular biomechanics and mechanobiology in vitro has fueled an evolution in the technology used in these studies. Particularly noteworthy are some of the new biomicroelectromechanical systems (BioMEMs) devices and techniques that have been introduced to the field. We describe some of the cellular micromechanical techniques and methods that have been developed for in vitro studies, and provide summaries of the ranges of measured values of various biomechanical quantities. We also briefly address some of our experiences in using these methods and include modifications we have introduced in order to improve them. PMID:18445766

  18. The Mre11-Nbs1 interface is essential for viability and tumor suppression

    PubMed Central

    Kim, Jun Hyun; Grosbart, Malgorzata; Anand, Roopesh; Wyman, Claire; Cejka, Petr; Petrini, John H.J.

    2016-01-01

    SUMMARY The Mre11 complex (Mre11, Rad50 and Nbs1) is integral to both DNA repair and ATM-dependent DNA damage signaling. All three Mre11 complex components are essential for viability at the cellular and organismal level. To delineate essential and non-essential Mre11 complex functions that are mediated by Nbs1, we used TALEN-based genome editing to derive Nbs1 mutant mice (Nbs1mid mice) which harbor mutations in the Mre11 interaction domain of Nbs1. Nbs1mid alleles that abolished interaction were incompatible with viability. Conversely, a 108 amino acid Nbs1 fragment comprising the Mre11 interface was sufficient to rescue viability and ATM activation in cultured cells and to support differentiation of hematopoietic cells in vivo. These data indicate that the essential role of Nbs1 is via its interaction with Mre11, that most of the Nbs1 protein is dispensable for Mre11 complex functions, and suggest that Mre11 and Rad50 directly activate ATM. PMID:28076792

  19. Effect of smokeless tobacco products on human oral bacteria growth and viability.

    PubMed

    Liu, Min; Jin, Jinshan; Pan, Hongmiao; Feng, Jinhui; Cerniglia, Carl E; Yang, Maocheng; Chen, Huizhong

    2016-12-01

    To evaluate the toxicity of smokeless tobacco products (STPs) on oral bacteria, seven smokeless tobacco aqueous extracts (STAEs) from major brands of STPs and three tobacco-specific N-nitrosamines (TSNAs) were used in a growth and viability test against 38 oral bacterial species or subspecies. All seven STAEs showed concentration-dependent effects on the growth and viability of tested oral bacteria under anaerobic culture conditions, although there were strain-to-strain variations. In the presence of 1 mg/ml STAEs, the growth of 4 strains decreased over 0.32-2.14 log10 fold, while 14 strains demonstrated enhanced growth of 0.3-1.76 log10 fold, and the growth of 21 strains was not significantly affected. In the presence of 10 mg/ml STAEs, the growth of 17 strains was inhibited 0.3-2.11 log10 fold, 18 strains showed enhanced growth of 0.3-0.97 log10 fold, and 4 strains were not significantly affected. In the presence of 50 mg/ml STAEs, the growth of 32 strains was inhibited 0.3-2.96 log10 fold, 8 strains showed enhanced growth of 0.3-1.0 log10 fold, and 2 strains were not significantly affected. All seven STAEs could promote the growth of 4 bacterial strains, including Eubacterium nodatum, Peptostreptococcus micros, Streptococcus anginosus, and Streptococcus constellatus. Exposure to STAEs modulated the viability of some bacterial strains, with 21.1-66.5% decrease for 4 strains at 1 mg/ml, 20.3-85.7% decrease for 10 strains at 10 mg/ml, 20.0-93.3% decrease for 27 strains at 50 mg/ml, and no significant effect for 11 strains at up to 50 mg/ml. STAEs from snuffs inhibited more tested bacterial strains than those from snus indicating that the snuffs may be more toxic to the oral bacteria than snus. For TSNAs, cell growth and viability of 34 tested strains were not significantly affected at up to 100 μg/ml; while the growth of P. micros was enhanced 0.31-0.54 log10 fold; the growth of Veillonella parvula was repressed 0.33-0.36 log10 fold; and the cell

  20. Polyaspartamide derivative nanoparticles with tunable surface charge achieve highly efficient cellular uptake and low cytotoxicity.

    PubMed

    Xu, Min; Zhao, Yuefang; Feng, Min

    2012-08-07

    Cationic nanocarrier mediated intracellular therapeutic agent delivery acts as a double-edged sword: the carriers promote cellular uptake, but interact nonspecifically and strongly with negatively charged endogenic proteins and cell membranes, which results in aggregates and high cytotoxicity. The present study was aimed at exploring zwitterionic polyaspartamide derivative nanoparticles for efficient intracellular delivery with low cytotoxicity. Poly(aspartic acid) partially grafted tetraethylenepentamine (PASP-pg-TEPA) with different isoelectric points (IEPs) was synthesized. The PASP-pg-TEPA formed zwitterionic nanoparticles with an irregular core and a well-defined shell structure in aqueous medium. Their particle size decreased from about 300 to 80 nm with an increase of the IEP from 7.5 to 9.1. The surface charge of the PASP-pg-TEPA nanoparticles could be tuned from positive to negative with a change of the pH of the medium. The nanoparticles with an IEP above 8.5 exhibited good stability under simulated physiological conditions. It was noted that the zwitterionic PASP-pg-TEPA nanoparticles displayed highly efficient cellular uptake in HeLa cells (approximately 99%) in serum-containing medium and did not adversely affect the cell viability at concentrations up to 1 mg/mL. Furthermore, thermodynamic analysis using isothermal titration calorimetry provided direct evidence that these zwitterionic nanoparticles had low binding affinities for serum protein. Therefore, the zwitterionic PASP-pg-TEPA nanoparticles could overcome limitations of cationic nanocarriers and achieve efficient intracellular delivery with low cytotoxicity.

  1. Bioabsorbable zinc ion induced biphasic cellular responses in vascular smooth muscle cells

    PubMed Central

    Ma, Jun; Zhao, Nan; Zhu, Donghui

    2016-01-01

    Bioabsorbable metal zinc (Zn) is a promising new generation of implantable scaffold for cardiovascular and orthopedic applications. In cardiovascular stent applications, zinc ion (Zn2+) will be gradually released into the surrounding vascular tissues from such Zn-containing scaffolds after implantation. However, the interactions between vascular cells and Zn2+ are still largely unknown. We explored the short-term effects of extracellular Zn2+ on human smooth muscle cells (SMCs) up to 24 h, and an interesting biphasic effect of Zn2+ was observed. Lower concentrations (<80 μM) of Zn2+ had no adverse effects on cell viability but promoted cell adhesion, cell spreading, cell proliferation, cell migration, and enhanced the expression of F-actin and vinculin. Cells treated with such lower concentrations of Zn2+ displayed an elongated shape compared to controls without any treatment. In contrast, cells treated with higher Zn2+ concentrations (80–120 μM) had opposite cellular responses and behaviors. Gene expression profiles revealed that the most affected functional genes were related to angiogenesis, inflammation, cell adhesion, vessel tone, and platelet aggregation. Results indicated that Zn has interesting concentration-dependent biphasic effects on SMCs with low concentrations being beneficial to cellular functions. PMID:27248371

  2. Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

    PubMed Central

    Perdomo-Celis, Federico; Salgado, Doris M.; Castañeda, Diana M.

    2016-01-01

    Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-γ]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r = 0.74; P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P ≤ 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3+ CD8+ amine-positive cells was found before cryopreservation (P = 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-γ-producing CD3+ cells after stimulation compared with healthy controls and convalescent-phase patients (P ≤ 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho = −0.56; P = 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs. PMID:26961858

  3. Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue.

    PubMed

    Perdomo-Celis, Federico; Salgado, Doris M; Castañeda, Diana M; Narváez, Carlos F

    2016-05-01

    Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-γ]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r = 0.74; P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P ≤ 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3(+) CD8(+) amine-positive cells was found before cryopreservation (P = 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-γ-producing CD3(+) cells after stimulation compared with healthy controls and convalescent-phase patients (P ≤ 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho = -0.56; P = 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs.

  4. Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

    PubMed

    Poutiainen, Pekka K; Rönkkö, Teemu; Hinkkanen, Ari E; Palvimo, Jorma J; Närvänen, Ale; Turhanen, Petri; Laatikainen, Reino; Weisell, Janne; Pulkkinen, Juha T

    2014-01-15

    In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 μM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.

  5. Sensitivity of eastern oyster (Crassostrea virginica) spermatozoa and oocytes to dispersed oil: Cellular responses and impacts on fertilization and embryogenesis.

    PubMed

    Vignier, J; Volety, A K; Rolton, A; Le Goïc, N; Chu, F-L E; Robert, R; Soudant, P

    2017-03-23

    The 2010 Deepwater Horizon (DWH) oil spill released millions of barrels of oil and dispersant into the Gulf of Mexico. The timing of the spill coincided with the spawning season of Crassostrea virginica. Consequently, gametes released in the water were likely exposed to oil and dispersant. This study aimed to (i) evaluate the cellular effects of acute exposure of spermatozoa and oocytes to surface slick oil, dispersed mechanically (HEWAF) and chemically (CEWAF), using flow-cytometric (FCM) analyses, and (ii) determine whether the observed cellular effects relate to impairments of fertilization and embryogenesis of gametes exposed to the same concentrations of CEWAF and HEWAF. Following a 30-min exposure, the number of spermatozoa and their viability were reduced due to a physical action of oil droplets (HEWAF) and a toxic action of CEWAF respectively. Additionally, reactive oxygen species (ROS) production in exposed oocytes tended to increase with increasing oil concentrations suggesting that exposure to dispersed oil resulted in an oxidative stress. The decrease in fertilization success (1-h), larval survival (24-h) and increase in abnormalities (6-h and 24-h) may be partly related to altered cellular characteristics. FCM assays are a good predictor of sublethal effects especially on fertilization success. These data suggest that oil/dispersant are cytotoxic to gametes, which may affect negatively the reproduction success and early development of oysters.

  6. Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro

    PubMed Central

    Li, Jie; Zhang, Donglan; Ward, Kathleen M.; Prendergast, George C.; Ayene, Iraimoudi S.

    2012-01-01

    Cell viability assays have a variety of well known practical and technical limitations. All the available approaches have disadvantages, such as non-linearity, high background and cumbersome protocols. Several commonly used tetrazolium chemicals rely upon generation of a colored formazan product formed by mitochondrial reduction of these compounds via phenazine methosulfate (PMS). However, sensitivity is inherently limited because their reduction relies on mitochondrial bioreduction and cellular transport of PMS, as well as accessibility to tetrazolium chemicals. In this study, we identify hydroxethyldisulfide (HEDS) as an inexpensive probe that can measure cellular metabolic activity without the need of PMS. In tissue culture medium, HEDS accurately quantitated metabolically active live cells in a linear manner superior to tetrazolium based and other assays. Cell toxicity produced by chemotherapeutics (cisplatin, etoposide), oxidants (hydrogen peroxide, acetaminophen), toxins (Phenyl arsine oxide, arsenite) or ionizing radiation was rapidly determined by the HEDS assay. We found that HEDS was superior to other commonly used assays for cell viability determinations in its solubility, membrane permeability, and intracellular conversion to a metabolic reporter that is readily transported into the extracellular medium. Our findings establish the use of HEDS in a simple, rapid and low cost assay to accurately quantify viable cells. PMID:22321380

  7. Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro.

    PubMed

    Li, Jie; Zhang, Donglan; Ward, Kathleen M; Prendergast, George C; Ayene, Iraimoudi S

    2012-06-01

    Cell viability assays have a variety of well known practical and technical limitations. All the available approaches have disadvantages, such as non-linearity, high background and cumbersome protocols. Several commonly used tetrazolium chemicals rely upon generation of a colored formazan product formed by mitochondrial reduction of these compounds via phenazine methosulfate (PMS). However, sensitivity is inherently limited because their reduction relies on mitochondrial bioreduction and cellular transport of PMS, as well as accessibility to tetrazolium chemicals. In this study, we identify hydroxethyldisulfide (HEDS) as an inexpensive probe that can measure cellular metabolic activity without the need of PMS. In tissue culture medium, HEDS accurately quantitated metabolically active live cells in a linear manner superior to tetrazolium based and other assays. Cell toxicity produced by chemotherapeutics (cisplatin, etoposide), oxidants (hydrogen peroxide, acetaminophen), toxins (phenyl arsine oxide, arsenite) or ionizing radiation was rapidly determined by the HEDS assay. We found that HEDS was superior to other commonly used assays for cell viability determinations in its solubility, membrane permeability, and intracellular conversion to a metabolic reporter that is readily transported into the extracellular medium. Our findings establish the use of HEDS in a simple, rapid and low cost assay to accurately quantify viable cells.

  8. Protein kinase C is essential for viability of the rice blast fungus M agnaporthe oryzae

    PubMed Central

    Penn, Tina J.; Wood, Mark E.; Soanes, Darren M.; Csukai, Michael; Corran, Andrew John

    2015-01-01

    Summary Protein kinase C constitutes a family of serine–threonine kinases found in all eukaryotes and implicated in a wide range of cellular functions, including regulation of cell growth, cellular differentiation and immunity. Here, we present three independent lines of evidence which indicate that protein kinase C is essential for viability of M agnaporthe oryzae. First, all attempts to generate a target deletion of PKC 1, the single copy protein kinase C‐encoding gene, proved unsuccessful. Secondly, conditional gene silencing of PKC 1 by RNA interference led to severely reduced growth of the fungus, which was reversed by targeted deletion of the Dicer2‐encoding gene, MDL 2. Finally, selective kinase inhibition of protein kinase C by targeted allelic replacement with an analogue‐sensitive PKC 1AS allele led to specific loss of fungal viability in the presence of the PP1 inhibitor. Global transcriptional profiling following selective PKC inhibition identified significant changes in gene expression associated with cell wall re‐modelling, autophagy, signal transduction and secondary metabolism. When considered together, these results suggest protein kinase C is essential for growth and development of M . oryzae with extensive downstream targets in addition to the cell integrity pathway. Targeting protein kinase C signalling may therefore prove an effective means of controlling rice blast disease. PMID:26192090

  9. Circulatory shear flow alters the viability and proliferation of circulating colon cancer cells

    NASA Astrophysics Data System (ADS)

    Fan, Rong; Emery, Travis; Zhang, Yongguo; Xia, Yuxuan; Sun, Jun; Wan, Jiandi

    2016-06-01

    During cancer metastasis, circulating tumor cells constantly experience hemodynamic shear stress in the circulation. Cellular responses to shear stress including cell viability and proliferation thus play critical roles in cancer metastasis. Here, we developed a microfluidic approach to establish a circulatory microenvironment and studied circulating human colon cancer HCT116 cells in response to a variety of magnitude of shear stress and circulating time. Our results showed that cell viability decreased with the increase of circulating time, but increased with the magnitude of wall shear stress. Proliferation of cells survived from circulation could be maintained when physiologically relevant wall shear stresses were applied. High wall shear stress (60.5 dyne/cm2), however, led to decreased cell proliferation at long circulating time (1 h). We further showed that the expression levels of β-catenin and c-myc, proliferation regulators, were significantly enhanced by increasing wall shear stress. The presented study provides a new insight to the roles of circulatory shear stress in cellular responses of circulating tumor cells in a physiologically relevant model, and thus will be of interest for the study of cancer cell mechanosensing and cancer metastasis.

  10. The effects of six root-end filling materials and their leachable components on cell viability.

    PubMed

    Al-Sa'eed, Oula R; Al-Hiyasat, Ahmad S; Darmani, Homa

    2008-11-01

    This study investigated the effect of six root-end filling materials (Retroplast [Retroplast Trading, Dybesøvej, Denmark], Geristore, [DEN-MAT Corporation, Santa Maria, CA], Ketac Fil [3M ESPE, Seefeld, Germany], IRM [Caulk-Dentsply, Milford, DE], Super EBA [Bosworth Company, Skokie, IL], and MTA [Dentsply-Tulsa Dental, Johnson City, TN]) on the viability of Balb/C 3T3 fibroblasts using the [3-4, 5-dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide succinate (MTT) assay. Ten discs (5 mm x 2 mm) of each material were eluted in tissue culture medium for 24 hours at 37 degrees C for 3 successive days and the elutes used for cell viability testing and for determination of leached components. The results showed that Retroplast, Geristore, and Ketac Fil increased cell proliferation, whereas Super-EBA decreased cell viability. The proliferative effect of Retroplast and Geristore increased with the eluting time (24 hours, 48 hours, and 72 hours), whereas the effects of the other materials did not significantly change. IRM and MTA did not affect cell viability. High-performance liquid chromatography analysis and atomic absorption spectroscopy showed that there was variation in the amount of leached components from the materials. Our results indicate that the reaction of cells to root-end filling materials varies considerably between materials.

  11. Viability, Advantages and Design Methodologies of M-Learning Delivery

    ERIC Educational Resources Information Center

    Zabel, Todd W.

    2010-01-01

    The purpose of this study was to examine the viability and principle design methodologies of Mobile Learning models in developing regions. Demographic and market studies were utilized to determine the viability of M-Learning delivery as well as best uses for such technologies and methods given socioeconomic and political conditions within the…

  12. Commercialization of cellular immunotherapies for cancer.

    PubMed

    Walker, Anthony; Johnson, Robert

    2016-04-15

    Successful commercialization of a cell therapy requires more than proving safety and efficacy to the regulators. The inherent complexity of cellular products delivers particular manufacturing, logistical and reimbursement hurdles that threaten commercial viability for any therapy with a less than spectacular clinical profile that truly changes the standard of care. This is particularly acute for autologous cell therapies where patients receive bespoke treatments manufactured from a sample of their own cells and where economies of scale, which play an important role in containing the production costs for small molecule and antibody therapeutics, are highly limited. Nevertheless, the promise of 'game-changing' efficacy, as exemplified by very high levels of complete responses in refractory haematological malignancies, has attracted capital investments on a vast scale, and the attendant pace of technology development provides promising indicators for future clinical and commercial success.

  13. Fatigue of cellular materials

    SciTech Connect

    Huang, J.S.; Lin, J.Y.

    1996-01-01

    The fatigue of cellular materials is analyzed using dimensional arguments. When the first unbroken cell wall ahead of the macrocrack tip fails after some cycles of loading, the macrocrack advances one cell diameter, giving the macrocrack growth rate of cellular materials. Paris law for microcrack propagation, Basquin law for high cycle fatigue and Coffin-Manson law for low cycle fatigue are employed in calculating the number of cycles to failure of the first unbroken cell wall ahead of the macrocrack tip. It is found that fatigue of cellular materials depends on cyclic stress intensity range, cell size, relative density and the fatigue parameters of the solid from which they are made. Theoretical modelling of fatigue of foams is compared to data in polymer foams; agreement is good.

  14. Irregular Cellular Learning Automata.

    PubMed

    Esnaashari, Mehdi; Meybodi, Mohammad Reza

    2015-08-01

    Cellular learning automaton (CLA) is a recently introduced model that combines cellular automaton (CA) and learning automaton (LA). The basic idea of CLA is to use LA to adjust the state transition probability of stochastic CA. This model has been used to solve problems in areas such as channel assignment in cellular networks, call admission control, image processing, and very large scale integration placement. In this paper, an extension of CLA called irregular CLA (ICLA) is introduced. This extension is obtained by removing the structure regularity assumption in CLA. Irregularity in the structure of ICLA is needed in some applications, such as computer networks, web mining, and grid computing. The concept of expediency has been introduced for ICLA and then, conditions under which an ICLA becomes expedient are analytically found.

  15. Epigenetics and Cellular Metabolism

    PubMed Central

    Xu, Wenyi; Wang, Fengzhong; Yu, Zhongsheng; Xin, Fengjiao

    2016-01-01

    Living eukaryotic systems evolve delicate cellular mechanisms for responding to various environmental signals. Among them, epigenetic machinery (DNA methylation, histone modifications, microRNAs, etc.) is the hub in transducing external stimuli into transcriptional response. Emerging evidence reveals the concept that epigenetic signatures are essential for the proper maintenance of cellular metabolism. On the other hand, the metabolite, a main environmental input, can also influence the processing of epigenetic memory. Here, we summarize the recent research progress in the epigenetic regulation of cellular metabolism and discuss how the dysfunction of epigenetic machineries influences the development of metabolic disorders such as diabetes and obesity; then, we focus on discussing the notion that manipulating metabolites, the fuel of cell metabolism, can function as a strategy for interfering epigenetic machinery and its related disease progression as well. PMID:27695375

  16. Origins of cellular geometry

    PubMed Central

    2011-01-01

    Cells are highly complex and orderly machines, with defined shapes and a startling variety of internal organizations. Complex geometry is a feature of both free-living unicellular organisms and cells inside multicellular animals. Where does the geometry of a cell come from? Many of the same questions that arise in developmental biology can also be asked of cells, but in most cases we do not know the answers. How much of cellular organization is dictated by global cell polarity cues as opposed to local interactions between cellular components? Does cellular structure persist across cell generations? What is the relationship between cell geometry and tissue organization? What ensures that intracellular structures are scaled to the overall size of the cell? Cell biology is only now beginning to come to grips with these questions. PMID:21880160

  17. The use of biodegradable polymers in design of cellular scaffolds.

    PubMed

    Orłowska, Joanna; Kurczewska, Urszula; Derwińska, Katarzyna; Orłowski, Wojciech; Orszulak-Michalak, Daria

    2015-03-05

    The objective of this work was to demonstrate the usage of biodegradable polymers, made of calcium alginate and dibutyrylchitin, in the design of cellular scaffolds having broad application in reconstructive therapy (dentistry, orthopedics). To visualize cells seeded on calcium alginate and dibutyrylchitin polymers DAPI staining of fibroblasts nuclei was used. The cytotoxicity of the materials and microscopic evaluation of the viability of seeded cells was tested with a PKH 67 fluorescent dye. To assess the cellular toxicity the proliferation of fibroblasts adjacent to the tested polymers was examined. The vitability of cells seeded on polymers was also evaluated by measuring the fluorescence intensity of calcein which binds only to live cells. The conducted experiments (DAPI and PKH 67 staining) show that the tested materials have a positive influence on cell adhesion crucial for wound healing - fibroblasts. The self-made dibutyrylchitin dressing do not cause the reduction of viability of cells seeded on them. The in vitro study illustrated the interactions between the tested materials, constructed of calcium alginate or dibutyrylchitin and mouse fibroblasts and proved their usefulness in the design of cellular scaffolds. Examined polymers turned out to be of great interest and promise for cellular scaffolds design.

  18. Ebselen Alters Mitochondrial Physiology and Reduces Viability of Rat Hippocampal Astrocytes

    PubMed Central

    Santofimia-Castaño, Patricia; Salido, Ginés M.

    2013-01-01

    The seleno-organic compound and radical scavenger ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) have been extensively employed as an anti-inflammatory and neuroprotective compound. However, its glutathione peroxidase activity at the expense of cellular thiols groups could underlie certain deleterious actions of the compound on cell physiology. In this study, we have analyzed the effect of ebselen on rat hippocampal astrocytes in culture. Cellular viability, the intracellular free-Ca2+ concentration ([Ca2+]c), the mitochondrial free-Ca2+ concentration ([Ca2+]m), and mitochondrial membrane potential (ψm) were analyzed. The caspase-3 activity was also assayed. Our results show that cell viability was reduced by treatment of cells with ebselen, depending on the concentration employed. In the presence of ebselen, we observed an initial transient increase in [Ca2+]c that was then followed by a progressive increase to an elevated plateau. We also observed a transient increase in [Ca2+]m in the presence of ebselen that returned toward a value over the prestimulation level. The compound induced depolarization of ψm and altered the permeability of the mitochondrial membrane. Additionally, a disruption of the mitochondrial network was observed. Finally, we did not detect changes in caspase-3 activation in response to ebselen treatment. Collectively, these data support the likelihood of ebselen, depending on the concentration employed, reduces viability of rat hippocampal astrocytes via its action on the mitochondrial activity. These may be early effects that do not involve caspase-3 activation. We conclude that, depending on the concentration used, ebselen might exert deleterious actions on astrocyte physiology that could compromise cell function. PMID:23496767

  19. The Location of Viability Genes within a Testcross Framework.

    PubMed

    Chapco, W

    1972-02-01

    The maximum likelihood method is applied to the problem of estimating the positions and effects of viability genes. Whenever testcross linkage data indicate the presence of differential viability, it is hypothesized that there exists one viability gene between each marker. Estimation is possible only for two-point data since the number of independent expectation expressions is less than the number of parameters for three or more markers. It is pointed out that within the two-point testcross system, it is impossible to distinguish between pleiotropic effects of the marker genes and the effect of a middle viability gene, if existent. The methods outlined will be useful in their application to experiments specifically designed to locate induced viability genes.

  20. Assessment of tissue viability by polarization spectroscopy

    NASA Astrophysics Data System (ADS)

    Nilsson, G.; Anderson, C.; Henricson, J.; Leahy, M.; O'Doherty, J.; Sjöberg, F.

    2008-09-01

    A new and versatile method for tissue viability imaging based on polarization spectroscopy of blood in superficial tissue structures such as the skin is presented in this paper. Linearly polarized light in the visible wavelength region is partly reflected directly by the skin surface and partly diffusely backscattered from the dermal tissue matrix. Most of the directly reflected light preserves its polarization state while the light returning from the deeper tissue layers is depolarized. By the use of a polarization filter positioned in front of a sensitive CCD-array, the light directly reflected from the tissue surface is blocked, while the depolarized light returning from the deeper tissue layers reaches the detector array. By separating the colour planes of the detected image, spectroscopic information about the amount of red blood cells (RBCs) in the microvascular network of the tissue under investigation can be derived. A theory that utilizes the differences in light absorption of RBCs and bloodless tissue in the red and green wavelength region forms the basis of an algorithm for displaying a colour coded map of the RBC distribution in a tissue. Using a fluid model, a linear relationship (cc. = 0.99) between RBC concentration and the output signal was demonstrated within the physiological range 0-4%. In-vivo evaluation using transepidermal application of acetylcholine by the way of iontophoresis displayed the heterogeneity pattern of the vasodilatation produced by the vasoactive agent. Applications of this novel technology are likely to be found in drug and skin care product development as well as in the assessment of skin irritation and tissue repair processes and even ultimately in a clinic case situation.

  1. The 37/67kDa laminin receptor (LR) inhibitor, NSC47924, affects 37/67kDa LR cell surface localization and interaction with the cellular prion protein

    PubMed Central

    Sarnataro, Daniela; Pepe, Anna; Altamura, Gennaro; De Simone, Imma; Pesapane, Ada; Nitsch, Lucio; Montuori, Nunzia; Lavecchia, Antonio; Zurzolo, Chiara

    2016-01-01

    The 37/67 kDa laminin receptor (LR) is a non-integrin protein, which binds both laminin-1 of the extracellular matrix and prion proteins, that hold a central role in prion diseases. The 37/67 kDa LR has been identified as interactor for the prion protein (PrPC) and to be required for pathological PrP (PrPSc) propagation in scrapie-infected neuronal cells, leading to the possibility that 37/67 kDa LR-PrPC interaction is related to the pathogenesis of prion diseases. A relationship between 37/67 kDa LR and PrPC in the presence of specific LR inhibitor compounds has not been investigated yet. We have characterized the trafficking of 37/67 kDa LR in both neuronal and non-neuronal cells, finding the receptor on the cell surface and nuclei, and identified the 67 kDa LR as the almost exclusive isoform interacting with PrPC. Here, we show that the treatment with the 37/67 kDa LR inhibitor, NSC47924, affects both the direct 37/67 kDa LR-PrPC interaction in vitro and the formation of the immunocomplex in live cells, inducing a progressive internalization of 37/67 kDa LR and stabilization of PrPC on the cell surface. These data reveal NSC47924 as a useful tool to regulate PrPC and 37/67 kDa LR trafficking and degradation, representing a novel small molecule to be tested against prion diseases. PMID:27071549

  2. The viability of photovoltaics on the Martian surface

    NASA Technical Reports Server (NTRS)

    Gaier, James R.; Perez-Davis, Marla E.

    1994-01-01

    The viability of photovoltaics (PV) on the Martian surface may be determined by their ability to withstand significant degradation in the Martian environment. Probably the greatest threat is posed by fine dust particles which are continually blown about the surface of the planet. In an effort to determine the extent of the threat, and to investigate some abatement strategies, a series of experiments were conducted in the Martian Surface Wind Tunnel (MARSWIT) at NASA Ames Research Center. The effects of dust composition, particle size, wind velocity, angle of attack, and protective coatings on the transmittance of light through PV coverglass were determined. Both initially clear and initially dusted samples were subjected both to clear winds and simulated dust storms in the MARSWIT. It was found that wind velocity, particle size, and angle of attack are important parameters affecting occlusion of PV surfaces, while dust composition and protective coatings were not. Neither induced turbulence nor direct current biasing up to 200 volts were effective abatement techniques. Abrasion diffused the light impinging on the PV cells, but did not reduce total coverglass transmittance by more than a few percent.

  3. Chemicals and lemon essential oil effect on Alicyclobacillus acidoterrestris viability

    PubMed Central

    Maldonado, Maria Cristina; Aban, Marina Paola; Navarro, Antonio Roberto

    2013-01-01

    Alicyclobacillus acidoterrestris is considered to be one of the important target microorganisms in the quality control of acidic canned foods. There is an urgent need to develop a suitable method for inhibiting or controlling the germination and outgrowth of A.acidoterrestris in acidic drinks. The aim of this work was to evaluate the chemicals used in the lemon industry (sodium benzoate, potassium sorbate), and lemon essential oil as a natural compound, against a strain of A.acidoterrestris in MEB medium and in lemon juice concentrate. The results pointed out that sodium benzoate (500–1000–2000 ppm) and lemon essential oil (0.08–0.12–0.16%) completely inhibited the germination of A. acidoterrestris spores in MEB medium and LJC for 11 days. Potassium sorbate (600–1200 ppm) was more effective to inhibit the growth of the microbial target in lemon juice than in MEB medium. The effect of sodium benzoate, potassium sorbate and essential oil was sporostatic in MEB and LJC as they did not affect spore viability. PMID:24688502

  4. Using the pollen viability and morphology for fluoride pollution biomonitoring.

    PubMed

    Malayeri, Behrooz Eshghi; Noori, Mitra; Jafari, Mehrana

    2012-06-01

    The methods using plants for biomonitoring of air and soil quality are simple, cheap, and fast and can supplement the classical physicochemical methods. In this study, biological pollen characterization of some collected legume species from an aluminum smelter area in Iran (IRALCO) was carried out to determine the actual value of pollen as a bioindicator of the effects of soil and atmospheric pollution. Young buds and flowers of six legumes (Cercis siliquastrum L., Medicago sativa L., Robinia pseudoacacia L., Melilotus officinalis (L.) lam, Trifolium repens L., and Sophora alopecuroides L.) in polluted and control plants were removed and compared. Studies of light and electron microscopic preparation showed some abnormalities during pollen development in affect of fluoride pollution. The viability of pollen grains estimated by staining with acetocarmine shows sharp differences in smearing advanced pollen grains from abnormal ones. Except M. officinalis, the pollen grains of C. siliquastrum, M. sativa, R. pseudoacacia, T. repens, and S. alopecuroides in polluted areas showed light, partial, or no staining with acetocarmine, whereas almost all of the control ones clearly stained. Observation of the pollen grains by light microscopy and scanning electron microscopy showed the significant effect of fluoride on shapes and sizes of pollen grains. The stimulation and inhibition of these pollen characteristics depend on the pollen species as well as on the pollutant and its concentration. Therefore, pollen grains provide essential information on biological impact of pollutants and they are good candidates for biomonitoring the atmospheric and edaphic pollutions.

  5. Chemicals and lemon essential oil effect on Alicyclobacillus acidoterrestris viability.

    PubMed

    Maldonado, Maria Cristina; Aban, Marina Paola; Navarro, Antonio Roberto

    2013-12-01

    Alicyclobacillus acidoterrestris is considered to be one of the important target microorganisms in the quality control of acidic canned foods. There is an urgent need to develop a suitable method for inhibiting or controlling the germination and outgrowth of A.acidoterrestris in acidic drinks. The aim of this work was to evaluate the chemicals used in the lemon industry (sodium benzoate, potassium sorbate), and lemon essential oil as a natural compound, against a strain of A.acidoterrestris in MEB medium and in lemon juice concentrate. The results pointed out that sodium benzoate (500-1000-2000 ppm) and lemon essential oil (0.08-0.12-0.16%) completely inhibited the germination of A. acidoterrestris spores in MEB medium and LJC for 11 days. Potassium sorbate (600-1200 ppm) was more effective to inhibit the growth of the microbial target in lemon juice than in MEB medium. The effect of sodium benzoate, potassium sorbate and essential oil was sporostatic in MEB and LJC as they did not affect spore viability.

  6. Silver Nanoparticles Decrease the Viability of Cryptosporidium parvum Oocysts

    PubMed Central

    Gaiser, Birgit K.; Bhandari, Bidha; Bartley, Paul M.; Katzer, Frank; Bridle, Helen

    2015-01-01

    Oocysts of the waterborne protozoan parasite Cryptosporidium parvum are highly resistant to chlorine disinfection. We show here that both silver nanoparticles (AgNPs) and silver ions significantly decrease oocyst viability, in a dose-dependent manner, between concentrations of 0.005 and 500 μg/ml, as assessed by an excystation assay and the shell/sporozoite ratio. For percent excystation, the results are statistically significant for 500 μg/ml of AgNPs, with reductions from 83% for the control to 33% with AgNPs. For Ag ions, the results were statistically significant at 500 and 5,000 μg/ml, but the percent excystation values were reduced only to 66 and 62%, respectively, from 86% for the control. The sporozoite/shell ratio was affected to a greater extent following AgNP exposure, presumably because sporozoites are destroyed by interaction with NPs. We also demonstrated via hyperspectral imaging that there is a dual mode of interaction, with Ag ions entering the oocyst and destroying the sporozoites while AgNPs interact with the cell wall and, at high concentrations, are able to fully break the oocyst wall. PMID:26497464

  7. An optimized SYBR Green I/PI assay for rapid viability assessment and antibiotic susceptibility testing for Borrelia burgdorferi.

    PubMed

    Feng, Jie; Wang, Ting; Zhang, Shuo; Shi, Wanliang; Zhang, Ying

    2014-01-01

    Lyme disease caused by Borrelia burgdorferi is the most common tick-borne disease in the US and Europe. Unlike most bacteria, measurements of growth and viability of B. burgdorferi are challenging. The current B. burgdorferi viability assays based on microscopic counting and PCR are cumbersome and tedious and cannot be used in a high throughput format. Here, we evaluated several commonly used viability assays including MTT and XTT assays, fluorescein diacetate assay, Sytox Green/Hoechst 33342 assay, the commercially available LIVE/DEAD BacLight assay, and SYBR Green I/PI assay by microscopic counting and by automated 96-well plate reader for rapid viability assessment of B. burgdorferi. We found that the optimized SYBR Green I/PI assay based on green to red fluorescence ratio is superior to all the other assays for measuring the viability of B. burgdorferi in terms of sensitivity, accuracy, reliability, and speed in automated 96-well plate format and in comparison with microscopic counting. The BSK-H medium which produced a high background for the LIVE/DEAD BacLight assay did not affect the SYBR Green I/PI assay, and the viability of B. burgdorferi culture could be directly measured using a microtiter plate reader. The SYBR Green I/PI assay was found to reliably assess the viability of planktonic as well as biofilm B. burgdorferi and could be used as a rapid antibiotic susceptibility test. Thus, the SYBR Green I/PI assay provides a more sensitive, rapid and convenient method for evaluating viability and antibiotic susceptibility of B. burgdorferi and can be used for high-throughput drug screens.

  8. Primate-specific oestrogen-responsive long non-coding RNAs regulate proliferation and viability of human breast cancer cells

    PubMed Central

    Lin, Chin-Yo; Dachet, Fabien; Cai, Juan; Ju, Donghong; Goldstone, Amanda; Wood, Emily J.; Liu, Ka; Jia, Hui; Kosir, Mary A.; Thepsuwan, Pattaraporn

    2016-01-01

    Long non-coding RNAs (lncRNAs) are transcripts of a recently discovered class of genes which do not code for proteins. LncRNA genes are approximately as numerous as protein-coding genes in the human genome. However, comparatively little remains known about lncRNA functions. We globally interrogated changes in the lncRNA transcriptome of oestrogen receptor positive human breast cancer cells following treatment with oestrogen, and identified 127 oestrogen-responsive lncRNAs. Consistent with the emerging evidence that most human lncRNA genes lack homologues outside of primates, our evolutionary analysis revealed primate-specific lncRNAs downstream of oestrogen signalling. We demonstrate, using multiple functional assays to probe gain- and loss-of-function phenotypes in two oestrogen receptor positive human breast cancer cell lines, that two primate-specific oestrogen-responsive lncRNAs identified in this study (the oestrogen-repressed lncRNA BC041455, which reduces cell viability, and the oestrogen-induced lncRNA CR593775, which increases cell viability) exert previously unrecognized functions in cell proliferation and growth factor signalling pathways. The results suggest that oestrogen-responsive lncRNAs are capable of altering the proliferation and viability of human breast cancer cells. No effects on cellular phenotypes were associated with control transfections. As heretofore unappreciated components of key signalling pathways in cancers, including the MAP kinase pathway, lncRNAs hence represent a novel mechanism of action for oestrogen effects on cellular proliferation and viability phenotypes. This finding warrants further investigation in basic and translational studies of breast and potentially other types of cancers, has broad relevance to lncRNAs in other nuclear hormone receptor pathways, and should facilitate exploiting and targeting these cell viability modulating lncRNAs in post-genomic therapeutics. PMID:28003470

  9. The New Cellular Immunology

    ERIC Educational Resources Information Center

    Claman, Henry N.

    1973-01-01

    Discusses the nature of the immune response and traces many of the discoveries that have led to the present state of knowledge in immunology. The new cellular immunology is directing its efforts toward improving health by proper manipulation of the immune mechanisms of the body. (JR)

  10. Cellular genetic therapy.

    PubMed

    Del Vecchio, F; Filareto, A; Spitalieri, P; Sangiuolo, F; Novelli, G

    2005-01-01

    Cellular genetic therapy is the ultimate frontier for those pathologies that are consequent to a specific nonfunctional cellular type. A viable cure for there kinds of diseases is the replacement of sick cells with healthy ones, which can be obtained from the same patient or a different donor. In fact, structures can be corrected and strengthened with the introduction of undifferentiated cells within specific target tissues, where they will specialize into the desired cellular types. Furthermore, consequent to the recent results obtained with the transdifferentiation experiments, a process that allows the in vitro differentiation of embryonic and adult stem cells, it has also became clear that many advantages may be obtained from the use of stem cells to produce drugs, vaccines, and therapeutic molecules. Since stem cells can sustain lineage potentials, the capacity for differentiation, and better tolerance for the introduction of exogenous genes, they are also considered as feasible therapeutic vehicles for gene therapy. In fact, it is strongly believed that the combination of cellular genetic and gene therapy approaches will definitely allow the development of new therapeutic strategies as well as the production of totipotent cell lines to be used as experimental models for the cure of genetic disorders.

  11. Cellular senescence: when bad things happen to good cells.

    PubMed

    Campisi, Judith; d'Adda di Fagagna, Fabrizio

    2007-09-01

    Cells continually experience stress and damage from exogenous and endogenous sources, and their responses range from complete recovery to cell death. Proliferating cells can initiate an additional response by adopting a state of permanent cell-cycle arrest that is termed cellular senescence. Understanding the causes and consequences of cellular senescence has provided novel insights into how cells react to stress, especially genotoxic stress, and how this cellular response can affect complex organismal processes such as the development of cancer and ageing.

  12. Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa.

    PubMed

    Aparicio, I M; Bragado, M J; Gil, M C; Garcia-Herreros, M; Gonzalez-Fernandez, L; Tapia, J A; Garcia-Marin, L J

    2007-08-01

    Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.

  13. Nanodiamonds on tetrahedral amorphous carbon significantly enhance dopamine detection and cell viability.

    PubMed

    Peltola, Emilia; Wester, Niklas; Holt, Katherine B; Johansson, Leena-Sisko; Koskinen, Jari; Myllymäki, Vesa; Laurila, Tomi

    2017-02-15

    We hypothesize that by using integrated carbon nanostructures on tetrahedral amorphous carbon (ta-C), it is possible to take the performance and characteristics of these bioelectrodes to a completely new level. The integrated carbon electrodes were realized by combining nanodiamonds (NDs) with ta-C thin films coated on Ti-coated Si-substrates. NDs were functionalized with mixture of carboxyl and amine groups NDandante or amine NDamine, carboxyl NDvox or hydroxyl groups NDH and drop-casted or spray-coated onto substrate. By utilizing these novel structures we show that (i) the detection limit for dopamine can be improved by two orders of magnitude [from 10µM to 50nM] in comparison to ta-C thin film electrodes and (ii) the coating method significantly affects electrochemical properties of NDs and (iii) the ND coatings selectively promote cell viability. NDandante and NDH showed most promising electrochemical properties. The viability of human mesenchymal stem cells and osteoblastic SaOS-2 cells was increased on all ND surfaces, whereas the viability of mouse neural stem cells and rat neuroblastic cells was improved on NDandante and NDH and reduced on NDamine and NDvox. The viability of C6 cells remained unchanged, indicating that these surfaces will not cause excess gliosis. In summary, we demonstrated here that by using functionalized NDs on ta-C thin films we can significantly improve sensitivity towards dopamine as well as selectively promote cell viability. Thus, these novel carbon nanostructures provide an interesting concept for development of various in vivo targeted sensor solutions.

  14. Preincubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function upon thawing.

    PubMed

    Terry, Claire; Dhawan, Anil; Mitry, Ragai R; Lehec, Sharon C; Hughes, Robin D

    2005-12-01

    Cryopreservation of human hepatocytes is important for the treatment of liver disease by hepatocyte transplantation and also for the use of hepatocytes as an in vitro model of the liver. One factor in the success of cryopreservation is the quality of cells before freezing. Preincubation of hepatocytes with cytoprotective compounds to allow recovery from the isolation process prior to cryopreservation, such as those that will boost cellular adenosine triphosphate (ATP) content or antioxidants, may improve the viability and function of cells upon thawing. Rat hepatocytes were used to investigate the effects of preincubation with 10 compounds: precursors (glucose, fructose, glutathione, and S-adenosyl-L-methionine), antioxidants (ascorbic acid and alpha-lipoic acid), and compounds with multiple effects (N-acetylcysteine, pentoxifylline, prostaglandin E(1), and tauroursodeoxycholic acid). Human hepatocytes were then used to investigate 5 of the original 10 compounds (glucose, fructose, alpha-lipoic acid, S-adenosyl-L-methionine, and pentoxifylline). Glucose preincubation (100-300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the viability and reduced lactate dehydrogenase (LDH) leakage of human hepatocytes. Fructose preincubation (100-300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the attachment efficiency of human hepatocytes. alpha-lipoic acid preincubation (0.5-5 mM) improved the viability and attachment efficiency of both rat and human hepatocytes. At a concentration of 2.5 mM alpha-lipoic acid also improved the albumin production of human hepatocytes. In conclusion, preincubation of hepatocytes prior to cryopreservation can improve the viability and function of thawed cells and may provide a method of obtaining better-quality cryopreserved hepatocytes for transplantation.

  15. A microfluidic-based cell encapsulation platform to achieve high long-term cell viability in photopolymerized PEGNB hydrogel microspheres.

    PubMed

    Jiang, Zhongliang; Xia, Bingzhao; McBride, Ralph; Oakey, John

    2017-01-07

    Cell encapsulation within photopolymerized polyethylene glycol (PEG)-based hydrogel scaffolds has been demonstrated as a robust strategy for cell delivery, tissue engineering, regenerative medicine, and developing in vitro platforms to study cellular behavior and fate. Strategies to achieve spatial and temporal control over PEG hydrogel mechanical properties, chemical functionalization, and cytocompatibility have advanced considerably in recent years. Recent microfluidic technologies have enabled the miniaturization of PEG hydrogels, thus enabling the fabrication of miniaturized cell-laden vehicles. However, rapid oxygen diffusive transport times on the microscale dramatically inhibit chain growth photopolymerization of polyethylene glycol diacrylate (PEGDA), thus decreasing the viability of cells encapsulated within these microstructures. Another promising PEG-based scaffold material, PEG norbornene (PEGNB), is formed by a step-growth photopolymerization and is not inhibited by oxygen. PEGNB has also been shown to be more cytocompatible than PEGDA and allows for orthogonal addition reactions. The step-growth kinetics, however, are slow and therefore challenging to fully polymerize within droplets flowing through microfluidic devices. Here, we describe a microfluidic-based droplet fabrication platform that generates consistently monodisperse cell-laden water-in-oil emulsions. Microfluidically generated PEGNB droplets are collected and photopolymerized under UV exposure in bulk emulsions. In this work, we compare this microfluidic-based cell encapsulation platform with a vortex-based method on the basis of microgel size, uniformity, post-encapsulation cell viability and long-term cell viability. Several factors that influence post-encapsulation cell viability were identified. Finally, long-term cell viability achieved by this platform was compared to a similar cell encapsulation platform using PEGDA. We show that this PEGNB microencapsulation platform is capable of

  16. Cellular ageing mechanisms in osteoarthritis.

    PubMed

    Sacitharan, P K; Vincent, T L

    2016-08-01

    Age is the strongest independent risk factor for the development of osteoarthritis (OA) and for many years this was assumed to be due to repetitive microtrauma of the joint surface over time, the so-called 'wear and tear' arthritis. As our understanding of OA pathogenesis has become more refined, it has changed our appreciation of the role of ageing on disease. Cartilage breakdown in disease is not a passive process but one involving induction and activation of specific matrix-degrading enzymes; chondrocytes are exquisitely sensitive to changes in the mechanical, inflammatory and metabolic environment of the joint; cartilage is continuously adapting to these changes by altering its matrix. Ageing influences all of these processes. In this review, we will discuss how ageing affects tissue structure, joint use and the cellular metabolism. We describe what is known about pathways implicated in ageing in other model systems and discuss the potential value of targeting these pathways in OA.

  17. Mitotane reduces human and mouse ACTH-secreting pituitary cell viability and function.

    PubMed

    Gentilin, Erica; Tagliati, Federico; Terzolo, Massimo; Zoli, Matteo; Lapparelli, Marcello; Minoia, Mariella; Ambrosio, Maria Rosaria; Degli Uberti, Ettore C; Zatelli, Maria Chiara

    2013-09-01

    Medical therapy for Cushing's disease (CD) is currently based on agents mainly targeting adrenocortical function. Lately, pituitary-directed drugs have been developed, with limited efficacy. Mitotane, a potent adrenolytic drug, has been recently investigated for the treatment of CD, but the direct pituitary effects have not been clarified so far. The aim of our study was to investigate whether mitotane may affect corticotroph function and cell survival in the mouse pituitary cell line AtT20/D16v-F2 and in the primary cultures of human ACTH-secreting pituitary adenomas, as an in vitro model of pituitary corticotrophs. We found that in the AtT20/D16v-F2 cell line and in primary cultures, mitotane reduces cell viability by inducing caspase-mediated apoptosis and reduces ACTH secretion. In the AtT20/D16v-F2 cell line, mitotane reduces Pomc expression and blocks the stimulatory effects of corticotropin-releasing hormone on cell viability, ACTH secretion, and Pomc expression. These effects were apparent at mitotane doses greater than those usually necessary for reducing cortisol secretion in Cushing's syndrome, but still in the therapeutic window for adrenocortical carcinoma treatment. In conclusion, our results demonstrate that mitotane affects cell viability and function of human and mouse ACTH-secreting pituitary adenoma cells. These data indicate that mitotane could have direct pituitary effects on corticotroph cells.

  18. Stable cellular senescence is associated with persistent DDR activation.

    PubMed

    Fumagalli, Marzia; Rossiello, Francesca; Mondello, Chiara; d'Adda di Fagagna, Fabrizio

    2014-01-01

    The DNA damage response (DDR) is activated upon DNA damage generation to promote DNA repair and inhibit cell cycle progression in the presence of a lesion. Cellular senescence is a permanent cell cycle arrest characterized by persistent DDR activation. However, some reports suggest that DDR activation is a feature only of early cellular senescence that is then lost with time. This challenges the hypothesis that cellular senescence is caused by persistent DDR activation. To address this issue, we studied DDR activation dynamics in senescent cells. Here we show that normal human fibroblasts retain DDR markers months after replicative senescence establishment. Consistently, human fibroblasts from healthy aged donors display markers of DDR activation even three years in culture after entry into replicative cellular senescence. However, by extending our analyses to different human cell strains, we also observed an apparent DDR loss with time following entry into cellular senescence. This though correlates with the inability of these cell strains to survive in culture upon replicative or irradiation-induced cellular senescence. We propose a model to reconcile these results. Cell strains not suffering the prolonged in vitro culture stress retain robust DDR activation that persists for years, indicating that under physiological conditions persistent DDR is causally involved in senescence establishment and maintenance. However, cell strains unable to maintain cell viability in vitro, due to their inability to cope with prolonged cell culture-associated stress, show an only-apparent reduction in DDR foci which is in fact due to selective loss of the most damaged cells.

  19. Combinatorial approaches to evaluate nanodiamond uptake and induced cellular fate

    PubMed Central

    Eldawud, Reem; Reitzig, Manuela; Opitz, Jörg; Rojansakul, Yon; Jiang, Wenjuan; Nangia, Shikha; Dinu, Cerasela Zoica

    2016-01-01

    Nanodiamonds (NDs) are an emerging class of engineered nanomaterials that hold great promise for the next generation of bionanotechnological products to be used for drug and gene delivery, or for bio-imaging and biosensing. Previous studies have shown that upon their cellular uptake, NDs exhibit high biocompatibility in various in vitro and in vivo set-ups. Herein we hypothesized that the increased NDs biocompatibility is a result of minimum membrane perturbations and their reduced ability to induce disruption or damage during cellular translocation. Using multi-scale combinatorial approaches that simulate ND-membrane interactions, we correlated NDs real-time cellular uptake and kinetics with the ND-induced membrane fluctuations to derive energy requirements for the uptake to occur. Our discrete and real-time analyses showed that the majority of NDs internalization occurs within 2 h of cellular exposure, however, with no effects on cellular viability, proliferation or cellular behavior. Furthermore, our simulation analyses using coarse-grained models identified key changes in the energy profile, membrane deformation and recovery time, all functions of the average ND or ND-based agglomerate size. Understanding the mechanisms responsible for ND-cell membrane interactions could possibly advance their implementation in various biomedical applications. PMID:26820775

  20. Combinatorial approaches to evaluate nanodiamond uptake and induced cellular fate.

    PubMed

    Eldawud, Reem; Reitzig, Manuela; Opitz, Jörg; Rojansakul, Yon; Jiang, Wenjuan; Nangia, Shikha; Dinu, Cerasela Zoica

    2016-02-26

    Nanodiamonds (NDs) are an emerging class of engineered nanomaterials that hold great promise for the next generation of bionanotechnological products to be used for drug and gene delivery, or for bio-imaging and biosensing. Previous studies have shown that upon their cellular uptake, NDs exhibit high biocompatibility in various in vitro and in vivo set-ups. Herein we hypothesized that the increased NDs biocompatibility is a result of minimum membrane perturbations and their reduced ability to induce disruption or damage during cellular translocation. Using multi-scale combinatorial approaches that simulate ND-membrane interactions, we correlated NDs real-time cellular uptake and kinetics with the ND-induced membrane fluctuations to derive energy requirements for the uptake to occur. Our discrete and real-time analyses showed that the majority of NDs internalization occurs within 2 h of cellular exposure, however, with no effects on cellular viability, proliferation or cellular behavior. Furthermore, our simulation analyses using coarse-grained models identified key changes in the energy profile, membrane deformation and recovery time, all functions of the average ND or ND-based agglomerate size. Understanding the mechanisms responsible for ND-cell membrane interactions could possibly advance their implementation in various biomedical applications.

  1. Combinatorial approaches to evaluate nanodiamond uptake and induced cellular fate

    NASA Astrophysics Data System (ADS)

    Eldawud, Reem; Reitzig, Manuela; Opitz, Jörg; Rojansakul, Yon; Jiang, Wenjuan; Nangia, Shikha; Zoica Dinu, Cerasela

    2016-02-01

    Nanodiamonds (NDs) are an emerging class of engineered nanomaterials that hold great promise for the next generation of bionanotechnological products to be used for drug and gene delivery, or for bio-imaging and biosensing. Previous studies have shown that upon their cellular uptake, NDs exhibit high biocompatibility in various in vitro and in vivo set-ups. Herein we hypothesized that the increased NDs biocompatibility is a result of minimum membrane perturbations and their reduced ability to induce disruption or damage during cellular translocation. Using multi-scale combinatorial approaches that simulate ND-membrane interactions, we correlated NDs real-time cellular uptake and kinetics with the ND-induced membrane fluctuations to derive energy requirements for the uptake to occur. Our discrete and real-time analyses showed that the majority of NDs internalization occurs within 2 h of cellular exposure, however, with no effects on cellular viability, proliferation or cellular behavior. Furthermore, our simulation analyses using coarse-grained models identified key changes in the energy profile, membrane deformation and recovery time, all functions of the average ND or ND-based agglomerate size. Understanding the mechanisms responsible for ND-cell membrane interactions could possibly advance their implementation in various biomedical applications.

  2. The combined influence of sub-optimal temperature and salinity on the in vitro viability of Perkinsus marinus, a protistan parasite of the eastern oyster Crassostrea virginica

    USGS Publications Warehouse

    La Peyre, M.K.; Casas, S.M.; Gayle, W.; La Peyre, Jerome F.

    2010-01-01

    Perkinsus marinus is a major cause of mortality in eastern oysters along the Gulf of Mexico and Atlantic coasts. It is also well documented that temperature and salinity are the primary environmental factors affecting P. marinus viability and proliferation. However, little is known about the effects of combined sub-optimal temperatures and salinities on P. marinus viability. This in vitro study examined those effects by acclimating P. marinus at three salinities (7, 15, 25. ppt) to 10 ??C to represent the lowest temperatures generally reached in the Gulf of Mexico, and to 2 ??C to represent the lowest temperatures reached along the mid-Atlantic coasts and by measuring changes in cell viability and density on days 1, 30, 60 and 90 following acclimation. Cell viability and density were also measured in 7. ppt cultures acclimated to each temperature and then transferred to 3.5. ppt. The largest decreases in cell viability occurred only with combined low temperature and salinity, indicating that there is clearly a synergistic effect. The largest decreases in cell viability occurred only with both low temperature and salinity after 30. days (3.5. ppt, 2 ??C: 0% viability), 60. days (3.5. ppt, 10 ??C: 0% viability) and 90. days (7. ppt, 2 ??C: 0.6 ?? 0.7%; 7. ppt, 10 ??C: 0.2 ?? 0.2%). ?? 2010 .

  3. Effect of cellular inositol content on ethanol tolerance of Saccharomyces cerevisiae in sake brewing.

    PubMed

    Furukawa, Keiji; Kitano, Hideyuki; Mizoguchi, Haruhiko; Hara, Shodo

    2004-01-01

    The effect of cellular inositol content on the ethanol tolerance of sake yeast was investigated. In a static culture of strain K901 in a synthetic medium, when cells were grown in the presence of inositol in limited amount (L-cells), the inositol content of cells decreased by one-third that of cells grown in the presence of inositol in sufficient amount (H-cells). L-cells exhibited a higher death rate constant than H-cells in the presence of 12-20% ethanol, while no difference in specific ethanol production rate in the presence of 0-18% ethanol between the two cell types was observed. L-cells leaked more intracellular components, such as nucleotides, phosphate and potassium, in the presence of ethanol than H-cells. L-cells exhibited a lower intracellular pH value than H-cells, which represented the lowering of cell vitality by the decrease in H(+) extrusion activity. Furthermore, the plasma membrane H(+)-ATPase activity of L-cells was approximately one-half of that of H-cells. Therefore, it was considered that the decrease in viability in the presence of ethanol due to inositol limitation results from the lowering of H(+)-ATPase activity, which maintains the permeability barrier of the yeast membrane, ensuring the homeostasis of ions in the cytoplasm of yeast cells. It is assumed that the lowering of H(+)-ATPase activity due to inositol limitation is caused by the change in lipid environment of the enzyme, which is affected by inositol-containing glycerophospholipids such as phosphatidylinositol (PI), because in the PI-saturated mixed micellar assay system, the difference in H(+)-ATPase activity between L- and H-cells disappeared. In the early stage of sake mash, inositol limitation lowers the ethanol tolerance due to the decrease in H(+)-ATPase activity as in static culture. In the final stage of sake mash, the disruption of the ino1 gene responsible for inositol synthesis, resulted in a decrease in cell density. Furthermore, the ino1 disruptant, which was not

  4. Phenylboronic acid selectively inhibits human prostate and breast cancer cell migration and decreases viability.

    PubMed

    Bradke, Tiffany M; Hall, Casey; Carper, Stephen W; Plopper, George E

    2008-01-01

    We compared the in vitro effect of boric acid (BA) versus phenylboronic acid (PBA) on the migration of prostate and breast cancer cell lines and non-tumorigenic cells from the same tissues. Treatment at 24 hours with BA (< or =500 microM) did not inhibit chemotaxis on fibronectin in any cell line. However, treatment over the same time course with concentrations of PBA as low as 1 muM significantly inhibited cancer cell migration without effecting non-tumorigenic cell lines. The compounds did not affect cell adhesion or viability at 24 hours but did alter morphology; both decreased cancer cell viability at eight days. These results suggest that PBA is more potent than BA in targeting the metastatic and proliferative properties of cancer cells.

  5. The mouse dead-end gene isoform α is necessary for germ cell and embryonic viability

    PubMed Central

    Bhattacharya, Chitralekha; Aggarwal, Sita; Zhu, Rui; Kumar, Madhu; Zhao, Ming; Meistrich, Marvin L.; Matin, Angabin

    2007-01-01

    Inactivation of the dead-end (Dnd1) gene in the Ter mouse strain results in depletion of primordial germ cells (PGCs) so that mice become sterile. However, on the 129 mouse strain background, loss of Dnd1 also increases testicular germ cell tumor incidence in parallel to PGC depletion. We report that inactivation of Dnd1 also affects embryonic viability in the 129 strain. Mouse Dnd1 encodes two protein isoforms, DND1-isoform α (DND1- α) and DND1-isoform β (DND1-β). Using isoform specific antibodies, we determined DND1-α is expressed in embryos and embryonic gonads whereas DND1-β expression is restricted to germ cells of the adult testis. Our data implicates DND1-α isoform to be necessary for germ cell viability and therefore its loss in Ter mice results in PGC depletion, germ cell tumor development and partial embryonic lethality in the 129 strain. PMID:17291453

  6. The market viability of nuclear hydrogen technologies.

    SciTech Connect

    Botterud, A.; Conzelmann, G.; Petri, M. C.; Yildiz, B.

    2007-04-06

    The Department of Energy Office of Nuclear Energy is supporting system studies to gain a better understanding of nuclear power's potential role in a hydrogen economy and what hydrogen production technologies show the most promise. This assessment includes identifying commercial hydrogen applications and their requirements, comparing the characteristics of nuclear hydrogen systems to those market requirements, evaluating nuclear hydrogen configuration options within a given market, and identifying the key drivers and thresholds for market viability of nuclear hydrogen options. One of the objectives of the current analysis phase is to determine how nuclear hydrogen technologies could evolve under a number of different futures. The outputs of our work will eventually be used in a larger hydrogen infrastructure and market analysis conducted for DOE-EE using a system-level market simulation tool now underway. This report expands on our previous work by moving beyond simple levelized cost calculations and looking at profitability, risk, and uncertainty from an investor's perspective. We analyze a number of technologies and quantify the value of certain technology and operating characteristics. Our model to assess the profitability of the above technologies is based on Real Options Theory and calculates the discounted profits from investing in each of the production facilities. We use Monte-Carlo simulations to represent the uncertainty in hydrogen and electricity prices. The model computes both the expected value and the distribution of discounted profits from a production plant. We also quantify the value of the option to switch between hydrogen and electricity production in order to maximize investor profits. Uncertainty in electricity and hydrogen prices can be represented with two different stochastic processes: Geometric Brownian Motion (GBM) and Mean Reversion (MR). Our analysis finds that the flexibility to switch between hydrogen and electricity leads to

  7. Exploring the frontier between life and death in Escherichia coli: evaluation of different viability markers in live and heat- or UV-killed cells.

    PubMed

    Villarino, A; Bouvet, O M; Regnault, B; Martin-Delautre, S; Grimont PAD

    2000-11-01

    A number of methods have been proposed to assess the viability of cells without culture. Each method is based on criteria that reflect different levels of cellular integrity or functionality. As a consequence, the interpretation of viability is often ambiguous. The purposes of this work were to evaluate the capacity of current viability markers to distinguish between live and dead Escherichia coli K-12 cells. Methods that assess 'viability' by the demonstration of metabolic activities (esterase activity, active electron transport chain, transport of glucose), cellular integrity (membrane integrity, presence of nucleic acids) or the building up of cellular material (cell elongation) have been evaluated in live and UV- or heat-killed cells. With live cells, viability markers detected cells in counts similar to the colony count. However, these so-called viability markers could stain dead cells for some time after the lethal treatment. For the UV-killed cells, residual activities were detected even after 48 h of storage at 20 degrees C. However, for heat-treated cells, these activities disappeared within hours after heat treatment. Only a combination of fluorescence in situ hybridization with rRNA probes and cell elongation in response to nutrients (in the presence of an inhibitor of cell division) had the ability to differentiate live from dead cells. Problems in the definition of a viable but nonculturable state are in part due to the lack of a clear definition of bacterial death. We consider death as an irreversible state where no growth, cell elongation or protein synthesis may occur.

  8. The effects of arginine glutamate, a promising excipient for protein formulation, on cell viability: Comparisons with NaCl

    PubMed Central

    Kheddo, Priscilla; Golovanov, Alexander P.; Mellody, Kieran T.; Uddin, Shahid; van der Walle, Christopher F.; Dearman, Rebecca J.

    2016-01-01

    The effects of an equimolar mixture of l-arginine and l-glutamate (Arg·Glu) on cell viability and cellular stress using in vitro cell culture systems are examined with reference to NaCl, in the context of monoclonal antibody formulation. Cells relevant to subcutaneous administration were selected: the human monocyte cell line THP-1, grown as a single cell suspension, and adherent human primary fibroblasts. For THP-1 cells, the mechanism of cell death caused by relatively high salt concentrations was investigated and effects on cell activation/stress assessed as a function of changes in membrane marker and cytokine (interleukin-8) expression. These studies demonstrated that Arg·Glu does not have any further detrimental effects on THP-1 viability in comparison to NaCl at equivalent osmolalities, and that both salts at higher concentrations cause cell death by apoptosis; there was no significant effect on measures of THP-1 cellular stress/activation. For adherent fibroblasts, both salts caused significant toxicity at ~ 400 mOsm/kg, although Arg·Glu caused a more precipitous subsequent decline in viability than did NaCl. These data indicate that Arg·Glu is of equivalent toxicity to NaCl and that the mechanism of toxicity is such that cell death is unlikely to trigger inflammation upon subcutaneous injection in vivo. PMID:26873863

  9. Predictability in cellular automata.

    PubMed

    Agapie, Alexandru; Andreica, Anca; Chira, Camelia; Giuclea, Marius

    2014-01-01

    Modelled as finite homogeneous Markov chains, probabilistic cellular automata with local transition probabilities in (0, 1) always posses a stationary distribution. This result alone is not very helpful when it comes to predicting the final configuration; one needs also a formula connecting the probabilities in the stationary distribution to some intrinsic feature of the lattice configuration. Previous results on the asynchronous cellular automata have showed that such feature really exists. It is the number of zero-one borders within the automaton's binary configuration. An exponential formula in the number of zero-one borders has been proved for the 1-D, 2-D and 3-D asynchronous automata with neighborhood three, five and seven, respectively. We perform computer experiments on a synchronous cellular automaton to check whether the empirical distribution obeys also that theoretical formula. The numerical results indicate a perfect fit for neighbourhood three and five, which opens the way for a rigorous proof of the formula in this new, synchronous case.

  10. Probabilistic cellular automata.

    PubMed

    Agapie, Alexandru; Andreica, Anca; Giuclea, Marius

    2014-09-01

    Cellular automata are binary lattices used for modeling complex dynamical systems. The automaton evolves iteratively from one configuration to another, using some local transition rule based on the number of ones in the neighborhood of each cell. With respect to the number of cells allowed to change per iteration, we speak of either synchronous or asynchronous automata. If randomness is involved to some degree in the transition rule, we speak of probabilistic automata, otherwise they are called deterministic. With either type of cellular automaton we are dealing with, the main theoretical challenge stays the same: starting from an arbitrary initial configuration, predict (with highest accuracy) the end configuration. If the automaton is deterministic, the outcome simplifies to one of two configurations, all zeros or all ones. If the automaton is probabilistic, the whole process is modeled by a finite homogeneous Markov chain, and the outcome is the corresponding stationary distribution. Based on our previous results for the asynchronous case-connecting the probability of a configuration in the stationary distribution to its number of zero-one borders-the article offers both numerical and theoretical insight into the long-term behavior of synchronous cellular automata.

  11. Effect of Butyrate on Collagen Expression, Cell Viability, Cell Cycle Progression and Related Proteins Expression of MG-63 Osteoblastic Cells

    PubMed Central

    Chang, Mei-Chi; Tsai, Yi-Ling; Liou, Eric Jein-Wein; Tang, Chia-Mei; Wang, Tong-Mei; Liu, Hsin-Cheng; Liao, Ming-Wei; Yeung, Sin-Yuet; Chan, Chiu-Po; Jeng, Jiiang-Huei

    2016-01-01

    Aims Butyric acid is one major metabolic product generated by anaerobic Gram-negative bacteria of periodontal and root canal infection. Butyric acid affects the activity of periodontal cells such as osteoblasts. The purposes of this study were to investigate the effects of butyrate on MG-63 osteoblasts. Methods MG-63 cells were exposed to butyrate and cell viability was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mRNA and protein expression of type I collagen and cell cycle-related proteins were measured by reverse-transcriptase polymerase chain reaction (RT-PCR), western blotting or immunofluorescent staining. Cellular production of reactive oxygen species (ROS) was analyzed by 2',7'-dichlorofluorescein (DCF) fluorescence flow cytometry. Results Exposure to butyrate suppressed cell proliferation, and induced G2/M (8 and 16 mM) cell cycle arrest of MG-63 cells. Some cell apoptosis was noted. The mRNA expression of cdc2 and cyclin-B1 decreased after exposure to butyrate. The protein expression of type I collagen, cdc2 and cyclin B1 were decreased, whereas the expression of p21, p27 and p57 was stimulated. Under the treatment of butyrate, ROS production in MG-63 cells markedly increased. Conclusions The secretion of butyric acid by periodontal and root canal microorganisms may inhibit bone cell growth and matrix turnover. This is possibly due to induction of cell cycle arrest and ROS generation and inhibition of collagen expression. These results suggest the involvement of butyric acid in the pathogenesis of periodontal and periapical tissue destruction by impairing bone healing responses. PMID:27893752

  12. An Endosperm-Associated Cuticle Is Required for Arabidopsis Seed Viability, Dormancy and Early Control of Germination

    PubMed Central

    Loubery, Sylvain; Utz-Pugin, Anne; Bailly, Christophe; Mène-Saffrané, Laurent; Lopez-Molina, Luis

    2015-01-01

    Cuticular layers and seeds are prominent plant adaptations to terrestrial life that appeared early and late during plant evolution, respectively. The cuticle is a waterproof film covering plant aerial organs preventing excessive water loss and protecting against biotic and abiotic stresses. Cutin, consisting of crosslinked fatty acid monomers, is the most abundant and studied cuticular component. Seeds are dry, metabolically inert structures promoting plant dispersal by keeping the plant embryo in an arrested protected state. In Arabidopsis thaliana seeds, the embryo is surrounded by a single cell endosperm layer itself surrounded by a seed coat layer, the testa. Whole genome analyses lead us to identify cutin biosynthesis genes as regulatory targets of the phytohormones gibberellins (GA) and abscisic acid (ABA) signaling pathways that control seed germination. Cutin-containing layers are present in seed coats of numerous species, including Arabidopsis, where they regulate permeability to outer compounds. However, the role of cutin in mature seed physiology and germination remains poorly understood. Here we identify in mature seeds a thick cuticular film covering the entire outer surface of the endosperm. This seed cuticle is defective in cutin-deficient bodyguard1 seeds, which is associated with alterations in endospermic permeability. Furthermore, mutants affected in cutin biosynthesis display low seed dormancy and viability levels, which correlates with higher levels of seed lipid oxidative stress. Upon seed imbibition cutin biosynthesis genes are essential to prevent endosperm cellular expansion and testa rupture in response to low GA synthesis. Taken together, our findings suggest that in the course of land plant evolution cuticular structures were co-opted to achieve key physiological seed properties. PMID:26681322

  13. An Endosperm-Associated Cuticle Is Required for Arabidopsis Seed Viability, Dormancy and Early Control of Germination.

    PubMed

    De Giorgi, Julien; Piskurewicz, Urszula; Loubery, Sylvain; Utz-Pugin, Anne; Bailly, Christophe; Mène-Saffrané, Laurent; Lopez-Molina, Luis

    2015-12-01

    Cuticular layers and seeds are prominent plant adaptations to terrestrial life that appeared early and late during plant evolution, respectively. The cuticle is a waterproof film covering plant aerial organs preventing excessive water loss and protecting against biotic and abiotic stresses. Cutin, consisting of crosslinked fatty acid monomers, is the most abundant and studied cuticular component. Seeds are dry, metabolically inert structures promoting plant dispersal by keeping the plant embryo in an arrested protected state. In Arabidopsis thaliana seeds, the embryo is surrounded by a single cell endosperm layer itself surrounded by a seed coat layer, the testa. Whole genome analyses lead us to identify cutin biosynthesis genes as regulatory targets of the phytohormones gibberellins (GA) and abscisic acid (ABA) signaling pathways that control seed germination. Cutin-containing layers are present in seed coats of numerous species, including Arabidopsis, where they regulate permeability to outer compounds. However, the role of cutin in mature seed physiology and germination remains poorly understood. Here we identify in mature seeds a thick cuticular film covering the entire outer surface of the endosperm. This seed cuticle is defective in cutin-deficient bodyguard1 seeds, which is associated with alterations in endospermic permeability. Furthermore, mutants affected in cutin biosynthesis display low seed dormancy and viability levels, which correlates with higher levels of seed lipid oxidative stress. Upon seed imbibition cutin biosynthesis genes are essential to prevent endosperm cellular expansion and testa rupture in response to low GA synthesis. Taken together, our findings suggest that in the course of land plant evolution cuticular structures were co-opted to achieve key physiological seed properties.

  14. Gene Essentiality Is a Quantitative Property Linked to Cellular Evolvability.

    PubMed

    Liu, Gaowen; Yong, Mei Yun Jacy; Yurieva, Marina; Srinivasan, Kandhadayar Gopalan; Liu, Jaron; Lim, John Soon Yew; Poidinger, Michael; Wright, Graham Daniel; Zolezzi, Francesca; Choi, Hyungwon; Pavelka, Norman; Rancati, Giulia

    2015-12-03

    Gene essentiality is typically determined by assessing the viability of the corresponding mutant cells, but this definition fails to account for the ability of cells to adaptively evolve to genetic perturbations. Here, we performed a stringent screen to assess the degree to which Saccharomyces cerevisiae cells can survive the deletion of ~1,000 individual "essential" genes and found that ~9% of these genetic perturbations could in fact be overcome by adaptive evolution. Our analyses uncovered a genome-wide gradient of gene essentiality, with certain essential cellular functions being more "evolvable" than others. Ploidy changes were prevalent among the evolved mutant strains, and aneuploidy of a specific chromosome was adaptive for a class of evolvable nucleoporin mutants. These data justify a quantitative redefinition of gene essentiality that incorporates both viability and evolvability of the corresponding mutant cells and will enable selection of therapeutic targets associated with lower risk of emergence of drug resistance.

  15. 24 CFR 971.5 - Long-term viability.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... ASSESSMENT OF THE REASONABLE REVITALIZATION POTENTIAL OF CERTAIN PUBLIC HOUSING REQUIRED BY LAW § 971.5 Long... ensure viability and to sustain the operating costs that are described in paragraph (a)(2) of...

  16. 24 CFR 971.5 - Long-term viability.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... ASSESSMENT OF THE REASONABLE REVITALIZATION POTENTIAL OF CERTAIN PUBLIC HOUSING REQUIRED BY LAW § 971.5 Long... ensure viability and to sustain the operating costs that are described in paragraph (a)(2) of...

  17. 24 CFR 971.5 - Long-term viability.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... ASSESSMENT OF THE REASONABLE REVITALIZATION POTENTIAL OF CERTAIN PUBLIC HOUSING REQUIRED BY LAW § 971.5 Long... ensure viability and to sustain the operating costs that are described in paragraph (a)(2) of...

  18. AMP-activated protein kinase supports the NGF-induced viability of human HeLa cells to glucose starvation.

    PubMed

    Ting, Luo; Bo, Wan; Li, Ruwei; Chen, Xinya; Wang, Yingli; Jun, Zhou; Yu, Long

    2010-07-01

    As an important cellular energy regulation kinase, AMP-activated protein kinase (AMPK) has been demonstrated as a key molecule in the development of tolerance to nutrient starvation. Activation of AMPK includes the phosphorylation of Thr172 of the alpha-subunit. Nerve growth factor (NGF) was originally isolated for its ability to stimulate both survival and differentiation in peripheral neurons, but many investigations have shown that the NGF also plays an important role in survival, growth and invasion of many human cancers. In this study, we used CCK-8 cell viability assay to find that NGF could facilitate the viability of HeLa cells following glucose deprivation while not in glucose-normal control groups. This effect of NGF-induced viability promotion to glucose starvation can be suppressed by Compound C, a specific inhibitor of AMPK. Meanwhile, western blot analysis showed that AMPKalpha1/alpha2 Thr172 phosphorylation level in HeLa cells was up-regulated after NGF treatment under glucose starvation, and Compound C was able to reduce the AMPKalpha1/alpha2 Thr172 phosphorylation level which was up-regulated by NGF in HeLa cells. Taken together, these results indicate that AMP-activated protein kinase supports the NGF-induced viability of human HeLa cells to glucose starvation.

  19. Mesoporous silica nanoparticles inhibit cellular respiration.

    PubMed

    Tao, Zhimin; Morrow, Matthew P; Asefa, Tewodros; Sharma, Krishna K; Duncan, Cole; Anan, Abhishek; Penefsky, Harvey S; Goodisman, Jerry; Souid, Abdul-Kader

    2008-05-01

    We studied the effect of two types of mesoporous silica nanoparticles, MCM-41 and SBA-15, on mitochondrial O 2 consumption (respiration) in HL-60 (myeloid) cells, Jurkat (lymphoid) cells, and isolated mitochondria. SBA-15 inhibited cellular respiration at 25-500 microg/mL; the inhibition was concentration-dependent and time-dependent. The cellular ATP profile paralleled that of respiration. MCM-41 had no noticeable effect on respiration rate. In cells depleted of metabolic fuels, 50 microg/mL SBA-15 delayed the onset of glucose-supported respiration by 12 min and 200 microg/mL SBA-15 by 34 min; MCM-41 also delayed the onset of glucose-supported respiration. Neither SBA-15 nor MCM-41 affected cellular glutathione. Both nanoparticles inhibited respiration of isolated mitochondria and submitochondrial particles.

  20. Crack Propagation in Bamboo's Hierarchical Cellular Structure

    PubMed Central

    Habibi, Meisam K.; Lu, Yang

    2014-01-01

    Bamboo, as a natural hierarchical cellular material, exhibits remarkable mechanical properties including excellent flexibility and fracture toughness. As far as bamboo as a functionally graded bio-composite is concerned, the interactions of different constituents (bamboo fibers; parenchyma cells; and vessels.) alongside their corresponding interfacial areas with a developed crack should be of high significance. Here, by using multi-scale mechanical characterizations coupled with advanced environmental electron microscopy (ESEM), we unambiguously show that fibers' interfacial areas along with parenchyma cells' boundaries were preferred routes for crack growth in both radial and longitudinal directions. Irrespective of the honeycomb structure of fibers along with cellular configuration of parenchyma ground, the hollow vessels within bamboo culm affected the crack propagation too, by crack deflection or crack-tip energy dissipation. It is expected that the tortuous crack propagation mode exhibited in the present study could be applicable to other cellular natural materials as well. PMID:24998298

  1. Crack propagation in bamboo's hierarchical cellular structure.

    PubMed

    Habibi, Meisam K; Lu, Yang

    2014-07-07

    Bamboo, as a natural hierarchical cellular material, exhibits remarkable mechanical properties including excellent flexibility and fracture toughness. As far as bamboo as a functionally graded bio-composite is concerned, the interactions of different constituents (bamboo fibers; parenchyma cells; and vessels.) alongside their corresponding interfacial areas with a developed crack should be of high significance. Here, by using multi-scale mechanical characterizations coupled with advanced environmental electron microscopy (ESEM), we unambiguously show that fibers' interfacial areas along with parenchyma cells' boundaries were preferred routes for crack growth in both radial and longitudinal directions. Irrespective of the honeycomb structure of fibers along with cellular configuration of parenchyma ground, the hollow vessels within bamboo culm affected the crack propagation too, by crack deflection or crack-tip energy dissipation. It is expected that the tortuous crack propagation mode exhibited in the present study could be applicable to other cellular natural materials as well.

  2. [Selection on viability of individuals heterozygous for the temperature-sensitive lethal mutation l(2)M167(DTS) in experimental populations of Drosophila melanogaster].

    PubMed

    Kulikov, A M; Marec, F; Mitrofanov, V G

    2005-06-01

    In experiments on introduction of mutation l(2)M167(DTS) in Drosophila melanogaster populations, larval and pupal viability and developmental rate are limiting factors determining the intensity of selection on the l(2)M167(DTS) mutation. Notwithstanding the rapid elimination of the mutation from the population, positive selection for viability was shown, which increased fitness of the mutation carriers in generations. The fitness component viability was estimated in individuals l(2)M167(DTS)/+; relative to that of wild-type individuals, it varied from 0.1 to 1. Factors affecting this trait in overcrowded populations were found.

  3. Adherent cell assay results affected by variable z-position mixing.

    PubMed

    Carramanzana, Nelson; Ross, Sandra; Biddlecombe, Gloria; Lin, Chi-Hwei; Johnson, Michael

    2010-04-01

    We demonstrate that modifying mixing dynamics after addition of organic solute into aqueous buffers dramatically affects cell morphology and protein expression. Variable z-position (VZP) or varying the height of aspiration and dispense positions during mixing eliminates artifactual effects. Here, we tested 4 adherent cell types and show effects of VZP on quantitative imaging, protein expression, viability, and morphology. The result: The quantitation of cytoplasmic fluorescence within the fields of interest of the phalloidin-actin stain assay improved by 47% and fluorescence variability emitted by cells expressing green fluorescence protein (GFP) fusion proteins decreased by 15%. Assays that perform measurement by averaged reading of the entire well are somewhat susceptible. For example, protein production decreased 8% on the hypoxia response element (HRE)-luciferase assay. VZP did not affect quantitative cell viability, deviate the half maximal effective dose concentration (EC(50)) values or alter expected curve patterns. VZP is a valuable systematic process for cellular assay workflows as it efficiently folds organic solute into the aqueous solution.

  4. Fast intracellular dissolution and persistent cellular uptake of silver nanoparticles in CHO-K1 cells: implication for cytotoxicity.

    PubMed

    Jiang, Xiumei; Miclăuş, Teodora; Wang, Liming; Foldbjerg, Rasmus; Sutherland, Duncan S; Autrup, Herman; Chen, Chunying; Beer, Christiane

    2015-03-01

    Toxicity of silver nanoparticles (Ag NPs) has been reported both in vitro and in vivo. However, the intracellular stability and chemical state of Ag NPs are still not very well studied. In this work, we systematically investigated the cellular uptake pathways, intracellular dissolution and chemical species, and cytotoxicity of Ag NPs (15.9 ± 7.6 nm) in Chinese hamster ovary cell subclone K1 cells, a cell line recommended by the OECD for genotoxicity studies. Quantification of intracellular nanoparticle uptake and ion release was performed through inductively coupled plasma mass spectrometry. X-ray absorption near-edge structure (XANES) was employed to assess the chemical state of intracellular silver. The toxic potential of Ag NPs and Ag(+) was evaluated by cell viability, reactive oxygen species (ROS) production and live-dead cell staining. The results suggest that cellular uptake of Ag NPs involves lipid-raft-mediated endocytosis and energy-independent diffusion. The degradation study shows that Ag NPs taken up into cells dissolved quickly and XANES results directly indicated that the internalized Ag was oxidized to Ag-O- species and then stabilized in silver-sulfur (Ag-S-) bonds within the cells. Subsequent cytotoxicity studies show that Ag NPs decrease cell viability and increase ROS production. Pre-incubation with N-acetyl-L-cysteine, an efficient antioxidant and Ag(+) chelator, diminished the cytotoxicity caused by Ag NPs or Ag(+) exposure. Our study suggests that the cytotoxicity mechanism of Ag NPs is related to the intracellular release of silver ions, followed by their binding to SH-groups, presumably coming from amino acids or proteins, and affecting protein functions and the antioxidant defense system of cells.

  5. Cellular phones: are they detrimental?

    PubMed

    Salama, Osama E; Abou El Naga, Randa M

    2004-01-01

    The issue of possible health effects of cellular phones is very much alive in the public's mind where the rapid increase in the number of the users of cell phones in the last decade has increased the exposure of people to the electromagnetic fields (EMFs). Health consequences of long term use of mobile phones are not known in detail but available data indicates the development of non specific annoying symptoms on acute exposure to mobile phone radiations. In an attempt to determine the prevalence of such cell phones associated health manifestations and the factors affecting their occurrence, a cross sectional study was conducted in five randomly selected faculties of Alexandria University. Where, 300 individuals including teaching staff, students and literate employee were equally allocated and randomly selected among the five faculties. Data about mobile phone's users and their medical history, their pattern of mobile usage and the possible deleterious health manifestations associated with cellular phone use was collected. The results revealed 68% prevalence of mobile phone usage, nearly three quarters of them (72.5%) were complainers of the health manifestations. They suffered from headache (43%), earache (38.3%), sense of fatigue (31.6%), sleep disturbance (29.5%), concentration difficulty (28.5%) and face burning sensation (19.2%). Both univariate and multivariate analysis were consistent in their findings. Symptomatic users were found to have significantly higher frequency of calls/day, longer call duration and longer total duration of mobile phone usage/day than non symptomatic users. For headache both call duration and frequency of calls/day were the significant predicting factors for its occurrence (chi2 = 18.208, p = 0.0001). For earache, in addition to call duration, the longer period of owning the mobile phone were significant predictors (chi2 = 16.996, p = 0.0002). Sense of fatigue was significantly affected by both call duration and age of the user

  6. Biomass viability: An experimental study and the development of an empirical mathematical model for submerged membrane bioreactor.

    PubMed

    Zuthi, M F R; Ngo, H H; Guo, W S; Nghiem, L D; Hai, F I; Xia, S Q; Zhang, Z Q; Li, J X

    2015-08-01

    This study investigates the influence of key biomass parameters on specific oxygen uptake rate (SOUR) in a sponge submerged membrane bioreactor (SSMBR) to develop mathematical models of biomass viability. Extra-cellular polymeric substances (EPS) were considered as a lumped parameter of bound EPS (bEPS) and soluble microbial products (SMP). Statistical analyses of experimental results indicate that the bEPS, SMP, mixed liquor suspended solids and volatile suspended solids (MLSS and MLVSS) have functional relationships with SOUR and their relative influence on SOUR was in the order of EPS>bEPS>SMP>MLVSS/MLSS. Based on correlations among biomass parameters and SOUR, two independent empirical models of biomass viability were developed. The models were validated using results of the SSMBR. However, further validation of the models for different operating conditions is suggested.

  7. Demographic rates and population viability of black bears in Louisiana

    USGS Publications Warehouse

    Laufenberg, Jared S.; Clark, Joseph D.; Hooker, Michael J.; Lowe, Carrie L.; O'Connell-Goode, Kaitlin C.; Troxler, Jesse C.; Davidson, Maria M.; Chamberlain, Michael J.; Chandler, Richard B.

    2015-01-01

    The Louisiana black bear (Ursus americanus luteolus) was reduced to a few small, fragmented, and isolated subpopulations in the Lower Mississippi Alluvial Valley by the mid-twentieth century resulting from loss and fragmentation of habitat. In 1992, the United States Fish and Wildlife Service (USFWS) granted the Louisiana black bear threatened status under the United States Endangered Species Act of 1973. Since that time, a recovery plan was developed, a reintroduced population was established, and habitat recovery has occurred. The Recovery Plan states that a minimum of 2 populations must be viable (i.e., persistence probabilities over 100 years >0.95), 1 in the Tensas River Basin and 1 in the Atchafalaya River Basin. Consequently, our objectives were to 1) estimate demographic rates of Louisiana black bear subpopulations, 2) develop data-driven stochastic population projection models, and 3) determine how different projection model assumptions affect population trajectories and predictions about long-term persistence. Our overall goal was to assess long-term persistence of the bear subpopulations in Louisiana, individually and as a whole. We collected data using varying combinations of non-invasive DNA sampling, live capture, winter den visits, and radio monitoring from 2002 to 2012 in the 4 areas currently supporting breeding subpopulations in Louisiana: Tensas River Basin (TRB), Upper Atchafalaya River Basin (UARB), Lower Atchafalaya River Basin (LARB), and a recently reintroduced population at the Three Rivers Complex (TRC). From 2002 to 2012, we radio monitored fates of 86 adult females within the TRB and 43 in the TRC. Mean estimates of annual adult survival for the TRB and TRC were 0.997 and 0.990, respectively, when unknown fates were assumed alive and 0.970 and 0.926 when unknown fates were assumed dead. From 2003 to 2013, we observed 130 cub litters from 74 females in the TRB, and 74 cub litters from 45 females in the TRC. During the same period, we

  8. Formin’ cellular structures

    PubMed Central

    Bogdan, Sven; Schultz, Jörg; Grosshans, Jörg

    2014-01-01

    Members of the Diaphanous (Dia) protein family are key regulators of fundamental actin driven cellular processes, which are conserved from yeast to humans. Researchers have uncovered diverse physiological roles in cell morphology, cell motility, cell polarity, and cell division, which are involved in shaping cells into tissues and organs. The identification of numerous binding partners led to substantial progress in our understanding of the differential functions of Dia proteins. Genetic approaches and new microscopy techniques allow important new insights into their localization, activity, and molecular principles of regulation. PMID:24719676

  9. Cellular mechanics and motility

    NASA Astrophysics Data System (ADS)

    Hénon, Sylvie; Sykes, Cécile

    2015-10-01

    The term motility defines the movement of a living organism. One widely known example is the motility of sperm cells, or the one of flagellar bacteria. The propulsive element of such organisms is a cilium(or flagellum) that beats. Although cells in our tissues do not have a flagellum in general, they are still able to move, as we will discover in this chapter. In fact, in both cases of movement, with or without a flagellum, cell motility is due to a dynamic re-arrangement of polymers inside the cell. Let us first have a closer look at the propulsion mechanism in the case of a flagellum or a cilium, which is the best known, but also the simplest, and which will help us to define the hydrodynamic general conditions of cell movement. A flagellum is sustained by cellular polymers arranged in semi-flexible bundles and flagellar beating generates cell displacement. These polymers or filaments are part of the cellular skeleton, or "cytoskeleton", which is, in this case, external to the cellular main body of the organism. In fact, bacteria move in a hydrodynamic regime in which viscosity dominates over inertia. The system is thus in a hydrodynamic regime of low Reynolds number (Box 5.1), which is nearly exclusively the case in all cell movements. Bacteria and their propulsion mode by flagella beating are our unicellular ancestors 3.5 billion years ago. Since then, we have evolved to form pluricellular organisms. However, to keep the ability of displacement, to heal our wounds for example, our cells lost their flagellum, since it was not optimal in a dense cell environment: cells are too close to each other to leave enough space for the flagella to accomplish propulsion. The cytoskeleton thus developed inside the cell body to ensure cell shape changes and movement, and also mechanical strength within a tissue. The cytoskeleton of our cells, like the polymers or filaments that sustain the flagellum, is also composed of semi-flexible filaments arranged in bundles, and also in

  10. Oral Cellular Neurothekeoma

    PubMed Central

    Emami, Nader; Zawawi, Faisal; Ywakim, Rania; Daniel, Sam J.

    2013-01-01

    Cellular neurothekeoma is known as a cutaneous tumor with uncertain histogenesis. Very little involvement of mucosal membrane has been reported in the literature so far. This is a case report of an intraoral lesion in a 15-years-old girl. Histopathologic evaluation showed a tumor-consists of spindle to epitheloid cells forming micronodules in a concentric whorled shape pattern. Tumor cells were positive for CD63, vimentin, and NKI-C3. Total excision was performed and no recurrence happened after 16-month followup. PMID:23691398

  11. The effect of turbulent strain rate on the viability of E.coli in simulated wastewater discharge

    NASA Astrophysics Data System (ADS)

    Cotel, Aline; Battani, Brian; Semrau, Jeremy

    2003-11-01

    During storm events, release of combined sewage overflows can and does occur throughout the United States, most notably in the Great Lakes. Such overflows can deteriorate overall water quality and also lead to the closure of recreational beaches for many reasons, including due to the presence of harmful microorganisms. Here we report on how different mixing regimes created by varying the Reynolds number of simulated wastewater discharges affect the concentration and viability of microorganisms. A laboratory model was created to simulate a typical discharge containing free-floating Escherichia coli. From the experimental results, it was apparent that in the near field (five diameters from the point of discharge) the viability of E. coli was reduced as the Reynolds number of discharge increased, and such viability was more than could be explained by dilution alone. The discrepancy between observed cell viability and dilution can be attributed to the Kolmogorov strain rate. Such an effect on cell viability was only observed in the near field and did not occur in the far field, suggesting that one possible strategy to mitigate the impact of wastewater discharges, particularly that of combined sewer overflows would be to increase the Kolmogorov strain rate in the near field.

  12. Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser.

    PubMed

    Alexsandra da Silva Neto Trajano, Larissa; da Silva, Camila Luna; de Carvalho, Simone Nunes; Cortez, Erika; Mencalha, André Luiz; de Souza da Fonseca, Adenilson; Stumbo, Ana Carolina

    2016-07-01

    Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells.

  13. Inducers of chemical hypoxia act in a gender- and brain region-specific manner on primary astrocyte viability and cytochrome C oxidase.

    PubMed

    Roemgens, André; Singh, Shilpee; Beyer, Cordian; Arnold, Susanne

    2011-07-01

    Oxygen is the ultimate electron acceptor for mitochondrial respiration, a process catalyzed by cytochrome c oxidase (COX). In mammals, oxygen concentration regulates gene transcription of COX subunit IV isoforms. Here, we demonstrate that chemical hypoxia, i.e. inhibition of mitochondrial respiration by application of the COX inhibitors cobalt, cyanide, and azide, affects COX isoform IV-1 and IV-2 transcription in a gender- and brain region-specific way. After treatment with cyanide and cobalt, female cortical and mesencephalic astrocytes, respectively, revealed an up-regulation of COX IV-2 which was accompanied by increased ROS production and necrotic cell death. In male astrocytes, the ratio of COX IV-1/COX IV-2 was lowest after treatment with cobalt and paralleled by highest levels of ROS production and necrosis. These results support the view of a causal correlation of COX IV-2 transcription with cellular oxidative stress and cell death and highlight a gender specificity of these effects. By comparing three toxins, cobalt represented the most potent inducer of overall cell death and resembled most closely the previously observed effects of oxygen deprivation on decreasing the cox4i1/cox4i2 ratio. Overall, an increased sensitivity of male compared with female cell viability towards the toxins was detected. These regulatory responses might be causative for the known gender specificity of toxic and neurodegenerative processes in the brain.

  14. Inhibition of mitochondrial 2-oxoglutarate dehydrogenase impairs viability of cancer cells in a cell-specific metabolism-dependent manner

    PubMed Central

    Bunik, Victoria I.; Mkrtchyan, Garik; Grabarska, Aneta; Oppermann, Henry; Daloso, Danilo; Araujo, Wagner L.; Juszczak, Malgorzata; Rzeski, Wojciech; Bettendorff, Lucien; Fernie, Alisdair R.; Meixensberger, Jürgen; Stepulak, Andrzej; Gaunitz, Frank

    2016-01-01

    2-Oxoglutarate dehydrogenase (OGDH) of the tricarboxylic acid (TCA) cycle is often implied to be inactive in cancer, but this was not experimentally tested. We addressed the question through specific inhibition of OGDH by succinyl phosphonate (SP). SP action on different cancer cells was investigated using indicators of cellular viability and reactive oxygen species (ROS), metabolic profiling and transcriptomics. Relative sensitivity of various cancer cells to SP changed with increasing SP exposure and could differ in the ATP- and NAD(P)H-based assays. Glioblastoma responses to SP revealed metabolic sub-types increasing or decreasing cellular ATP/NAD(P)H ratio under OGDH inhibition. Cancer cell homeostasis was perturbed also when viability indicators were SP-resistant, e.g. in U87 and N2A cells. The transcriptomics database analysis showed that the SP-sensitive cells, such as A549 and T98G, exhibit the lowest expression of OGDH compared to other TCA cycle enzymes, associated with higher expression of affiliated pathways utilizing 2-oxoglutarate. Metabolic profiling confirmed the dependence of cellular SP reactivity on cell-specific expression of the pathways. Thus, oxidative decarboxylation of 2-oxoglutarate is significant for the interdependent homeostasis of NAD(P)H, ATP, ROS and key metabolites in various cancer cells. Assessment of cell-specific responses to OGDH inhibition is of diagnostic value for anticancer strategies. PMID:27027236

  15. Procalcitonin Impairs Liver Cell Viability and Function In Vitro: A Potential New Mechanism of Liver Dysfunction and Failure during Sepsis?

    PubMed Central

    Ehler, Johannes; Wagner, Nana-Maria

    2017-01-01

    Purpose. Liver dysfunction and failure are severe complications of sepsis and result in poor outcome and increased mortality. The underlying pathologic mechanisms of hepatocyte dysfunction and necrosis during sepsis are only incompletely understood. Here, we investigated whether procalcitonin, a biomarker of sepsis, modulates liver cell function and viability. Materials and Methods. Employing a previously characterized and patented biosensor system evaluating hepatocyte toxicity in vitro, human hepatocellular carcinoma cells (HepG2/C3A) were exposed to 0.01–50 ng/mL procalcitonin for 2 × 72 h and evaluated for proliferation, necrosis, metabolic activity, cellular integrity, microalbumin synthesis, and detoxification capacity. Acetaminophen served as positive control. For further standardization, procalcitonin effects were confirmed in a cellular toxicology assay panel employing L929 fibroblasts. Data were analyzed using ANOVA/Tukey's test. Results. Already at concentrations as low as 0.25 ng/mL, procalcitonin induced HepG2/C3A necrosis (P < 0.05) and reduced metabolic activity, cellular integrity, synthesis, and detoxification capacity (all P < 0.001). Comparable effects were obtained employing L929 fibroblasts. Conclusion. We provide evidence for procalcitonin to directly impair function and viability of human hepatocytes and exert general cytotoxicity in vitro. Therapeutical targeting of procalcitonin could thus display a novel approach to reduce incidence of liver dysfunction and failure during sepsis and lower morbidity and mortality of septic patients. PMID:28255555

  16. Procalcitonin Impairs Liver Cell Viability and Function In Vitro: A Potential New Mechanism of Liver Dysfunction and Failure during Sepsis?

    PubMed

    Sauer, Martin; Doß, Sandra; Ehler, Johannes; Mencke, Thomas; Wagner, Nana-Maria

    2017-01-01

    Purpose. Liver dysfunction and failure are severe complications of sepsis and result in poor outcome and increased mortality. The underlying pathologic mechanisms of hepatocyte dysfunction and necrosis during sepsis are only incompletely understood. Here, we investigated whether procalcitonin, a biomarker of sepsis, modulates liver cell function and viability. Materials and Methods. Employing a previously characterized and patented biosensor system evaluating hepatocyte toxicity in vitro, human hepatocellular carcinoma cells (HepG2/C3A) were exposed to 0.01-50 ng/mL procalcitonin for 2 × 72 h and evaluated for proliferation, necrosis, metabolic activity, cellular integrity, microalbumin synthesis, and detoxification capacity. Acetaminophen served as positive control. For further standardization, procalcitonin effects were confirmed in a cellular toxicology assay panel employing L929 fibroblasts. Data were analyzed using ANOVA/Tukey's test. Results. Already at concentrations as low as 0.25 ng/mL, procalcitonin induced HepG2/C3A necrosis (P < 0.05) and reduced metabolic activity, cellular integrity, synthesis, and detoxification capacity (all P < 0.001). Comparable effects were obtained employing L929 fibroblasts. Conclusion. We provide evidence for procalcitonin to directly impair function and viability of human hepatocytes and exert general cytotoxicity in vitro. Therapeutical targeting of procalcitonin could thus display a novel approach to reduce incidence of liver dysfunction and failure during sepsis and lower morbidity and mortality of septic patients.

  17. RF-EMF exposure at 1800 MHz did not elicit DNA damage or abnormal cellular behaviors in different neurogenic cells.

    PubMed

    Su, Liling; Wei, Xiaoxia; Xu, Zhengping; Chen, Guangdi

    2017-04-01

    Despite many years of studies, the debate on genotoxic effects of radiofrequency electromagnetic fields (RF-EMF) continues. To systematically evaluate genotoxicity of RF-EMF, this study examined effects of RF-EMF on DNA damage and cellular behavior in different neurogenic cells. Neurogenic A172, U251, and SH-SY5Y cells were intermittently (5 min on/10 min off) exposed to 1800 MHz RF-EMF at an average specific absorption rate (SAR) of 4.0 W/kg for 1, 6, or 24 h. DNA damage was evaluated by quantification of γH2AX foci, an early marker of DNA double-strand breaks. Cell cycle progression, cell proliferation, and cell viability were examined by flow cytometry, hemocytometer, and cell counting kit-8 assay, respectively. Results showed that exposure to RF-EMF at an SAR of 4.0 W/kg neither significantly induced γH2AX foci formation in A172, U251, or SH-SY5Y cells, nor resulted in abnormal cell cycle progression, cell proliferation, or cell viability. Furthermore, prolonged incubation of these cells for up to 48 h after exposure did not significantly affect cellular behavior. Our data suggest that 1800 MHz RF-EMF exposure at 4.0 W/kg is unlikely to elicit DNA damage or abnormal cellular behaviors in neurogenic cells. Bioelectromagnetics. 38:175-185, 2017. © 2016 Wiley Periodicals, Inc.

  18. Amyloid Beta Peptides Affect Pregnenolone and Pregnenolone Sulfate Levels in PC-12 and SH-SY5Y Cells Depending on Cholesterol.

    PubMed

    Calan, Ozlem Gursoy; Akan, Pinar; Cataler, Aysenur; Dogan, Cumhur; Kocturk, Semra

    2016-07-01

    Increased amyloid beta (AB) peptide concentration is one of the initiating factors in the neurodegeneration process. It has been suggested that cholesterol induces the synthesis of AB peptide from amyloid precursor protein or facilitates the formation of amyloid plaque by lowering the aggregation threshold of the peptide. It is also shown that AB peptides may affect cholesterol metabolism and the synthesis of steroid hormones such as progesterone and estradiol. Pregnenolone (P) and pregnenolone sulfate (PS) are the major steroids produced from cholesterol in neural tissue. In toxicity conditions, the effect of AB peptides on P and PS levels has not yet been determined. Furthermore, it has not been clearly defined how changes in cellular P and PS levels affect neuronal cell survival. The aim of this study was to determine the effects of AB peptides on cellular changes in P and PS levels depending on the level of their main precursor, cholesterol. Cholesterol and toxic concentrations of AB fragments (AB 25-35, AB 1-40 and AB 1-42) were applied to PC-12 and SH-SY5Y cells. Changes in cellular cholesterol, P and PS levels were determined simultaneously in a dose-and time-dependent manner. The cell viability and cell death types were also evaluated. AB peptides affected both cell viability and P/PS levels. Steroid levels were altered depending on AB fragment type and the cholesterol content of the cells. Treatment with each of the AB fragments alone increased P levels by twofold. However, combined treatment with AB peptides and cholesterol increased P levels by approximately sixfold, while PS levels were increased only about 2.5 fold in both cell lines. P levels in the groups treated with AB 25-35 were higher than those in AB 1-40 and AB 1-42 groups. The cell viabilities were significantly low in the group treated by AB and cholesterol (9 mM). The effect of AB peptides on P levels might be a result of cellular self-defense. On the other hand, the rate of P increase

  19. Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements.

    PubMed

    Bhatia, Sujata K; Yetter, Ann B

    2008-08-01

    Medical devices and implanted biomaterials are often assessed for biological reactivity using visual scores of cell-material interactions. In such testing, biomaterials are assigned cytotoxicity ratings based on visual evidence of morphological cellular changes, including cell lysis, rounding, spreading, and proliferation. For example, ISO 10993 cytotoxicity testing of medical devices allows the use of a visual grading scale. The present study compared visual in vitro cytotoxicity ratings to quantitative in vitro cytotoxicity measurements for biomaterials to determine the level of correlation between visual scoring and a quantitative cell viability assay. Biomaterials representing a spectrum of biological reactivity levels were evaluated, including organo-tin polyvinylchloride (PVC; a known cytotoxic material), ultra-high molecular weight polyethylene (a known non-cytotoxic material), and implantable tissue adhesives. Each material was incubated in direct contact with mouse 3T3 fibroblast cell cultures for 24 h. Visual scores were assigned to the materials using a 5-point rating scale; the scorer was blinded to the material identities. Quantitative measurements of cell viability were performed using a 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay; again, the assay operator was blinded to material identities. The investigation revealed a high degree of correlation between visual cytotoxicity ratings and quantitative cell viability measurements; a Pearson's correlation gave a correlation coefficient of 0.90 between the visual cytotoxicity score and the percent viable cells. An equation relating the visual cytotoxicity score and the percent viable cells was derived. The results of this study are significant for the design and interpretation of in vitro cytotoxicity studies of novel biomaterials.

  20. Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.

    PubMed

    Duellman, Sarah J; Zhou, Wenhui; Meisenheimer, Poncho; Vidugiris, Gediminas; Cali, James J; Gautam, Prson; Wennerberg, Krister; Vidugiriene, Jolanta

    2015-10-01

    Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z' = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.

  1. Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening

    PubMed Central

    Zhou, Wenhui; Meisenheimer, Poncho; Vidugiris, Gediminas; Cali, James J.; Gautam, Prson; Wennerberg, Krister; Vidugiriene, Jolanta

    2015-01-01

    Abstract Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z′ = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples. PMID:26383544

  2. Revisiting Cardiac Cellular Composition

    PubMed Central

    Pinto, Alexander R.; Ilinykh, Alexei; Ivey, Malina J.; Kuwabara, Jill T.; D'Antoni, Michelle L.; Debuque, Ryan; Chandran, Anjana; Wang, Lina; Arora, Komal; Rosenthal, Nadia; Tallquist, Michelle D.

    2015-01-01

    Rationale Accurate knowledge of the cellular composition of the heart is essential to fully understand the changes that occur during pathogenesis and to devise strategies for tissue engineering and regeneration. Objective To examine the relative frequency of cardiac endothelial cells, hematopoietic-derived cells and fibroblasts in the mouse and human heart. Methods and Results Using a combination of genetic tools and cellular markers, we examined the occurrence of the most prominent cell types in the adult mouse heart. Immunohistochemistry revealed that endothelial cells constitute over 60%, hematopoietic-derived cells 5–10%, and fibroblasts under 20% of the non-myocytes in the heart. A refined cell isolation protocol and an improved flow cytometry approach provided an independent means of determining the relative abundance of non-myocytes. High dimensional analysis and unsupervised clustering of cell populations confirmed that endothelial cells are the most abundant cell population. Interestingly, fibroblast numbers are smaller than previously estimated, and two commonly assigned fibroblast markers, Sca-1 and CD90, underrepresent fibroblast numbers. We also describe an alternative fibroblast surface marker that more accurately identifies the resident cardiac fibroblast population. Conclusions This new perspective on the abundance of different cell types in the heart demonstrates that fibroblasts comprise a relatively minor population. By contrast, endothelial cells constitute the majority of non-cardiomyocytes and are likely to play a greater role in physiologic function and response to injury than previously appreciated. PMID:26635390

  3. Multifunctional periodic cellular metals.

    PubMed

    Wadley, Haydn N G

    2006-01-15

    Periodic cellular metals with honeycomb and corrugated topologies are widely used for the cores of light weight sandwich panel structures. Honeycombs have closed cell pores and are well suited for thermal protection while also providing efficient load support. Corrugated core structures provide less efficient and highly anisotropic load support, but enable cross flow heat exchange opportunities because their pores are continuous in one direction. Recent advances in topology design and fabrication have led to the emergence of lattice truss structures with open cell structures. These three classes of periodic cellular metals can now be fabricated from a wide variety of structural alloys. Many topologies are found to provide adequate stiffness and strength for structural load support when configured as the cores of sandwich panels. Sandwich panels with core relative densities of 2-10% and cell sizes in the millimetre range are being assessed for use as multifunctional structures. The open, three-dimensional interconnected pore networks of lattice truss topologies provide opportunities for simultaneously supporting high stresses while also enabling cross flow heat exchange. These highly compressible structures also provide opportunities for the mitigation of high intensity dynamic loads created by impacts and shock waves in air or water. By filling the voids with polymers and hard ceramics, these structures have also been found to offer significant resistance to penetration by projectiles.

  4. Cellular Array Processing Simulation

    NASA Astrophysics Data System (ADS)

    Lee, Harry C.; Preston, Earl W.

    1981-11-01

    The Cellular Array Processing Simulation (CAPS) system is a high-level image language that runs on a multiprocessor configuration. CAPS is interpretively decoded on a conventional minicomputer with all image operation instructions executed on an array processor. The synergistic environment that exists between the minicomputer and the array processor gives CAPS its high-speed throughput, while maintaining a convenient conversational user language. CAPS was designed to be both modular and table driven so that it can be easily maintained and modified. CAPS uses the image convolution operator as one of its primitives and performs this cellular operation by decomposing it into parallel image steps that are scheduled to be executed on the array processor. Among its features is the ability to observe the imagery in real time as a user's algorithm is executed. This feature reduces the need for image storage space, since it is feasible to retain only original images and produce resultant images when needed. CAPS also contains a language processor that permits users to develop re-entrant image processing subroutines or algorithms.

  5. Population viability analysis: using a modeling tool to assess the viability of tapir populations in fragmented landscapes.

    PubMed

    Medici, Emília Patrícia; Desbiez, Arnaud Leonard Jean

    2012-12-01

    A population viability analysis (PVA) was conducted of the lowland tapir populations in the Atlantic Forest of the Pontal do Paranapanema region, Brazil, including Morro do Diabo State Park (MDSP) and surrounding forest fragments. Results from the model projected that the population of 126 tapirs in MDSP is likely to persist over the next 100 years; however, 200 tapirs would be required to maintain a viable population. Sensitivity analysis showed that sub-adult mortality and adult mortality have the strongest influence on the dynamics of lowland tapir populations. High road-kill has a major impact on the MDSP tapir population and can lead to population extinction. Metapopulation modeling showed that dispersal of tapirs from MDSP to the surrounding fragments can be detrimental to the overall metapopulation, as fragments act as sinks. Nevertheless, the model showed that under certain conditions the maintenance of the metapopulation dynamics might be determinant for the persistence of tapirs in the region, particularly in the smaller fragments. The establishment of corridors connecting MDSP to the forest fragments models resulted in an increase in the stochastic growth rate, making tapirs more resilient to threats and catastrophes, but only if rates of mortality were not increased when using corridors. The PVA showed that the conservation of tapirs in the Pontal region depends on: the effective protection of MDSP; maintenance and, whenever possible, enhancement of the functional connectivity of the landscape, reducing mortality during dispersal and threats in the unprotected forest fragments; and neutralization of all threats affecting tapirs in the smaller forest fragments.

  6. Effect of 1-Minute Fluoride Treatment on Potential Virulence and Viability of a Cariogenic Biofilm.

    PubMed

    Pandit, Santosh; Cai, Jian-Na; Jung, Ji-Eun; Jeon, Jae-Gyu

    2015-01-01

    Fluoride is a well-studied and widely used agent for the prevention of dental caries. Although dental caries is strongly related to cariogenic biofilms, the effect of brief fluoride treatment on the virulence properties of biofilms has not been well studied. This study evaluated the effect of a 1-min fluoride treatment on the virulence properties and viability of cariogenic biofilms, using a Streptococcus mutans biofilm model. For this study, 46-hour-old S. mutans biofilms were formed on saliva-coated hydroxyapatite discs and were treated with fluoride (0, 30, 300, 1,000, and 2,000 ppm F(-)) for 1 min. Viability and changes in acidogenicity, aciduricity and extracellular polysaccharide (EPS) formation of the biofilms were analyzed using biochemical and microbiological methods (pH drop, H(+) permeability, acid killing, and bacterial colony-forming unit assays). Laser scanning confocal fluorescence microscopy study was also performed. After the 1-min fluoride treatment, acidogenicity, aciduricity, and EPS formation of 46-hour-old S. mutans biofilms were significantly reduced when treated with concentrations ≥300 ppm F(-). The antivirulence activities of the 1-min fluoride treatment increased in a concentration-dependent pattern. However, the 1-min fluoride treatments did not affect viability, biovolume, and microcolony appearance of biofilm bacteria, even at high concentrations. These results suggest that the brief treatment with fluoride at concentrations ≥300 ppm F(-) is an effective measure for controlling cariogenic biofilms.

  7. Identification of neurotoxic cytokines by profiling Alzheimer's disease tissues and neuron culture viability screening.

    PubMed

    Wood, Levi B; Winslow, Ashley R; Proctor, Elizabeth A; McGuone, Declan; Mordes, Daniel A; Frosch, Matthew P; Hyman, Bradley T; Lauffenburger, Douglas A; Haigis, Kevin M

    2015-11-13

    Alzheimer's disease (AD) therapeutics based on the amyloid hypothesis have shown minimal efficacy in patients, suggesting that the activity of amyloid beta (Aβ) represents only one aspect of AD pathogenesis. Since neuroinflammation is thought to play an important role in AD, we hypothesized that cytokines may play a direct role in promoting neuronal death. Here, we profiled cytokine expression in a small cohort of human AD and control brain tissues. We identified AD-associated cytokines using partial least squares regression to correlate cytokine expression with quantified pathologic disease state and then used neuron cultures to test whether cytokines up-regulated in AD tissues could affect neuronal viability. This analysis identified cytokines that were associated with the pathological severity. Of the top correlates, only TNF-α reduced viability in neuron culture when applied alone. VEGF also reduced viability when applied together with Aβ, which was surprising because VEGF has been viewed as a neuro-protective protein. We found that this synthetic pro-death effect of VEGF in the context of Aβ was commensurate with VEGFR-dependent changes in multiple signaling pathways that govern cell fate. Our findings suggest that profiling of tissues combined with a culture-based screening approach can successfully identify new mechanisms driving neuronal death.

  8. A loss-of-function genetic screening identifies novel mediators of thyroid cancer cell viability

    PubMed Central

    Cantisani, Maria Carmela; Parascandolo, Alessia; Perälä, Merja; Allocca, Chiara; Fey, Vidal; Sahlberg, Niko; Merolla, Francesco; Basolo, Fulvio; Laukkanen, Mikko O.; Kallioniemi, Olli Pekka; Santoro, Massimo; Castellone, Maria Domenica

    2016-01-01

    RET, BRAF and other protein kinases have been identified as major molecular players in thyroid cancer. To identify novel kinases required for the viability of thyroid carcinoma cells, we performed a RNA interference screening in the RET/PTC1(CCDC6-RET)-positive papillary thyroid cancer cell line TPC1 using a library of synthetic small interfering RNAs (siRNAs) targeting the human kinome and related proteins. We identified 14 hits whose silencing was able to significantly reduce the viability and the proliferation of TPC1 cells; most of them were active also in BRAF-mutant BCPAP (papillary thyroid cancer) and 8505C (anaplastic thyroid cancer) and in RAS-mutant CAL62 (anaplastic thyroid cancer) cells. These included members of EPH receptor tyrosine kinase family as well as SRC and MAPK (mitogen activated protein kinases) families. Importantly, silencing of the identified hits did not affect significantly the viability of Nthy-ori 3-1 (hereafter referred to as NTHY) cells derived from normal thyroid tissue, suggesting cancer cell specificity. The identified proteins are worth exploring as potential novel druggable thyroid cancer targets. PMID:27058903

  9. Seeking the environmental source of Leptospirosis reveals durable bacterial viability in river soils

    PubMed Central

    Thibeaux, Roman; Geroult, Sophie; Benezech, Claire; Chabaud, Stéphane; Soupé-Gilbert, Marie-Estelle; Girault, Dominique; Bierque, Emilie

    2017-01-01

    Background Leptospirosis is an important re-emerging infectious disease that affects humans worldwide. Infection occurs from indirect environment-mediated exposure to pathogenic leptospires through contaminated watered environments. The ability of pathogenic leptospires to persist in the aqueous environment is a key factor in transmission to new hosts. Hence, an effort was made to detect pathogenic leptospires in complex environmental samples, to genotype positive samples and to assess leptospiral viability over time. Methodology/Principal findings We focused our study on human leptospirosis cases infected with the New Caledonian Leptospira interrogans serovar Pyrogenes. Epidemiologically related to freshwater contaminations, this strain is responsible for ca. 25% of human cases in New Caledonia. We screened soil and water samples retrieved from suspected environmental infection sites for the pathogen-specific leptospiral gene lipL-32. Soil samples from all suspected infection sites tested showed detectable levels of pathogenic leptospiral DNA. More importantly, we demonstrated by viability qPCR that those pathogenic leptospires were viable and persisted in infection sites for several weeks after the index contamination event. Further, molecular phylogenetic analyses of the leptospiral lfb-1 gene successfully linked the identity of environmental Leptospira to the corresponding human-infecting strain. Conclusions/Significance Altogether, this study illustrates the potential of quantitative viability-PCR assay for the rapid detection of viable leptospires in environmental samples, which might open avenues to strategies aimed at assessing environmental risk. PMID:28241042

  10. Effects of biosurfactants on the viability and proliferation of human breast cancer cells.

    PubMed

    Duarte, Cristina; Gudiña, Eduardo J; Lima, Cristovao F; Rodrigues, Ligia R

    2014-01-01

    Biosurfactants are molecules with surface activity produced by microorganisms that can be used in many biomedical applications. The anti-tumour potential of these molecules is being studied, although results are still scarce and few data are available regarding the mechanisms underlying such activity. In this work, the anti-tumour activity of a surfactin produced by Bacillus subtilis 573 and a glycoprotein (BioEG) produced by Lactobacillus paracasei subsp. paracasei A20 was evaluated. Both biosurfactants were tested against two breast cancer cell lines, T47D and MDA-MB-231, and a non-tumour fibroblast cell line (MC-3 T3-E1), specifically regarding cell viability and proliferation. Surfactin was found to decrease viability of both breast cancer cell lines studied. A 24 h exposure to 0.05 g l(-1) surfactin led to inhibition of cell proliferation as shown by cell cycle arrest at G1 phase. Similarly, exposure of cells to 0.15 g l(-1) BioEG for 48 h decreased cancer cells' viability, without affecting normal fibroblasts. Moreover, BioEG induced the cell cycle arrest at G1 for both breast cancer cell lines. The biosurfactant BioEG was shown to be more active than surfactin against the studied breast cancer cells. The results gathered in this work are very promising regarding the biosurfactants potential for breast cancer treatment and encourage further work with the BioEG glycoprotein.

  11. Cell spreading and viability on zein films may be facilitated by transglutaminase.

    PubMed

    Cui, Hemiao; Liu, Gang L; Padua, Graciela W

    2016-09-01

    Zein is a biocompatible corn protein potentially useful in the development of biomaterials. In this study, the deposition of zein on oxygen plasma treated glass cover slips significantly enhanced cell spreading and viability. The mechanism for cellular response to zein coated surfaces was thought to involve the polyglutamine peptides on the zein structure. We hypothesized that zein was a substrate for tissue transglutaminase (tTG), an extracellular enzyme involved in cell-surface interactions. SDS-PAGE results suggested an interaction between zein and tTG, where zein was the glutamine donor. Cross-linking between zein and tTG may be the first step in successful cell adhesion and spreading.

  12. Short Peptides Enhance Single Cell Adhesion and Viability onMicroarrays

    SciTech Connect

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

    2007-01-19

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

  13. A possible regulatory role of glyoxalase I in cell viability of human prostate cancer.

    PubMed

    Davidson, Scott D; Milanesa, Dan M; Mallouh, Camille; Choudhury, Muhammad S; Tazaki, Hiroshi; Konno, Sensuke

    2002-05-01

    A role of glyoxalase I (Gly-I), a detoxifying enzyme, in cell viability of prostate cancer was investigated. Cell extracts obtained from 66 prostate tissue specimens and prostatic cancer PC-3 cells were assayed for Gly-I activity using the spectrophotometric method. Gly-I activity was consistently more than eightfold higher in prostate cancer (CAP) specimens (n = 37) than in non-cancerous (NCP) specimens (n = 29). To understand the importance of such a high Gly-I activity in CAP specimens, the effects of methylglyoxal (MG) on PC-3 cells were examined in vitro. MG, a putative toxic glycolytic metabolite, was capable of inducing severe (> 99%) cell death in 24 h, along with a significant reduction in activities of Gly-I as well as glyceraldehyde 3-phosphate dehydrogenase (G3PDH), a key glycolytic enzyme. However, such severe cell death was effectively (approximately 85%) prevented with N-acetylcysteine (NAC), a precursor of reduced glutathione (GSH) that is an essential cofactor for Gly-I, accompanied by the intact Gly-I and G3PDH activities. Therefore, Gly-I may play a critical detoxifying role in glycolysis to maintain cellular activity and viability of prostatic cancer cells.

  14. Application of cyclic biamperometry to viability and cytotoxicity assessment in human corneal epithelial cells.

    PubMed

    Rahimi, Mehdi; Youn, Hyun-Yi; McCanna, David J; Sivak, Jacob G; Mikkelsen, Susan R

    2013-05-01

    The application of cyclic biamperometry to viability and cytotoxicity assessments of human corneal epithelial cells has been investigated. Electrochemical measurements have been compared in PBS containing 5.0 mM glucose and minimal essential growth medium. Three different lipophilic mediators including dichlorophenol indophenol, 2-methyl-1,4-naphthoquinone (also called menadione or vitamin K3) and N,N,N',N'-tetramethyl-p-phenylenediamine have been evaluated for shuttling electrons across the cell membrane to the external medium. Transfer of these electrons to ferricyanide in the extra cellular medium results in the accumulation of ferrocyanide. The amount of ferrocyanide is then determined using cyclic biamperometry and is related to the extent of cell metabolic activity and therefore cell viability. To illustrate cytotoxicity assessment of chemicals, hydrogen peroxide, benzalkonium chloride and sodium dodecyl sulfate have been chosen as sample toxins, the cytotoxicities of which have been evaluated and compared to values reported in the literature. Similar values have been reported using colorimetric assays; however, the simplicity of this electrochemical assay can, in principle, open the way to miniaturization onto lab-on-chip devices and its incorporation into tiered-testing approaches for cytotoxicity assessment.

  15. Long-Term IGF-I Exposure Decreases Autophagy and Cell Viability

    PubMed Central

    Bitto, Alessandro; Lerner, Chad; Torres, Claudio; Roell, Michaela; Malaguti, Marco; Perez, Viviana; Lorenzini, Antonello; Hrelia, Silvana; Ikeno, Yuji; Matzko, Michelle Elizabeth; McCarter, Roger; Sell, Christian

    2010-01-01

    A reduction in IGF-I signaling has been found to increase lifespan in multiple organisms despite the fact that IGF-I is a trophic factor for many cell types and has been found to have protective effects against multiple forms of damage in acute settings. The increase in longevity seen in response to reduced IGF-I signaling suggests that there may be differences between the acute and chronic impact of IGF-I signaling. We have examined the possibility that long-term stimulation with IGF-I may have a negative impact at the cellular level using quiescent human fibroblasts. We find that fibroblast cells exposed to IGF-I for 14 days have reduced long-term viability as judged by colony forming assays, which is accompanied by an accumulation of senescent cells. In addition we observe an accumulation of cells with depolarized mitochondria and a reduction in autophagy in the long-term IGF-I treated cultures. An examination of mice with reduced IGF-I levels reveals evidence of enhanced autophagy and fibroblast cells derived from these mice have a larger mitochondrial mass relative to controls indicating that changes in mitochondrial turnover occurs in animals with reduced IGF-I. The results indicate that chronic IGF-I stimulation leads to mitochondrial dysfunction and reduced cell viability. PMID:20830296

  16. Exposure to the proton scavenger glycine under alkaline conditions induces Escherichia coli viability loss.

    PubMed

    Vanhauteghem, Donna; Janssens, Geert Paul Jules; Lauwaerts, Angelo; Sys, Stanislas; Boyen, Filip; Cox, Eric; Meyer, Evelyne

    2013-01-01

    Our previous work described a clear loss of Escherichia coli (E. coli) membrane integrity after incubation with glycine or its N-methylated derivatives N-methylglycine (sarcosine) and N,N-dimethylglycine (DMG), but not N,N,N-trimethylglycine (betaine), under alkaline stress conditions. The current study offers a thorough viability analysis, based on a combination of real-time physiological techniques, of E. coli exposed to glycine and its N-methylated derivatives at alkaline pH. Flow cytometry was applied to assess various physiological parameters such as membrane permeability, esterase activity, respiratory activity and membrane potential. ATP and inorganic phosphate concentrations were also determined. Membrane damage was confirmed through the measurement of nucleic acid leakage. Results further showed no loss of esterase or respiratory activity, while an instant and significant decrease in the ATP concentration occurred upon exposure to either glycine, sarcosine or DMG, but not betaine. There was a clear membrane hyperpolarization as well as a significant increase in cellular inorganic phosphate concentration. Based on these results, we suggest that the inability to sustain an adequate level of ATP combined with a decrease in membrane functionality leads to the loss of bacterial viability when exposed to the proton scavengers glycine, sarcosine and DMG at alkaline pH.

  17. Viability of Booby Offspring is Maximized by Having One Young Parent and One Old Parent

    PubMed Central

    Drummond, Hugh; Rodríguez, Cristina

    2015-01-01

    It is widely expected that the quality of offspring will vary with the age of their parents and that this variation should influence animals’ choice of mates. However, theoretical predictions for age effects are contradictory and, to our knowledge, we do not know for any wild animal how the quality of offspring is affected by both parents’ ages across their lifespans, or whether mothers’ and fathers’ ages interact. We tackled this question using long-term data on a highly philopatric, insular population of the blue-footed booby (Sula nebouxii). In this species extra-pair paternity is most common in pairs of two young parents or two old parents, implying that these age combinations might prejudice offspring quality. Analysis of the viability of 3,361 offspring of parents up to 21 years old revealed that fledglings with two young parents or two old parents were least likely to become breeders, whereas fledglings with one young parent and one old parent were most likely to do so. For young parents of either sex, offspring viability increased with age of the other parent; for very old parents, it decreased. These effects could be mediated by parents flexibly modifying their investment in offspring in response to their own and their partners´ ages, but evidence for this was lacking. In 5,343 breeding attempts, although mothers’ and fathers’ ages independently affected four heavily care-dependent breeding traits at the clutch and nestling stages, their interaction did not affect any trait. The effects of parental age combinations on viability could also be mediated by genes: fledglings with one young parent and one old parent could benefit from greater heterozygosity or better genes. PMID:26221951

  18. Analyses of several seed viability markers in individual recalcitrant seeds of Eugenia stipitata McVaugh with totipotent germination.

    PubMed

    Calvi, G P; Aud, F F; Ferraz, I D K; Pritchard, H W; Kranner, I

    2017-01-01

    The use of biochemical seed viability markers is often compromised by the unknown partitioning of analytes in bulk seed lots consisting of inseparable populations of viable and nonviable seeds. We took advantage of an unusual morphological syndrome found in the recalcitrant, undifferentiated seeds of Eugenia stipitata: one seed can be cut into several parts, each of which can germinate and develop into seedlings. We used four seed parts from one individual seed to analyse seed moisture content (MC), seed viability and the antioxidant glutathione (γ-glutamyl-cysteinyl-glycine; GSH), glutathione disulphide (GSSG) and intermediates of glutathione synthesis and breakdown. Seeds were exposed to different environmental MC to induce various levels of desiccation stress. Upon storage at high seed MC, seed viability was maintained, while GSH concentration increased and the glutathione half-cell reduction potential (EGSSG/2GSH ) was less negative than -215 mV, indicating GSH production and highly reducing conditions. Storage at low seed MC led to loss of GSH, resulting in a shift in EGSSG/2GSH , and seed death. In contrast, the cyst(e)ine half-cell reduction potential (ECySS/2CYS ) could not distinguish between the viability categories. Previous studies on seed populations revealed that the probability for a seed being alive is 50% at EGSSG/2GSH values between -180 and -160 mV. The single seed approach revealed that the window in which seed viability was lost could be slightly shifted towards more negative values. We discuss the contribution of cellular pH to EGSSG/2GSH and recommend E. stipitata as a recalcitrant seed model to study stress response on a single seed basis.

  19. Negative effects of temperature and atmospheric depositions on the seed viability of common juniper (Juniperus communis)

    PubMed Central

    Gruwez, R.; De Frenne, P.; De Schrijver, A.; Leroux, O.; Vangansbeke, P.; Verheyen, K.

    2014-01-01

    Background and Aims Environmental change is increasingly impacting ecosystems worldwide. However, our knowledge about the interacting effects of various drivers of global change on sexual reproduction of plants, one of their key mechanisms to cope with change, is limited. This study examines populations of poorly regenerating and threatened common juniper (Juniperus communis) to determine the influence of four drivers of global change (rising temperatures, nitrogen deposition, potentially acidifying deposition and altering precipitation patterns) on two key developmental phases during sexual reproduction, gametogenesis and fertilization (seed phase two, SP2) and embryo development (seed phase three, SP3), and on the ripening time of seeds. Methods In 42 populations throughout the distribution range of common juniper in Europe, 11 943 seeds of two developmental phases were sampled. Seed viability was determined using seed dissection and related to accumulated temperature (expressed as growing degree-days), nitrogen and potentially acidifying deposition (nitrogen plus sulfur), and precipitation data. Key Results Precipitation had no influence on the viability of the seeds or on the ripening time. Increasing temperatures had a negative impact on the viability of SP2 and SP3 seeds and decreased the ripening time. Potentially acidifying depositions negatively influenced SP3 seed viability, while enhanced nitrogen deposition led to lower ripening times. Conclusions Higher temperatures and atmospheric deposition affected SP3 seeds more than SP2 seeds. However, this is possibly a delayed effect as juniper seeds develop practically independently, due to the absence of vascular communication with the parent plant from shortly after fertilization. It is proposed that the failure of natural regeneration in many European juniper populations might be attributed to climate warming as well as enhanced atmospheric deposition of nitrogen and sulfur. PMID:24284814

  20. Frog population viability under present and future climate conditions: a Bayesian state-space approach.

    PubMed

    McCaffery, R; Solonen, A; Crone, E

    2012-09-01

    1. World-wide extinctions of amphibians are at the forefront of the biodiversity crisis, with climate change figuring prominently as a potential driver of continued amphibian decline. As in other taxa, changes in both the mean and variability of climate conditions may affect amphibian populations in complex, unpredictable ways. In western North America, climate models predict a reduced duration and extent of mountain snowpack and increased variability in precipitation, which may have consequences for amphibians inhabiting montane ecosystems. 2. We used Bayesian capture-recapture methods to estimate survival and transition probabilities in a high-elevation population of the Columbia spotted frog (Rana luteiventris) over 10 years and related these rates to interannual variation in peak snowpack. Then, we forecasted frog population growth and viability under a range of scenarios with varying levels of change in mean and variance in snowpack. 3. Over a range of future scenarios, changes in mean snowpack had a greater effect on viability than changes in the variance of snowpack, with forecasts largely predicting an increase in population viability. Population models based on snowpack during our study period predicted a declining population. 4. Although mean conditions were more important for viability than variance, for a given mean snowpack depth, increases in variability could change a population from increasing to decreasing. Therefore, the influence of changing climate variability on populations should be accounted for in predictive models. The Bayesian modelling framework allows for the explicit characterization of uncertainty in parameter estimates and ecological forecasts, and thus provides a natural approach for examining relative contributions of mean and variability in climatic variables to population dynamics. 5. Longevity and heterogeneous habitat may contribute to the potential for this amphibian species to be resilient to increased climatic variation, and

  1. Rv3351c, a Mycobacterium tuberculosis gene that affects bacterial growth and alveolar epithelial cell viability.

    PubMed

    Pavlicek, Rebecca L; Fine-Coulson, Kari; Gupta, Tuhina; Quinn, Frederick D; Posey, James E; Willby, Melisa; Castro-Garza, Jorge; Karls, Russell K

    2015-12-01

    Despite the interactions known to occur between various lower respiratory tract pathogens and alveolar epithelial cells (AECs), few reports examine factors influencing the interplay between Mycobacterium tuberculosis bacilli and AECs during infection. Importantly, in vitro studies have demonstrated that the M. tuberculosis hbha and esxA gene products HBHA and ESAT6 directly or indirectly influence AEC survival. In this report, we identify Rv3351c as another M. tuberculosis gene that impacts the fate of both the pathogen and AEC host. Intracellular replication of an Rv3351c mutant in the human AEC type II pneumocyte cell line A549 was markedly reduced relative to the complemented mutant and parent strain. Deletion of Rv3351c diminished the release of lactate dehydrogenase and decreased uptake of trypan blue vital stain by host cells infected with M. tuberculosis bacilli, suggesting attenuated cytotoxic effects. Interestingly, an isogenic hbha mutant displayed reductions in AEC killing similar to those observed for the Rv3351c mutant. This opens the possibility that multiple M. tuberculosis gene products interact with AECs. We also observed that Rv3351c aids intracellular replication and survival of M. tuberculosis in macrophages. This places Rv3351c in the same standing as HBHA and ESAT6, which are important factors in AECs and macrophages. Defining the mechanism(s) by which Rv3351c functions to aid pathogen survival within the host may lead to new drug or vaccine targets.

  2. Factors affecting culturability, viability, and filterability of Dekkera bruxellensis in red wine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forty-eight commercial Washington red wines suspected of Dekkera bruxellensis contamination, determined by winemakers, were donated for this work. Only eight out of 48 wines were confirmed to contain D. bruxellensis by PCR analysis and DNA sequencing. Nine strains of D. bruxellensis, one Candida par...

  3. SCALE OF HABITAT FRAGMENTATION AFFECTS ECOLOGICAL PROCESSES AND BIRD POPULATION VIABILITY. (R827673)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  4. Sensitivity analysis to aid shelter management decisions: how does altering expenditure affect operational viability?

    PubMed

    Widmar, Nicole Olynk; Lord, Emily; Litster, Annette

    2015-01-01

    Streamlining purchasing in nonhuman animal shelters can provide multiple financial benefits. Streamlining shelter inputs and thus reducing shelter costs can include trading paid labor and management for fewer, more involved volunteers or purchasing large quantities of medical supplies from fewer vendors to take advantage of bulk-purchasing discounts. Beyond direct savings, time and energy spent on purchasing and inventory control can be reduced through careful management. Although cost-cutting measures may seem attractive, shelter managers are cautioned to consider the potential unintended consequences of short-term cost reduction measures that could limit revenues or increase costs in the future. This analysis illustrates an example of the impact of cost reductions in specific expense categories and the impact on shelter net revenue, as well as the share of expenses across categories. An in-depth discussion of labor and purchasing cost-reducing strategies in the real world of animal shelter management is provided.

  5. On the potential of hydrated storage for naturally derived ECMs and associated effects on mechanical and cellular performance.

    PubMed

    Davis, L M; Callanan, A; Carroll, G T; Doyle, B J; Walsh, M T; McGloughlin, T M

    2014-01-01

    Tissue engineered acellular vascular grafts are an emerging concept in the development of vascular prostheses for the minimally invasive treatment of cardiovascular diseases. Extracellular matrix (ECM) scaffolds, such as small intestinal submucosa (SIS) and urinary bladder matrix (UBM), offer many advantages over currently available synthetic devices. However, storage of such biomaterials can unduly influence the scaffold properties. This study evaluated the effects of up to 16 weeks hydrated storage on the mechanical and cellular performance of stented and unstented tubular scaffolds. This study aimed to demonstrate the viability, mechanical integrity, and bioactive potential of xenogeneic ECMs as potential off-the-shelf vascular prosthetic devices. Rehydrated ECM samples versus the lyophilized controls showed an increase in UTS and stiffness. The mechanical strength of all samples evaluated was above the average reported aortic tissue failure strength and more compliant than current synthetic materials employed. Post-storage cellular bioactivity investigations indicated that both ECM scaffolds tested were unaffected by increased hydrated storage duration when compared with the controls. Overall, the results indicate that the biomechanical and biologic properties of ECMs are not negatively affected by long-term hydrated storage. Therefore, with further investigations, naturally derived ECM materials may offer potential as an off-the-shelf therapeutic treatment of cardiovascular diseases.

  6. Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells: an in vitro study.

    PubMed

    Choi, B-H; Zhu, S-J; Kim, B-Y; Huh, J-Y; Lee, S-H; Jung, J-H

    2005-06-01

    Previous studies have shown that a combination of platelet-rich plasma (PRP) and autogenous bone graft can increase the rate of osteogenesis and enhance bone formation qualitatively. However, contradictory results were reported in a recent animal study. In order to clarify this inconsistency, this study examined the influence of the PRP concentrations on the viability and proliferation of alveolar bone cells in vitro. Bone cells obtained from the alveolar bone chips were exposed to various PRP concentrations. After a culture period of 7 days, cellular viability and proliferation were evaluated by counting the number of cells and a MTT assay. The results showed that the viability and proliferation of alveolar bone cells were suppressed by high PRP concentrations, but were stimulated by low PRP concentrations (1-5%). These in vitro results support the view that variations in the PRP concentrations might influence the bone formation within the PRP-treated bone grafts.

  7. The coxsackievirus A9 RGD motif is not essential for virus viability.

    PubMed Central

    Hughes, P J; Horsnell, C; Hyypiä, T; Stanway, G

    1995-01-01

    An RGD (arginine-glycine-aspartic acid) motif in coxsackievirus A9 has been implicated in internalization through an interaction with the integrin alpha v beta 3. We have produced a number of virus mutants, lacking the motif, which have a small-plaque phenotype in LLC-Mk2 and A-Vero cells and are phenotypically normal in RD cells. Substitution of flanking amino acids also affected plaque size. The results suggest that interaction between the RGD motif and alpha v beta 3 is not critical for virus viability in the cell lines tested and therefore that alternative regions of the CAV-9 capsid are involved in internalization. PMID:7494317

  8. [Senescence and cellular immortality].

    PubMed

    Trentesaux, C; Riou, J-F

    2010-11-01

    Senescence was originally described from the observation of the limited ability of normal cells to grow in culture, and may be generated by telomere erosion, accumulation of DNA damages, oxidative stress and modulation of oncogenes or tumor suppressor genes. Senescence corresponds to a cellular response aiming to control tumor progression by limiting cell proliferation and thus constitutes an anticancer barrier. Senescence is observed in pre-malignant tumor stages and disappears from malignant tumors. Agents used in standard chemotherapy also have the potential to induce senescence, which may partly explain their therapeutic activities. It is possible to restore senescence in tumors using targeted therapies that triggers telomere dysfunction or reactivates suppressor genes functions, which are essential for the onset of senescence.

  9. Cellular aggregate size as the critical factor for flavonoid production by suspension cultures of Saussurea medusa.

    PubMed

    Fu, Chun-xiang; Zhao, De-xiu; Huang, Yan; Ma, Feng-shan

    2005-01-01

    Three previously established cell lines (yellow, red and white) of Saussurea medusa were investigated for jaceosidin and hispidulin production. Maximum yields of the jaceosidin and hispidulin were obtained in the red cell line at 75+/-0.41 and 6.4+/-0.31 mg l-1. Production of jaceosidin and hispidulin correlated with the sizes of compact callus aggregates (CCA) and cellular viability. In the red cell line, the sizes of CCA were predominantly of 2-4 mm diameter and accounted for 64% biomass. This line had a sustained cell viability over 10 successive sub-cultures.

  10. Development of an on-line exposure system to determine freshly produced diesel engine emission-induced cellular effects.

    PubMed

    Oostingh, Gertie J; Papaioannou, Eleni; Chasapidis, Leonidas; Akritidis, Theofylaktos; Konstandopoulos, Athanasios G; Duschl, Albert

    2013-09-01

    Diesel engine emission particle filters are often placed at exhaust outlets to remove particles from the exhaust. The use of filters results in the exposure to a reduced number of nanometer-sized particles, which might be more harmful than the exposure to a larger number of micrometer-sized particles. An in vitro exposure system was established to expose human alveolar epithelial cells to freshly generated exhaust. Computer simulations were used to determine the optimal flow characteristics and ensure equal exposure conditions for each well of a 6-well plate. A selective particle size sampler was used to continuously deliver diesel soot particles with different particle size distributions to cells in culture. To determine, whether the system could be used for cellular assays, alterations in cytokine production and cell viability of human alveolar A549 cells were determined after 3h on-line exposure followed by a 21-h conventional incubation period. Data indicated that complete diesel engine emission slightly affected pre-stimulated cells, but naive cells were not affected. The fractions containing large or small particles never affected the cells. The experimental set-up allowed a reliable exposure of the cells to the complete exhaust fraction or to the fractions containing either large or small diesel engine emission particles.

  11. Viability and the moral status of the fetus.

    PubMed

    Campbell, A V

    1985-01-01

    The viability of the fetus has played a significant part in the framing of abortion legislation, but the use of viability as a criterion represents a compromise which is conceptually unclear and which fails to satisfy either side in the abortion debate. Both conservative and liberal views on abortion regard fetal development as morally irrelevant. For the moderate (or gradualist) view some point in the development must be found which indicates a change in the moral status of the fetus. Since viability changes according to available techniques for neonatal care it cannot be the criterion. Alternative criteria are surveyed and it is concluded that the onset of sensation is a significant transition point beyond which abortion and infanticide cannot be morally distinguished. It is suggested that either legislation should be amended to prohibit abortion after 18 to 20 weeks, or abortion to full term should be permitted and the possibility of legislation for infanticide be envisaged.

  12. Comparison of cellular toxicity between multi-walled carbon nanotubes and onion-like shell-shaped carbon nanoparticles

    NASA Astrophysics Data System (ADS)

    Kang, Seunghyon; Kim, Ji-Eun; Kim, Daegyu; Woo, Chang Gyu; Pikhitsa, Peter V.; Cho, Myung-Haing; Choi, Mansoo

    2015-09-01

    The cellular toxicity of multi-walled carbon nanotubes (MWCNTs) and onion-like shell-shaped carbon nanoparticles (SCNPs) was investigated by analyzing the comparative cell viability. For the reasonable comparison, physicochemical characteristics were controlled thoroughly such as crystallinity, carbon bonding characteristic, hydrodynamic diameter, and metal contents of the particles. To understand relation between cellular toxicity of the particles and generation of reactive oxygen species (ROS), we measured unpaired singlet electrons of the particles and intracellular ROS, and analyzed cellular toxicity with/without the antioxidant N-acetylcysteine (NAC). Regardless of the presence of NAC, the cellular toxicity of SCNPs was found to be lower than that of MWCNTs. Since both particles show similar crystallinity, hydrodynamic size, and Raman signal with negligible contribution of remnant metal particles, the difference in cell viability would be ascribed to the difference in morphology, i.e., spherical shape (aspect ratio of one) for SCNP and elongated shape (high aspect ratio) for MWCNT.

  13. Rethinking the approach to viability monitoring in seed genebanks.

    PubMed

    Hay, Fiona R; Whitehouse, Katherine J

    2017-01-01

    Seed viability monitoring, usually through a germination test, is a key aspect of genebank management; a low viability result triggers the regeneration of an accession in order to ensure that the genetic diversity of the accession is conserved and available for distribution. However, regular viability monitoring of large collections is costly in terms of seeds, labour and other resources. Genebanks differ in how they conduct their viability monitoring and how they collect, manage and store the data that are generated. In this article, we propose alternatives to the current norm of conducting an initial germination test soon after arrival at the genebank and then testing after regular, set storage intervals, as recommended in the Food and Agriculture Organization's Genebank Standards for Plant Genetic Resources for Food and Agriculture. We use real data from the International Rice Genebank (held at the International Rice Research Institute) to illustrate some of the issues regarding the accuracy and reliability of germination test results, in particular when they are used to predict the longevity of a seed lot in storage and to set viability monitoring intervals. We suggest the use of seed storage experiments on samples of seeds to identify which seed lots from a particular crop season to test first. We also give advice on the use of sequential testing schemes potentially to reduce the number of seeds used for viability testing; the use of tolerance tables to identify unlikely results when samples are subdivided into replicates; and what data to include in a genebank management database to improve the management of seed collections.

  14. Rethinking the approach to viability monitoring in seed genebanks

    PubMed Central

    Whitehouse, Katherine J.

    2017-01-01

    Abstract Seed viability monitoring, usually through a germination test, is a key aspect of genebank management; a low viability result triggers the regeneration of an accession in order to ensure that the genetic diversity of the accession is conserved and available for distribution. However, regular viability monitoring of large collections is costly in terms of seeds, labour and other resources. Genebanks differ in how they conduct their viability monitoring and how they collect, manage and store the data that are generated. In this article, we propose alternatives to the current norm of conducting an initial germination test soon after arrival at the genebank and then testing after regular, set storage intervals, as recommended in the Food and Agriculture Organization's Genebank Standards for Plant Genetic Resources for Food and Agriculture. We use real data from the International Rice Genebank (held at the International Rice Research Institute) to illustrate some of the issues regarding the accuracy and reliability of germination test results, in particular when they are used to predict the longevity of a seed lot in storage and to set viability monitoring intervals. We suggest the use of seed storage experiments on samples of seeds to identify which seed lots from a particular crop season to test first. We also give advice on the use of sequential testing schemes potentially to reduce the number of seeds used for viability testing; the use of tolerance tables to identify unlikely results when samples are subdivided into replicates; and what data to include in a genebank management database to improve the management of seed collections. PMID:28361000

  15. Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells.

    PubMed

    Ouyang, Liliang; Yao, Rui; Zhao, Yu; Sun, Wei

    2016-09-16

    3D cell printing is an emerging technology for fabricating complex cell-laden constructs with precise and pre-designed geometry, structure and composition to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. This technology enables spatial manipulation of cells and biomaterials, also referred to as 'bioink', and thus allows study of cellular interactions in a 3D microenvironment and/or in the formation of functional tissues and organs. Recently, many efforts have been made to develop new bioinks and to apply more cell sources for better biocompatibility and biofunctionality. However, the influences of printing parameters on the shape fidelity of 3D constructs as well as on cell viability after the cell printing process have been poorly characterized. Furthermore, parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensibility. In this study, we systematically studied the influence of bioink properties and printing parameters on bioink printability and embryonic stem cell (ESC) viability in the process of extrusion-based cell printing, also known as bioplotting. A novel method was established to determine suitable conditions for bioplotting ESCs to achieve both good printability and high cell viability. The rheological properties of gelatin/alginate bioinks were evaluated to determine the gelation properties under different bioink compositions, printing temperatures and holding times. The bioink printability was characterized by a newly developed semi-quantitative method. The results demonstrated that bioinks with longer gelation times would result in poorer printability. The live/dead assay showed that ESC viability increased with higher printing temperatures and lower gelatin concentrations. Furthermore, an exponential relationship was obtained between ESC viability and induced shear stress. By defining the proper printability and

  16. [Viability of facultative anaerobic bacteria in commercial transport systems].

    PubMed

    Gruber, I M; Dmitrieva, M N; Gavrilova, N A; Zhigunova, O V; Nisilevich, V F

    2001-01-01

    The possibilities of the transport systems manufactured by Copan (Italy) and Deltalab (Spain) were studied on 15 microbial strains: representatives of the family Enterobacteriaceae (enterobacterial swabs with Cary-Blair medium) and the genus Streptococcus, as well as the species Neisseria meningitidis, Haemophilus influenzae (universal swabs with charcoal-enriched Amies medium). Microorganisms were shown to retain their viability (in colony-forming units, %) for 48 hours in the systems of both firms. H. influenzae exhibited greater viability in the system manufactured by Copan than in that manufactured by Deltalab (respectively, 62% and 28% in 24 hours, 19% and 6% in 48 hours).

  17. Economic Viability of Brewery Spent Grain as a Biofuel

    SciTech Connect

    Morrow, Charles

    2016-01-01

    This report summarizes an investigation into the technical feasibility and economic viability of use grain wastes from the beer brewing process as fuel to generate the heat needed in subsequent brewing process. The study finds that while use of spent grain as a biofuel is technically feasible, the economics are not attractive. Economic viability is limited by the underuse of capital equipment. The investment in heating equipment requires a higher utilization that the client brewer currently anticipates. It may be possible in the future that changing factors may swing the decision to a more positive one.

  18. Population-specific life histories contribute to metapopulation viability

    USGS Publications Warehouse

    Halsey, Samniqueka J.; Bell, Timothy J.; McEachern, Kathryn; Pavlovic, Noel B.

    2016-01-01

    Restoration efforts can be improved by understanding how variations in life-history traits occur within populations of the same species living in different environments. This can be done by first understanding the demographic responses of natural occurring populations. Population viability analysis continues to be useful to species management and conservation with sensitivity analysis aiding in the understanding of population dynamics. In this study, using life-table response experiments and elasticity analyses, we investigated how population-specific life-history demographic responses contributed to the metapopulation viability of the Federally threatened Pitcher's thistle (Cirsium pitcheri). Specifically, we tested the following hypotheses: (1) Subpopulations occupying different environments within a metapopulation have independent demographic responses and (2) advancing succession results in a shift from a demographic response focused on growth and fecundity to one dominated by stasis. Our results showed that reintroductions had a positive contribution to the metapopulation growth rate as compared to native populations which had a negative contribution. We found no difference in succession on the contribution to metapopulation viability. In addition, we identified distinct population-specific contributions to metapopulation viability and were able to associate specific life-history demographic responses. For example, the positive impact of Miller High Dunes population on the metapopulation growth rate resulted from high growth contributions, whereas increased time of plant in stasis for the State Park Big Blowout population resulted in negative contributions. A greater understanding of how separate populations respond in their corresponding environment may ultimately lead to more effective management strategies aimed at reducing extinction risk. We propose the continued use of sensitivity analyses to evaluate population-specific demographic influences on

  19. FEM-based oxygen consumption and cell viability models for avascular pancreatic islets

    PubMed Central

    Buchwald, Peter

    2009-01-01

    Background The function and viability of cultured, transplanted, or encapsulated pancreatic islets is often limited by hypoxia because these islets have lost their vasculature during the isolation process and have to rely on gradient-driven passive diffusion, which cannot provide adequate oxygen transport. Pancreatic islets (islets of Langerhans) are particularly susceptible due to their relatively large size, large metabolic demand, and increased sensitivity to hypoxia. Here, finite element method (FEM) based multiphysics models are explored to describe oxygen transport and cell viability in avascular islets both in static and in moving culture media. Methods Two- and three-dimensional models were built in COMSOL Multiphysics using the convection and diffusion as well as the incompressible Navier-Stokes fluid dynamics application modes. Oxygen consumption was assumed to follow Michaelis-Menten-type kinetics and to cease when local concentrations fell below a critical threshold; in a dynamic model, it was also allowed to increase with increasing glucose concentration. Results Partial differential equation (PDE) based exploratory cellular-level oxygen consumption and cell viability models incorporating physiologically realistic assumptions have been implemented for fully scaled cell culture geometries with 100, 150, and 200 μm diameter islets as representative. Calculated oxygen concentrations and intra-islet regions likely to suffer from hypoxia-related necrosis obtained for traditional flask-type cultures, oxygen-permeable silicone-rubber membrane bottom cultures, and perifusion chambers with flowing media and varying incoming glucose levels are presented in detail illustrated with corresponding colour-coded figures and animations. Conclusion Results of the computational models are, as a first estimate, in good quantitative agreement with existing experimental evidence, and they confirm that during culture, hypoxia is often a problem for non-vascularised islet

  20. Scale of Carbon Nanomaterials Affects Neural Outgrowth and Adhesion

    PubMed Central

    Franca, Eric; Jao, PitFee; Fang, Sheng-Po; Alagapan, Sankaraleengam; Pan, Liangbin; Yoon, Jung Hae; Yoon, Yong-Kyu ‘YK’

    2016-01-01

    Carbon nanomaterials have become increasingly popular microelectrode materials for neuroscience applications. Here we study how the scale of carbon nanotubes and carbon nanofibers affect neural viability, outgrowth, and adhesion. Carbon nanotubes were deposited on glass coverslips via a layer-by-layer method with polyethylenimine (PEI). Carbonized nanofibers were fabricated by electrospinning SU-8 and pyrolyzing the nanofiber depositions. Additional substrates tested were carbonized and SU-8 thin films and SU-8 nanofibers. Surfaces were O2-plasma treated, coated with varying concentrations of PEI, seeded with E18 rat cortical cells, and examined at 3, 4, and 7 days in vitro (DIV). Neural adhesion was examined at 4 DIV utilizing a parallel plate flow chamber. At 3 DIV, neural viability was lower on the nanofiber and thin film depositions treated with higher PEI concentrations which corresponded with significantly higher zeta potentials (surface charge); this significance was drastically higher on the nanofibers suggesting that the nanostructure may collect more PEI molecules, causing increased toxicity. At 7 DIV, significantly higher neurite outgrowth was observed on SU-8 nanofiber substrates with nanofibers a significant fraction of a neuron’s size. No differences were detected for carbonized nanofibers or carbon nanotubes. Both carbonized and SU-8 nanofibers had significantly higher cellular adhesion post-flow in comparison to controls whereas the carbon nanotubes were statistically similar to control substrates. These data suggest a neural cell preference for larger-scale nanomaterials with specific surface treatments. These characteristics could be taken advantage of in the future design and fabrication of neural microelectrodes. PMID:26829799

  1. Role of p53 in the cellular response following oleic acid accumulation in Chang liver cells.

    PubMed

    Park, Eun-Jung; Lee, Ah Young; Chang, Seung-Hee; Yu, Kyeong-Nam; Kim, Jae-Ho; Cho, Myung-Haing

    2014-01-03

    Abnormal accumulation of fatty acids triggers the harmful cellular response called lipotoxicity. In this study, we investigated the cellular response following accumulation of oleic acid (OA), a monounsaturated fatty acid, in human Chang liver cells. OA droplets were distributed freely in the cytoplasm and/or degraded within lysosomes. OA exposure increased ATP production and concomitantly dilated mitochondria. At 24h after OA exposure, cell viability decreased slightly and was coupled with a reduction in mitochondrial Ca(2+) concentration, the alteration in cell viability was also associated with the generation of reactive oxygen species and changes in the cell cycle. Moreover, OA treatment increased the expression of autophagy- and apoptotic cell death-related proteins in a dose-dependent manner. Furthermore, we investigated the role of p53, a tumor suppressor protein, in the cellular response elicited by OA accumulation. OA-induced changes in cell viability and ATP production were rescued to control levels when cells were pretreated with pifithrin-alpha (PTA), a p53 inhibitor. By contrast, the expressions of LC3-II and perilipin, proteins required for lipophagy, were down-regulated by PTA pretreatment. Taken together, our results suggest that p53 plays a key role in the cellular response elicited by OA accumulation in Chang liver cells.

  2. Cellular neuropathology of tuberous sclerosis.

    PubMed

    Huttenlocher, P R; Wollmann, R L

    1991-01-01

    The study of cerebral lesions of TSC by special histologic methods suggests that two populations of neurons and glia occur in TSC brains. One is a population of normally differentiated cells that form a normally constituted cortical plate. The other is a group of cells that are poorly differentiated, fail to organize into a normal cortical architecture, and form a variety of abnormal cellular aggregates in cortex and in subcortical locations. The proportion of these abnormal cells varies greatly from patient to patient. In some the central nervous system appears to be entirely spared. In others, only one or a few islands of dysplastic cells occur, whereas in still others a large number, perhaps even a majority, of neuroectodermal cells in the forebrain may be affected. The proportion of total cells that undergo abnormal differentiation apparently is an important factor relative to cortical function in TSC. At present we have no explanation for this marked heterogeneity in expression of the TSC gene or genes, and it remains one of the many unsolved mysteries of this illness.

  3. Electrostatic Tuning of Cellular Excitability

    PubMed Central

    Börjesson, Sara I.; Parkkari, Teija; Hammarström, Sven; Elinder, Fredrik

    2010-01-01

    Abstract Voltage-gated ion channels regulate the electric activity of excitable tissues, such as the heart and brain. Therefore, treatment for conditions of disturbed excitability is often based on drugs that target ion channels. In this study of a voltage-gated K channel, we propose what we believe to be a novel pharmacological mechanism for how to regulate channel activity. Charged lipophilic substances can tune channel opening, and consequently excitability, by an electrostatic interaction with the channel's voltage sensors. The direction of the effect depends on the charge of the substance. This was shown by three compounds sharing an arachidonyl backbone but bearing different charge: arachidonic acid, methyl arachidonate, and arachidonyl amine. Computer simulations of membrane excitability showed that small changes in the voltage dependence of Na and K channels have prominent impact on excitability and the tendency for repetitive firing. For instance, a shift in the voltage dependence of a K channel with −5 or +5 mV corresponds to a threefold increase or decrease in K channel density, respectively. We suggest that electrostatic tuning of ion channel activity constitutes a novel and powerful pharmacological approach with which to affect cellular excitability. PMID:20141752

  4. Demographics of polycystic kidney disease and captive population viability in pygmy hippopotamus (Choeropsis liberiensis).

    PubMed

    Flacke, Gabriella L; Tomkins, Joseph L; Black, Robert; Steck, Beatrice

    2017-02-15

    Polycystic kidney disease (PKD) was previously diagnosed at necropsy in several pygmy hippopotami (Choeropsis liberiensis) from the Smithsonian National Zoo and Zoo Basel, suggesting a threat to the long-term viability of the captive population. We determined the incidence and demographics of PKD in the captive population historically; we tested if the condition is linked to pedigree; we investigated mode of inheritance; we examined effects of PKD on longevity; we conducted survival analysis; and we examined long-term population viability. Thirty-seven percent of 149 necropsied adult pygmy hippos were affected by PKD, and it was more common in females, controlling for the overall female-biased sex-ratio. Prevalence increased significantly with age, but most hippos were beyond their reproductive prime before developing clinical signs; thus fecundity was likely unaffected. PKD was linked to pedigree and may exhibit X-linked dominance, but further research is needed to definitively establish the mode of inheritance. PKD did not affect longevity, overall or within any age class. There was no significant correlation between inbreeding coefficient (F) and PKD, and the prevalence in wild-caught and captive-born animals was similar. Longevity for both captive-born and inbred hippos (F > 0) was significantly shorter than longevity for their wild-caught and non-inbred counterparts. Demographic projections indicated the extant population will likely experience a slow increase over time, provided there are no space constraints. We conclude that although PKD is an important cause of morbidity and mortality in pygmy hippos, the condition is not a primary concern for overall viability of the captive population.

  5. Fibre based cellular transfection.

    PubMed

    Tsampoula, X; Taguchi, K; Cizmár, T; Garces-Chavez, V; Ma, N; Mohanty, S; Mohanty, K; Gunn-Moore, F; Dholakia, K

    2008-10-13

    Optically assisted transfection is emerging as a powerful and versatile method for the delivery of foreign therapeutic agents to cells at will. In particular the use of ultrashort pulse lasers has proved an important route to transiently permeating the cell membrane through a multiphoton process. Though optical transfection has been gaining wider usage to date, all incarnations of this technique have employed free space light beams. In this paper we demonstrate the first system to use fibre delivery for the optical transfection of cells. We engineer a standard optical fibre to generate an axicon tip with an enhanced intensity of the remote output field that delivers ultrashort (~ 800 fs) pulses without requiring the fibre to be placed in very close proximity to the cell sample. A theoretical model is also developed in order to predict the light propagation from axicon tipped and bare fibres, in both air and water environments. The model proves to be in good agreement with the experimental findings and can be used to establish the optimum fibre parameters for successful cellular transfection. We readily obtain efficiencies of up to 57 % which are comparable with free space transfection. This advance paves the way for optical transfection of tissue samples and endoscopic embodiments of this technique.

  6. Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling

    SciTech Connect

    Zhdanov, Alexander V.; Waters, Alicia H.C.; Golubeva, Anna V.; Papkovsky, Dmitri B.

    2015-01-01

    Changes in availability and utilisation of O{sub 2} and metabolic substrates are common in ischemia and cancer. We examined effects of substrate deprivation on HIF signalling in PC12 cells exposed to different atmospheric O{sub 2}. Upon 2–4 h moderate hypoxia, HIF-α protein levels were dictated by the availability of glutamine and glucose, essential for deep cell deoxygenation and glycolytic ATP flux. Nuclear accumulation of HIF-1α dramatically decreased upon inhibition of glutaminolysis or glutamine deprivation. Elevation of HIF-2α levels was transcription-independent and associated with the activation of Akt and Erk1/2. Upon 2 h anoxia, HIF-2α levels strongly correlated with cellular ATP, produced exclusively via glycolysis. Without glucose, HIF signalling was suppressed, giving way to other regulators of cell adaptation to energy crisis, e.g. AMPK. Consequently, viability of cells deprived of O{sub 2} and glucose decreased upon inhibition of AMPK with dorsomorphin. The capacity of cells to accumulate HIF-2α decreased after 24 h glucose deprivation. This effect, associated with increased AMPKα phosphorylation, was sensitive to dorsomorphin. In chronically hypoxic cells, glutamine played no major role in HIF-2α accumulation, which became mainly glucose-dependent. Overall, the availability of O{sub 2} and metabolic substrates intricately regulates HIF signalling by affecting cell oxygenation, ATP levels and pathways involved in production of HIF-α. - Highlights: • Gln and Glc regulate HIF levels in hypoxic cells by maintaining low O{sub 2} and high ATP. • HIF-α levels under anoxia correlate with cellular ATP and critically depend on Glc. • Gln and Glc modulate activity of Akt, Erk and AMPK, regulating HIF production. • HIF signalling is differentially inhibited by prolonged Glc and Gln deprivation. • Unlike Glc, Gln plays no major role in HIF signalling in chronically hypoxic cells.

  7. Membrane organization and regulation of cellular Cholesterol homeostasis

    PubMed Central

    Jaureguiberry, María S.; Tricerri, M. Alejandra; Sanchez, Susana A; Garda, Horacio A; Finarelli, Gabriela S.; Gonzalez, Marina C.; Rimoldi, Omar J.

    2010-01-01

    An excess of intracellular free Cholesterol (Chol) is cytotoxic, and its homeostasis is crucial for cell viability. Apolipoprotein A–I (apoA-I) is a highly efficient Chol acceptor as it activates complex cellular pathways that tend to mobilize and export Chol from cellular depots. Here we hypothesize that membrane composition and/or organization is strongly involved in Chol homeostasis. To test this hypothesis, we constructed a cell line over expressing Stearoyl CoA desaturase (SCD-cells), which modifies plasma membrane (PM) composition by the enrichment of monounsaturated fatty,acids and determined this effect on membrane properties, cell viability and cholesterol homeostasis. PM in SCD-cells has a higher phospholipids/sphingomyelin ratio and is slightly enriched in Chol. These cells showed an increase in the cholesteryl esters/free Chol ratio, they were more resistant to Chol toxicity and in addition, they exported more caveolin than Control cells. The data suggest that cell functionality is preserved by regulating membrane fluidity and Chol exportation and storage. PMID:20336284

  8. Membrane organization and regulation of cellular cholesterol homeostasis.

    PubMed

    Jaureguiberry, María S; Tricerri, M Alejandra; Sanchez, Susana A; Garda, Horacio A; Finarelli, Gabriela S; Gonzalez, Marina C; Rimoldi, Omar J

    2010-04-01

    An excess of intracellular free cholesterol (Chol) is cytotoxic, and its homeostasis is crucial for cell viability. Apolipoprotein A-I (apoA-I) is a highly efficient Chol acceptor because it activates complex cellular pathways that tend to mobilize and export Chol from cellular depots. We hypothesize that membrane composition and/or organization is strongly involved in Chol homeostasis. To test this hypothesis, we constructed a cell line overexpressing stearoyl coenzyme A (CoA) desaturase (SCD cells), which modifies plasma membrane (PM) composition by the enrichment of monounsaturated fatty acids, and determined this effect on membrane properties, cell viability, and Chol homeostasis. PM in SCD cells has a higher ratio of phospholipids to sphingomyelin and is slightly enriched in Chol. These cells showed an increase in the ratio of cholesteryl esters to free Chol; they were more resistant to Chol toxicity, and they exported more caveolin than control cells. The data suggest that cell functionality is preserved by regulating membrane fluidity and Chol exportation and storage.

  9. Stn1 is critical for telomere maintenance and long-term viability of somatic human cells

    PubMed Central

    Boccardi, Virginia; Razdan, Neetu; Kaplunov, Jessica; Mundra, Jyoti J; Kimura, Masayuki; Aviv, Abraham; Herbig, Utz

    2015-01-01

    Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging-associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA-mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long-term viability of normal somatic mammalian cells. PMID:25684230

  10. Zinc availability during germline development impacts embryo viability in Caenorhabditis elegans.

    PubMed

    Mendoza, Adelita D; Woodruff, Teresa K; Wignall, Sarah M; O'Halloran, Thomas V

    2017-01-01

    Zinc is an essential metal that serves as a cofactor in a variety of cellular processes, including meiotic maturation. Cellular control of zinc uptake, availability and efflux is closely linked to meiotic progression in rodent and primate reproduction where large fluctuations in zinc levels are critical at several steps in the oocyte-to-embryo transition. Despite these well-documented roles of zinc fluxes during meiosis, only a few of the genes encoding key zinc receptors, membrane-spanning transporters, and downstream signaling pathway factors have been identified to date. Furthermore, little is known about analogous roles for zinc fluxes in the context of a whole organism. Here, we evaluate whether zinc availability regulates germline development and oocyte viability in the nematode Caenorhabditis elegans, an experimentally flexible model organism. We find that similar to mammals, mild zinc limitation in C. elegans profoundly impacts the reproductive axis: the brood size is significantly reduced under conditions of zinc limitation where other physiological functions are not perturbed. Zinc limitation in this organism has a more pronounced impact on oocytes than sperm and this leads to the decrease in viable embryo production. Moreover, acute zinc limitation of isolated zygotes prevents extrusion of the second polar body during meiosis and leads to aneuploid embryos. Thus, the zinc-dependent steps in C. elegans gametogenesis roughly parallel those described in meiotic-to-mitotic transitions in mammals.

  11. Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability.

    PubMed

    Munkley, Jennifer; Vodak, Daniel; Livermore, Karen E; James, Katherine; Wilson, Brian T; Knight, Bridget; Mccullagh, Paul; Mcgrath, John; Crundwell, Malcolm; Harries, Lorna W; Leung, Hing Y; Robson, Craig N; Mills, Ian G; Rajan, Prabhakar; Elliott, David J

    2016-06-01

    Steroid androgen hormones play a key role in the progression and treatment of prostate cancer, with androgen deprivation therapy being the first-line treatment used to control cancer growth. Here we apply a novel search strategy to identify androgen-regulated cellular pathways that may be clinically important in prostate cancer. Using RNASeq data, we searched for genes that showed reciprocal changes in expression in response to acute androgen stimulation in culture, and androgen deprivation in patients with prostate cancer. Amongst 700 genes displaying reciprocal expression patterns we observed a significant enrichment in the cellular process glycosylation. Of 31 reciprocally-regulated glycosylation enzymes, a set of 8 (GALNT7, ST6GalNAc1, GCNT1, UAP1, PGM3, CSGALNACT1, ST6GAL1 and EDEM3) were significantly up-regulated in clinical prostate carcinoma. Androgen exposure stimulated synthesis of glycan structures downstream of this core set of regulated enzymes including sialyl-Tn (sTn), sialyl Lewis(X) (SLe(X)), O-GlcNAc and chondroitin sulphate, suggesting androgen regulation of the core set of enzymes controls key steps in glycan synthesis. Expression of each of these enzymes also contributed to prostate cancer cell viability. This study identifies glycosylation as a global target for androgen control, and suggests loss of specific glycosylation enzymes might contribute to tumour regression following androgen depletion therapy.

  12. Stn1 is critical for telomere maintenance and long-term viability of somatic human cells.

    PubMed

    Boccardi, Virginia; Razdan, Neetu; Kaplunov, Jessica; Mundra, Jyoti J; Kimura, Masayuki; Aviv, Abraham; Herbig, Utz

    2015-06-01

    Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging-associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA-mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long-term viability of normal somatic mammalian cells.

  13. Cell Attachment and Viability Study of PCL Nano-fiber Modified by Cold Atmospheric Plasma.

    PubMed

    Atyabi, Seyed Mohammad; Sharifi, Fereshteh; Irani, Shiva; Zandi, Mojgan; Mivehchi, Houri; Nagheh, Zahra

    2016-06-01

    The field of tissue engineering is an emerging discipline which applies the basic principles of life sciences and engineering to repair and restore living tissues and organs. The purpose of this study was to investigate the effect of cold and non-thermal plasma surface modification of poly (ϵ-caprolactone) (PCL) scaffolds on fibroblast cell behavior. Nano-fiber PCL was fabricated through electrospinning technique, and some fibers were then treated by cold and non-thermal plasma. The cell-biomaterial interactions were studied by culturing the fibroblast cells on nano-fiber PCL. Scaffold biocompatibility test was assessed using an inverted microscope. The growth and proliferation of fibroblast cells on nano-fiber PCL were analyzed by MTT viability assay. Cellular attachment on the nano-fiber and their morphology were evaluated using scanning electron microscope. The result of cell culture showed that nano-fiber could support the cellular growth and proliferation by developing three-dimensional topography. The present study demonstrated that the nano-fiber surface modification with cold plasma sharply enhanced the fibroblast cell attachment. Thus, cold plasma surface modification greatly raised the bioactivity of scaffolds.

  14. Viability and fertilizing capacity of cryopreserved sperm from three North American acipenseriform species: A retrospective study

    USGS Publications Warehouse

    Horvath, A.; Wayman, W.R.; Dean, J.C.; Urbanyi, B.; Tiersch, T.R.; Mims, S.D.; Johnson, D.; Jenkins, J.A.

    2008-01-01

    Populations of sturgeon across the globe are threatened due to unregulated harvest and habitat loss, and the status varies among species across North America. Ready access to viable and functional sperm would contribute to recovery programmes for these species. In this study, we examined the motility, viability (cell membrane integrity) of cryopreserved sperm from three North American acipenseriform species and fertilizing capacity. Milt samples were collected from captive shortnose sturgeon (Acipenser brevirostrum), wild paddlefish (Polyodon spathula) and pallid sturgeon (Scaphirhynchus albus) and cryopreserved using combinations of Modified Tsvetkova's (MT) extender, Original Tsvetkova's extender, and modified Hanks' balanced salt solution, along with the cryoprotectants methanol (MeOH) or dimethyl sulfoxide (DMSO). A dual-staining technique using the fluorescent stains SYBR-14 and propidium iodide was employed with flow cytometry to determine the percentages of spermatozoa that were viable by virtue of having intact membranes. The percentage of viable spermatozoa ranged from 5% to 12% in shortnose sturgeon, 30-59% in paddlefish, and 44-58% in pallid sturgeon. In the first experiment with shortnose sturgeon sperm, methanol allowed for higher values for dependent variables than did DMSO, and sperm viability generally correlated with post-thaw motility. However, fertilization rate, neurulation, or hatching rates were independent from these factors. In the second experiment with shortnose sturgeon, 5% MeOH combined with MT yielded higher values for all parameters tested than the other combinations: viability was correlated with motility, fertilization rate, and hatching rate. Overall, viability and post-thaw motility was not affected by the use of hyperosmotic extenders (OT) or cryoprotectants (DMSO), but their use decreased fertilization percentages. For paddlefish sperm (experiment 3), MT combined with 10% MeOH was clearly a good choice for cryopreservation

  15. Evaluating the Viability of Mobile Learning to Enhance Management Training

    ERIC Educational Resources Information Center

    Macdonald, Iain; Chiu, Jason

    2011-01-01

    A qualitative research project was conducted to test the viability of augmenting an e-learning program for workplace learners using mobile content delivered through smart phones. Ten learners taking a six week web-based e-learning course were given smart phones which enabled them to access approximately 70% of the course content, in addition to…

  16. 37 CFR 1.807 - Viability of deposit.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2014-07-01 2014-07-01 false Viability of deposit. 1.807 Section 1.807 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE GENERAL RULES OF PRACTICE IN PATENT CASES Biotechnology Invention Disclosures Deposit...

  17. 37 CFR 1.807 - Viability of deposit.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2012-07-01 2012-07-01 false Viability of deposit. 1.807 Section 1.807 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE GENERAL RULES OF PRACTICE IN PATENT CASES Biotechnology Invention Disclosures Deposit...

  18. 37 CFR 1.807 - Viability of deposit.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2011-07-01 2011-07-01 false Viability of deposit. 1.807 Section 1.807 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE GENERAL RULES OF PRACTICE IN PATENT CASES Biotechnology Invention Disclosures Deposit...

  19. 37 CFR 1.807 - Viability of deposit.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false Viability of deposit. 1.807 Section 1.807 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE GENERAL RULES OF PRACTICE IN PATENT CASES Biotechnology Invention Disclosures Deposit...

  20. 37 CFR 1.807 - Viability of deposit.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2013-07-01 2013-07-01 false Viability of deposit. 1.807 Section 1.807 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE GENERAL RULES OF PRACTICE IN PATENT CASES Biotechnology Invention Disclosures Deposit...

  1. Economic viability of beef cattle grazing systems under prolonged drought

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prolonged drought in the Southern Great Plains of the USA in recent years has raised concerns about vulnerability of beef cattle grazing systems under adverse climate change. To help address the economic viability of beef grazing operations in the Southern Great Plains, this paper provides an econom...

  2. Sewage sludge pasteurization by gamma radiation: Financial viability case studies

    NASA Astrophysics Data System (ADS)

    Swinwood, Jean F.; Kotler, Jiri

    This paper examines the financial viability of sewage sludge pasteurization by gamma radiation, by examining the following three North American scenarios: 1) Small volume sewage treatment plant experiencing high sludge disposal costs. 2) Large volume sewage treatment plant experiencing low sludge disposal costs. 3) Large volume sewage treatment plant experiencing high sludge disposal costs.

  3. Proving the Viability of a School Choice Voucher. Policy Brief

    ERIC Educational Resources Information Center

    Haller, Scott

    2015-01-01

    A recent Pioneer Institute report written by Ken Ardon and Cara Stilling Candal, "Modeling Urban Scholarship Vouchers in Massachusetts," explores the viability of a school choice voucher program in the Commonwealth. Nationally, school choice has been shown to improve parent satisfaction and student achievement, reduce racial segregation,…

  4. A comparison of assays measuring the viability of Legionella ...

    EPA Pesticide Factsheets

    Background: The relatively high prevalence of Legionella pneumophila in premise plumbing systems has been widely reported. Published reports indicate Legionella has a comparatively high resistance to chlorine and moreover has the ability to grow in phagocytic amoeba which could provide additional protection in chlorinated drinking water distribution systems. Copper-Silver (Cu-Ag) ionization treatment systems are commercially available for use in large building water systems to help control the risks from Legionella bacteria. The objectives of this study were to develop and optimize Legionella viability assays and use them to investigate the viability of Legionella bacteria after exposure to water treated with coppper and silver ions. Methods: Log phase L. pneumophila cells were used in all experiments and were generated by incubation at 35C for 48 hours in buffered yeast extract broth. Viability assays used included plating on buffered charcoal yeast extract agar to determine the number of culturable cells and treating cells with propidium monoazide (PMA) or ethidium monoazide (EMA) followed by quantitative PCR targeting mip gene of L. pneumophila. The qPCR viability assays were optimized using L. pneumophila inactivated by heat treatment at 65C for 60 min. The effectiveness of Cu-Ag ionization treatment was studied by inoculating L. pneumonia at 105 CFU/mL in water collected directly from a building water system that employed this technology and incubat

  5. Influence of electroporation on chicken blastoderm cell viability in vitro.

    PubMed

    Wawrzynska, Magdalena; Bednarczyk, Marek; Łakota, Pawel; Lubiszewska, Marta

    2008-01-01

    The aim of this study was to compare two types of devices used for blastoderm cell (BC) transfection: the Nucleofector (Amaxa, Biosystems) and the Multiporator (Eppendorf). To assess the influence of electric current on BCs, different conditions of both nucleofection and electroporation were used. Next, the viability of cells was assessed. The highest number of cells (90.8%) was viable after nucleofection in the G10 program. After transfection in the presence of pmaxGFP, the A23 program was found to be most advantageous. The elecroporation experiment with the Multiporator (Eppendorf) showed a significant influence of osmotic pressure and voltage on BC viability. Namely, in the isoosmolar buffer BC viability was statistically higher (P < or = 0.05) in comparison to the hypoosmolar buffer. The, viability of cells was statistically higher (P < or = 0.05) after application of 25V as compared to 50V. The efficiency of transfection in the presence of EGFP-C 1 after electroporation in 2 pulses, 25V, 500 micros in the isoosmolar buffer was better than in the recommended conditions in the Amaxa Biosystems A23 program.

  6. Low-level waste vitrification contact maintenance viability study

    SciTech Connect

    Leach, C.E., Westinghouse Hanford

    1996-07-12

    This study investigates the economic viability of contact maintenance in the Low-Level Waste Vitrification Facility, which is part of the Hanford Site Tank Waste Remediation System. This document was prepared by Flour Daniel, Inc., and transmitted to Westinghouse Hanford Company in September 1995.

  7. Proof of Economic Viability of Blended Learning Business Models

    ERIC Educational Resources Information Center

    Druhmann, Carsten; Hohenberg, Gregor

    2014-01-01

    The discussion on economically sustainable business models with respect to information technology is lacking in many aspects of proven approaches. In the following contribution the economic viability is valued based on a procedural model for design and evaluation of e-learning business models in the form of a case study. As a case study object a…

  8. Biodegradable Magnetic Particles for Cellular MRI

    NASA Astrophysics Data System (ADS)

    Nkansah, Michael Kwasi

    Cell transplantation has the potential to treat numerous diseases and injuries. While magnetic particle-enabled, MRI-based cell tracking has proven useful for visualizing the location of cell transplants in vivo, current formulations of particles are either too weak to enable single cell detection or have non-degradable polymer matrices that preclude clinical translation. Furthermore, the off-label use of commercial agents like Feridex®, Bangs beads and ferumoxytol for cell tracking significantly stunts progress in the field, rendering it needlessly susceptible to market externalities. The recent phasing out of Feridex from the market, for example, heightens the need for a dedicated agent specifically designed for MRI-based cell tracking. To this end, we engineered clinically viable, biodegradable particles of iron oxide made using poly(lactide-co-glycolide) (PLGA) and demonstrated their utility in two MRI-based cell tracking paradigms in vivo. Both micro- and nanoparticles (2.1±1.1 μm and 105±37 nm in size) were highly magnetic (56.7-83.7 wt% magnetite), and possessed excellent relaxometry (r2* relaxivities as high as 614.1 s-1mM-1 and 659.1 s -1mM-1 at 4.7 T respectively). Magnetic PLGA micropartides enabled the in vivo monitoring of neural progenitor cell migration to the olfactory bulb in rat brains over 2 weeks at 11.7 T with ˜2-fold greater contrast-to-noise ratio and ˜4-fold better sensitivity at detecting migrated cells in the olfactory bulb than Bangs beads. Highly magnetic PLGA nanoparticles enabled MRI detection (at 11.7 T) of up to 10 rat mesenchymal cells transplanted into rat brain at 100-μm resolution. Highly magnetic PLGA particles were also shown to degrade by 80% in mice liver over 12 weeks in vivo. Moreover, no adverse effects were observed on cellular viability and function in vitro after labeling a wide range of cells. Magnetically labeled rat mesenchymal and neural stem cells retained their ability to differentiate into multiple

  9. Multistructural biomimetic substrates for controlled cellular differentiation

    NASA Astrophysics Data System (ADS)

    Orza, Anamaria I.; Mihu, Carmen; Soritau, Olga; Diudea, Mircea; Florea, Adrian; Matei, Horea; Balici, Stefana; Mudalige, Thilak; Kanarpardy, Ganesh K.; Biris, Alexandru S.

    2014-02-01

    Multidimensional scaffolds are considered to be ideal candidates for regenerative medicine and tissue engineering based on their potential to provide an excellent microenvironment and direct the fate of the cultured cells. More recently, the use of stem cells in medicine has opened a new technological opportunity for controlled tissue formation. However, the mechanism through which the substrate directs the differentiation of stem cells is still rather unclear. Data concerning its specific surface chemistry, topology, and its signaling ability need to be further understood and analyzed. In our study, atomic force microscopy was used to study the stiffness, roughness, and topology of the collagen (Coll) and metallized collagen (MC) substrates, proposed as an excellent substrate for regenerative medicine. The importance of signaling molecules was studied by constructing a new hybrid signaling substrate that contains both collagen and laminin extracellular matrix (ECM) proteins. The cellular response—such as attachment capability, proliferation and cardiac and neuronal phenotype expression on the metallized and non-metallized hybrid substrates (collagen + laminin)—was studied using MTT viability assay and immunohistochemistry studies. Our findings indicate that such hybrid materials could play an important role in the regeneration of complex tissues.

  10. Putting the Rit in cellular resistance

    PubMed Central

    Cai, Weikang; Shi, Geng-Xian; Andres, Douglas A.

    2013-01-01

    Cells mobilize diverse signaling pathways to protect against stress-mediated injury. Ras family GTPases play critical roles in this process, controlling the activation and integration of multiple regulatory cascades. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also promote cell survival. However, this functional dichotomy is incompletely understood, particularly regulation of p38-dependent survival. Here, we discuss our recent evidence that the Rit GTPase associates with and is required for stress-mediated activation of a scaffolded p38-MK2-HSP27-Akt pro-survival signaling cascade. Drosophila lacking D-Ric, a Rit homologue, are susceptible to a variety of environmental stresses, while embryonic fibroblasts derived from Rit knockout mice display blunted stress-dependent signaling and decreased viability. Conversely, expression of constitutively active Rit triggers p38-Akt-dependent cell survival. Together, our studies establish Rit as the central regulator of an evolutionarily conserved, p38-dependent signaling cascade that functions as a critical survival mechanism in response to stress. PMID:23802035

  11. The Impaired Viability of Prostate Cancer Cell Lines by the Recombinant Plant Kallikrein Inhibitor*

    PubMed Central

    Ferreira, Joana Gasperazzo; Diniz, Paula Malloy Motta; de Paula, Cláudia Alessandra Andrade; Lobo, Yara Aparecida; Paredes-Gamero, Edgar Julian; Paschoalin, Thaysa; Nogueira-Pedro, Amanda; Maza, Paloma Korehisa; Toledo, Marcos Sergio; Suzuki, Erika; Oliva, Maria Luiza Vilela

    2013-01-01

    Prostate cancer is the most common type of cancer, and kallikreins play an important role in the establishment of this disease. rBbKIm is the recombinant Bauhinia bauhinioides kallikreins inhibitor that was modified to include the RGD/RGE motifs of the inhibitor BrTI from Bauhinia rufa. This work reports the effects of rBbKIm on DU145 and PC3 prostate cancer cell lines. rBbKIm inhibited the cell viability of DU145 and PC3 cells but did not affect the viability of fibroblasts. rBbKIm caused an arrest of the PC3 cell cycle at the G0/G1 and G2/M phases but did not affect the DU145 cell cycle, although rBbKIm triggers apoptosis and cytochrome c release into the cytosol of both cell types. The differences in caspase activation were observed because rBbKIm treatment promoted activation of caspase-3 in DU145 cells, whereas caspase-9 but not caspase-3 was activated in PC3 cells. Because angiogenesis is important to the development of a tumor, the effect of rBbKIm in this process was also analyzed, and an inhibition of 49% was observed in in vitro endothelial cell capillary-like tube network formation. In summary, we demonstrated that different properties of the protease inhibitor rBbKIm may be explored for investigating the androgen-independent prostate cancer cell lines PC3 and DU145. PMID:23511635

  12. Mitochondrial DNA ligase is dispensable for the viability of cultured cells but essential for mtDNA maintenance.

    PubMed

    Shokolenko, Inna N; Fayzulin, Rafik Z; Katyal, Sachin; McKinnon, Peter J; Wilson, Glenn L; Alexeyev, Mikhail F

    2013-09-13

    Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ(0) phenotype.

  13. Viability of fibroblasts cultured under nutritional stress irradiated with red laser, infrared laser, and red light-emitting diode

    NASA Astrophysics Data System (ADS)

    Volpato, Luiz Evaristo Ricci; de Oliveira, Rodrigo Cardoso; Espinosa, Mariano Martinez; Bagnato, Vanderley Salvador; Machado, Maria A. A. M.

    2011-07-01

    Phototherapy is noninvasive, painless and has no known side effect. However, for its incorporation into clinical practice, more well-designed studies are necessary to define optimal parameters for its application. The viability of fibroblasts cultured under nutritional stress irradiated with either a red laser, an infrared laser, or a red light-emitting diode (LED) was analyzed. Irradiation parameters were: red laser (660 nm, 40 mW, 1 W/cm2), infrared laser (780 nm, 40 mW, 1 W/cm2), and red LED (637 +/- 15 nm, 40 mW, 1 W/cm2). All applications were punctual and performed with a spot with 0.4 mm2 of diameter for 4 or 8 s. The Kruskal-Wallis test and analysis of variance of the general linear model (p <= 0.05) were used for statistical analysis. After 72 h, phototherapy with low-intensity laser and LED showed no toxicity at the cellular level. It even stimulated methylthiazol tetrazolium assay (MTT) conversion and neutral red uptake of fibroblasts cultured under nutritional stress, especially in the group irradiated with infrared laser (p = 0.004 for MTT conversion and p < 0.001 for neutral red uptake). Considering the parameters and protocol of phototherapy used, it can be concluded that phototherapy stimulated the viability of fibroblasts cultured under nutritional deficit resembling those found in traumatized tissue in which cell viability is reduced.

  14. Mitochondrial DNA Ligase Is Dispensable for the Viability of Cultured Cells but Essential for mtDNA Maintenance*

    PubMed Central

    Shokolenko, Inna N.; Fayzulin, Rafik Z.; Katyal, Sachin; McKinnon, Peter J.; Wilson, Glenn L.; Alexeyev, Mikhail F.

    2013-01-01

    Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ0 phenotype. PMID:23884459

  15. Molecular Viability Testing of UV-Inactivated Bacteria.

    PubMed

    Weigel, Kris M; Nguyen, Felicia K; Kearney, Moira R; Meschke, John S; Cangelosi, Gerard A

    2017-03-10

    The polymerase chain reaction (PCR) is effective at detecting bacterial DNA in samples, but it is unable to differentiate viable bacteria from inactivated cells or free DNA fragments. New PCR-based analytical strategies have been developed to address this limitation. Molecular viability testing (MVT) correlates bacterial viability with the ability to rapidly synthesize species-specific ribosomal RNA precursor (pre-rRNA) in response to brief nutritional stimulation. Previous studies demonstrated that MVT can assess bacterial inactivation by chlorine, serum, and low-temperature pasteurization. Here, we demonstrate that MVT can detect inactivation of Escherichia coli, Aeromonas hydrophila, and Enterococcus faecalis cells by ultraviolet (UV) irradiation. Some UV-inactivated E. coli cells transiently retained the ability to synthesize pre-rRNA post-irradiation (generating false-positive MVT results), but this activity ceased within one hour following UV exposure. Viable but transiently undetectable (by culture) E. coli cells were consistently detected by MVT. An alternative viability testing method, viability PCR (vPCR), correlates viability with cell envelope integrity. This method did not distinguish viable from UV-inactivated bacteria under some conditions, indicating that the inactivated cells retained intact cell envelopes. MVT holds promise as a means to rapidly assess microbial inactivation by UV treatment.IMPORTANCE Ultraviolet (UV) irradiation is increasingly used to disinfect water, food, and other materials for human use. Confirming the effectiveness of UV disinfection remains a challenging task. In particular, microbiological methods that rely on rapid detection of microbial DNA can yield misleading results. This is due to the detection of "remnant" DNA associated with dead microbial cells. This report describes a novel method that rapidly distinguishes living from dead microbial cells after UV disinfection.

  16. Viability and infectivity of fresh and cryopreserved Nosema ceranae spores.

    PubMed

    McGowan, Janine; De la Mora, Alvaro; Goodwin, Paul H; Habash, Marc; Hamiduzzaman, Mollah Md; Kelly, Paul G; Guzman-Novoa, Ernesto

    2016-12-01

    The microsporidium fungus Nosema ceranae is an intracellular parasite that infects the midgut of the honey bee, Apis mellifera. A major limitation of research on N. ceranae is that the fungus is non-culturable and thus studying it depends on the seasonal availability of Nosema spores. Also, spore viability and infectivity can vary considerably, and thus there is a need for reliable methods for determining those traits. This study examined different conditions for N. ceranae spore cryopreservation at -70°C, assessing spore viability and infectivity. Viability was determined by a staining procedure counting total spores numbers with bright field microscopy and un-viable spore numbers with the fluorescent dye, propidium iodide. Spore infectivity was determined with a dilution inoculation assay. Infectivity was dependent on the inoculum dose for the proportion of bees with detectable Nosema infections based on the number of spores per bee at 18days after inoculation; 4000 spores per bee or higher were needed to get approx. 100% of the inoculated bees infected. The median infective dose (ID50) was 149 spores per bee, and the minimum dose capable of causing a detectable infection was 1.28 spores. The proportion of N. ceranae infected bees correlated significantly with the number of spores per bee (r=0.98, P<0.0001). N. ceranae spores cryopreserved in water or 10% glycerol did not differ in viability compared to fresh spores, but lost infectivity when inoculated into bees. This study shows that while cryopreservation of N. ceranae spores can preserve viability, the spores can have reduced infectivity.

  17. 47 CFR 22.909 - Cellular markets.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false Cellular markets. 22.909 Section 22.909... Cellular Radiotelephone Service § 22.909 Cellular markets. Cellular markets are standard geographic areas used by the FCC for administrative convenience in the licensing of cellular systems. Cellular...

  18. 47 CFR 22.909 - Cellular markets.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 2 2011-10-01 2011-10-01 false Cellular markets. 22.909 Section 22.909... Cellular Radiotelephone Service § 22.909 Cellular markets. Cellular markets are standard geographic areas used by the FCC for administrative convenience in the licensing of cellular systems. Cellular...

  19. 47 CFR 22.909 - Cellular markets.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 2 2012-10-01 2012-10-01 false Cellular markets. 22.909 Section 22.909... Cellular Radiotelephone Service § 22.909 Cellular markets. Cellular markets are standard geographic areas used by the FCC for administrative convenience in the licensing of cellular systems. Cellular...

  20. 47 CFR 22.909 - Cellular markets.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 2 2014-10-01 2014-10-01 false Cellular markets. 22.909 Section 22.909... Cellular Radiotelephone Service § 22.909 Cellular markets. Cellular markets are standard geographic areas used by the FCC for administrative convenience in the licensing of cellular systems. Cellular...

  1. 47 CFR 22.909 - Cellular markets.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 2 2013-10-01 2013-10-01 false Cellular markets. 22.909 Section 22.909... Cellular Radiotelephone Service § 22.909 Cellular markets. Cellular markets are standard geographic areas used by the FCC for administrative convenience in the licensing of cellular systems. Cellular...

  2. Cellular Structure Pattern in Dielectric Barrier Discharge

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Dong, Lifang; Liu, Weibo; Gao, Xing; Wei, Lingyan

    2015-12-01

    We report the observation of a cellular structure pattern in a dielectric barrier discharge system. The evolution sequence and phase diagram of the pattern are given. It is firstly observed that the "cell nucleus" fire three or even more times at a fixed location at the rising edge of the applied voltage, and that the "cell walls" which have the same discharge times with the "cell nucleus" are ignited slightly after the "cell nucleus". By observing a series of frames recorded by a high speed video camera, it is found that the cellular structure pattern consists of volume discharges (VDs) and surface discharges (SDs) corresponding to the "cell nucleus" and "cell walls" respectively. That VDs and SDs are ignited in turn for several times in each half cycle of the applied voltage confirms the fact that VDs induce the SDs and SDs also affect the following VDs.

  3. The effect of simultaneous exposure of HEMn-DP and HEMn-LP melanocytes to nicotine and UV-radiation on the cell viability and melanogenesis.

    PubMed

    Delijewski, Marcin; Wrześniok, Dorota; Beberok, Artur; Rok, Jakub; Otręba, Michał; Buszman, Ewa

    2016-11-01

    Nicotine is a main compound of tobacco plants and may affect more than a billion people all over the world that are permanently exposed to nicotine from cigarettes, various forms of smoking cessation therapies, electronic cigarettes or second-hand smoke. It is known that nicotine forms complexes with melanin what may lead to accumulation of this alkaloid in tissues of living organisms containing the pigment. This may affect the viability of cells and process of melanin biosynthesis that takes place in melanocytes. Although UV radiation is known to be a particular inductor of melanin biosynthesis, its simultaneous effect with nicotine on this process as well as the viability of human cells containing melanin have not been assessed so far. The aim of this study was to examine the simultaneous impact of nicotine and UV radiation on viability and melanogenesis in cultured normal human melanocytes dark (HEMn-DP) and light (HEMn-LP) pigmented. Nicotine together with UV radiation induced concentration-dependent loss in melanocytes viability. The higher cell loss was observed in dark pigmented melanocytes in comparison to light pigmented cells. Simultaneous exposure of cells to nicotine and UV radiation also caused changes in melanization process in both tested cell lines. The data suggest that simultaneous exposure of melanocytes to nicotine and UV radiation up-regulates melanogenesis and affects cell viability. Observed processes are more pronounced in dark pigmented cells.

  4. Cellular circadian clocks in mood disorders.

    PubMed

    McCarthy, Michael J; Welsh, David K

    2012-10-01

    Bipolar disorder (BD) and major depressive disorder (MDD) are heritable neuropsychiatric disorders associated with disrupted circadian rhythms. The hypothesis that circadian clock dysfunction plays a causal role in these disorders has endured for decades but has been difficult to test and remains controversial. In the meantime, the discovery of clock genes and cellular clocks has revolutionized our understanding of circadian timing. Cellular circadian clocks are located in the suprachiasmatic nucleus (SCN), the brain's primary circadian pacemaker, but also throughout the brain and peripheral tissues. In BD and MDD patients, defects have been found in SCN-dependent rhythms of body temperature and melatonin release. However, these are imperfect and indirect indicators of SCN function. Moreover, the SCN may not be particularly relevant to mood regulation, whereas the lateral habenula, ventral tegmentum, and hippocampus, which also contain cellular clocks, have established roles in this regard. Dysfunction in these non-SCN clocks could contribute directly to the pathophysiology of BD/MDD. We hypothesize that circadian clock dysfunction in non-SCN clocks is a trait marker of mood disorders, encoded by pathological genetic variants. Because network features of the SCN render it uniquely resistant to perturbation, previous studies of SCN outputs in mood disorders patients may have failed to detect genetic defects affecting non-SCN clocks, which include not only mood-regulating neurons in the brain but also peripheral cells accessible in human subjects. Therefore, reporters of rhythmic clock gene expression in cells from patients or mouse models could provide a direct assay of the molecular gears of the clock, in cellular clocks that are likely to be more representative than the SCN of mood-regulating neurons in patients. This approach, informed by the new insights and tools of modern chronobiology, will allow a more definitive test of the role of cellular circadian clocks

  5. Cellularized Bilayer Pullulan-Gelatin Hydrogel for Skin Regeneration.

    PubMed

    Nicholas, Mathew N; Jeschke, Marc G; Amini-Nik, Saeid

    2016-05-01

    Skin substitutes significantly reduce the morbidity and mortality of patients with burn injuries and chronic wounds. However, current skin substitutes have disadvantages related to high costs and inadequate skin regeneration due to highly inflammatory wounds. Thus, new skin substitutes are needed. By combining two polymers, pullulan, an inexpensive polysaccharide with antioxidant properties, and gelatin, a derivative of collagen with high water absorbency, we created a novel inexpensive hydrogel-named PG-1 for "pullulan-gelatin first generation hydrogel"-suitable for skin substitutes. After incorporating human fibroblasts and keratinocytes onto PG-1 using centrifugation over 5 days, we created a cellularized bilayer skin substitute. Cellularized PG-1 was compared to acellular PG-1 and no hydrogel (control) in vivo in a mouse excisional skin biopsy model using newly developed dome inserts to house the skin substitutes and prevent mouse skin contraction during wound healing. PG-1 had an average pore size of 61.69 μm with an ideal elastic modulus, swelling behavior, and biodegradability for use as a hydrogel for skin substitutes. Excellent skin cell viability, proliferation, differentiation, and morphology were visualized through live/dead assays, 5-bromo-2'-deoxyuridine proliferation assays, and confocal microscopy. Trichrome and immunohistochemical staining of excisional wounds treated with the cellularized skin substitute revealed thicker newly formed skin with a higher proportion of actively proliferating cells and incorporation of human cells compared to acellular PG-1 or control. Excisional wounds treated with acellular or cellularized hydrogels showed significantly less macrophage infiltration and increased angiogenesis 14 days post skin biopsy compared to control. These results show that PG-1 has ideal mechanical characteristics and allows ideal cellular characteristics. In vivo evidence suggests that cellularized PG-1 promotes skin regeneration and may

  6. High levels of inorganic nutrients affect fertilization kinetics, early development and settlement of the scleractinian coral Platygyra acuta

    NASA Astrophysics Data System (ADS)

    Lam, E. K. Y.; Chui, A. P. Y.; Kwok, C. K.; Ip, A. H. P.; Chan, S. W.; Leung, H. N.; Yeung, L. C.; Ang, P. O.

    2015-09-01

    Dose-response experiments were conducted to investigate the effects of ammonia nitrogen (NH3/NH4 +) and orthophosphate (PO4 3-) on four stages of larval development in Platygyra acuta, including fertilization, embryonic development and the survival, motility, and settlement of planula larvae. Fertilization success was reduced significantly under 200 μM NH3/NH4 + or PO4 3-. These high doses of NH3/NH4 + and PO4 - affected egg viability (or sperm viability and polyspermic block simultaneously) and polyspermic block, respectively. These results provide the first evidence to indicate the mechanisms of how inorganic nutrients might affect coral fertilization processes. For embryonic development, NH3/NH4 + at 25-200 μM caused delay in cell division after 2-h exposure and NH3/NH4 + at 100-200 μM resulted in larval death after 72 h. However, no significant differences were observed in the mobility and survivorship of either planula or competent larvae under different levels of NH3/NH4 + or PO4 3-. There was a significant (~30 %) drop in the settlement of competent larvae under the combined effect of 100 μM NH3/NH4 + and PO4 3-. The effects of elevated nutrients appeared to become more significant only on gametes or larvae undergoing active cellular activities at fertilization, early development, and settlement.

  7. Influence of marihuana on cellular structures and biochemical activities.

    PubMed

    Tahir, S K; Zimmerman, A M

    1991-11-01

    Cannabinoids are known to affect a number of cellular systems and functions, but the basis for their action is unclear. In this paper we review the current evidence describing cannabinoid effects on various levels of cellular structure and activity and we present our current studies on the influence of delta-9-tetrahydrocannabinol, cannabidiol and cannabinol on one cellular system, the cytoskeleton. The organization of two cytoskeletal structures, microtubules and microfilaments, were examined and the mRNA levels of tubulin and actin, the major protein components of microtubules and microfilaments, respectively, were analysed.

  8. On Patterns in Affective Media

    NASA Astrophysics Data System (ADS)

    ADAMATZKY, ANDREW

    In computational experiments with cellular automaton models of affective solutions, where chemical species represent happiness, anger, fear, confusion and sadness, we study phenomena of space time dynamic of emotions. We demonstrate feasibility of the affective solution paradigm in example of emotional abuse therapy. Results outlined in the present paper offer unconventional but promising technique to design, analyze and interpret spatio-temporal dynamic of mass moods in crowds.

  9. Rapid assessment of the toxicity of oil sands process-affected waters using fish cell lines.

    PubMed

    Sansom, Bryan; Vo, Nguyen T K; Kavanagh, Richard; Hanner, Robert; Mackinnon, Michael; Dixon, D George; Lee, Lucy E J

    2013-01-01

    Rapid and reliable toxicity assessment of oil sands process-affected waters (OSPW) is needed to support oil sands reclamation projects. Conventional toxicity tests using whole animals are relatively slow, costly, and often subjective, while at the same time requiring the sacrifice of test organisms as is the case with lethal dosage/concentration assays. A nonlethal alternative, using fish cell lines, has been developed for its potential use in supporting oil sands reclamation planning and to help predict the viability of aquatic reclamation models such as end-pit lakes. This study employed six fish cell lines (WF-2, GFSk-S1, RTL-W1, RTgill-W1, FHML, FHMT) in 24 h viability assays for rapid fluorometric assessment of cellular integrity and functionality. Forty-nine test water samples collected from the surface of oil sands developments in the Athabasca Oil Sands deposit, north of Fort McMurray, Alberta, Canada, were evaluated in blind. Small subsample volumes (8 ml) were mixed with 2 ml of 5× concentrated exposure media and used for direct cell exposures. All cell line responses in terms of viability as measured by Alamar blue assay, correlated well with the naphthenic acids (NA) content in the samples (R (2) between 0.4519 and 0.6171; p<0.0001) when data comparisons were performed after the bioassays. NA or total acid-extractable organics group has been shown to be responsible for most of the acute toxicity of OSPW and our results further corroborate this. The multifish cell line bioassay provides a strong degree of reproducibility among tested cell lines and good relative sensitivity of the cell line bioassay as compared to available in vivo data that could lead to cost effective, high-throughput screening assays.

  10. Drosophila Uri, a PP1α binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity

    PubMed Central

    Kirchner, Jasmin; Vissi, Emese; Gross, Sascha; Szoor, Balazs; Rudenko, Andrey; Alphey, Luke; White-Cooper, Helen

    2008-01-01

    Background Protein phosphatase 1 (PP1) is involved in diverse cellular processes, and is targeted to substrates via interaction with many different protein binding partners. PP1 catalytic subunits (PP1c) fall into PP1α and PP1β subfamilies based on sequence analysis, however very few PP1c binding proteins have been demonstrated to discriminate between PP1α and PP1β. Results URI (unconventional prefoldin RPB5 interactor) is a conserved molecular chaperone implicated in a variety of cellular processes, including the transcriptional response to nutrient signalling and maintenance of DNA integrity. We show that Drosophila Uri binds PP1α with much higher affinity than PP1β, and that this ability to discriminate between PP1c forms is conserved to humans. Most Uri is cytoplasmic, however we found some protein associated with active RNAPII on chromatin. We generated a uri loss of function allele, and show that uri is essential for viability in Drosophila. uri mutants have transcriptional defects, reduced cell viability and differentiation in the germline, and accumulate DNA damage in their nuclei. Conclusion Uri is the first PP1α specific binding protein to be described in Drosophila. Uri protein plays a role in transcriptional regulation. Activity of uri is required to maintain DNA integrity and cell survival in normal development. PMID:18412953

  11. MSAT and cellular hybrid networking

    NASA Technical Reports Server (NTRS)

    Baranowsky, Patrick W., II

    1993-01-01

    Westinghouse Electric Corporation is developing both the Communications Ground Segment and the Series 1000 Mobile Phone for American Mobile Satellite Corporation's (AMSC's) Mobile Satellite (MSAT) system. The success of the voice services portion of this system depends, to some extent, upon the interoperability of the cellular network and the satellite communication circuit switched communication channels. This paper will describe the set of user-selectable cellular interoperable modes (cellular first/satellite second, etc.) provided by the Mobile Phone and described how they are implemented with the ground segment. Topics including roaming registration and cellular-to-satellite 'seamless' call handoff will be discussed, along with the relevant Interim Standard IS-41 Revision B Cellular Radiotelecommunications Intersystem Operations and IOS-553 Mobile Station - Land Station Compatibility Specification.

  12. Egg Viability, Mating Frequency and Male Mating Ability Evolve in Populations of Drosophila melanogaster Selected for Resistance to Cold Shock

    PubMed Central

    Singh, Karan; Kochar, Ekta; Prasad, N. G.

    2015-01-01

    Background Ability to resist temperature shock is an important component of fitness of insects and other ectotherms. Increased resistance to temperature shock is known to affect life-history traits. Temperature shock is also known to affect reproductive traits such as mating ability and viability of gametes. Therefore selection for increased temperature shock resistance can affect the evolution of reproductive traits. Methods We selected replicate populations of Drosophila melanogaster for resistance to cold shock. We then investigated the evolution of reproductive behavior along with other components of fitness- larval survivorship, adult mortality, fecundity, egg viability in these populations. Results We found that larval survivorship, adult mortality and fecundity post cold shock were not significantly different between selected and control populations. However, compared to the control populations, the selected populations laid significantly higher percentage of fertile eggs (egg viability) 24 hours post cold shock. The selected populations had higher mating frequency both with and without cold shock. After being subjected to cold shock, males from the selected populations successfully mated with significantly more non-virgin females and sired significantly more progeny compared to control males. Conclusions A number of studies have reported the evolution of survivorship in response to selection for temperature shock resistance. Our results clearly indicate that adaptation to cold shock can involve changes in components of reproductive fitness. Our results have important implications for our understanding of how reproductive behavior can evolve in response to thermal stress. PMID:26065704

  13. Effect of fluoride on the cell viability, cell organelle potential, and photosynthetic capacity of freshwater and soil algae.

    PubMed

    Chae, Yooeun; Kim, Dokyung; An, Youn-Joo

    2016-12-01

    Although fluoride occurs naturally in the environment, excessive amounts of fluoride in freshwater and terrestrial ecosystems can be harmful. We evaluated the toxicity of fluoride compounds on the growth, viability, and photosynthetic capacity of freshwater (Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata) and terrestrial (Chlorococcum infusionum) algae. To measure algal growth inhibition, a flow cytometric method was adopted (i.e., cell size, granularity, and auto-fluorescence measurements), and algal yield was calculated to assess cell viability. Rhodamine123 and fluorescein diacetate were used to evaluate mitochondrial membrane potential (MMA, ΔΨm) and cell permeability. Nine parameters related to the photosynthetic capacity of algae were also evaluated. The results indicated that high concentrations of fluoride compounds affected cell viability, cell organelle potential, and photosynthetic functions. The cell viability measurements of the three algal species decreased, but apoptosis was only observed in C. infusionum. The MMA (ΔΨm) of cells exposed to fluoride varied among species, and the cell permeability of the three species generally decreased. The decrease in the photosynthetic activity of algae may be attributable to the combination of fluoride ions (F(-)) with magnesium ions (Mg(2+)) in chlorophyll. Our results therefore provide strong evidence for the potential risks of fluoride compounds to microflora and microfauna in freshwater and terrestrial ecosystems.

  14. Enhanced resveratrol production in Vitis vinifera cell suspension cultures by heavy metals without loss of cell viability.

    PubMed

    Cai, Zhenzhen; Kastell, Anja; Speiser, Claire; Smetanska, Iryna

    2013-09-01

    The effects of heavy metal ions (Co(2+), Ag(+), Cd(2+)) on cell viability and secondary metabolite production, particularly anthocyanins and phenolic acids in Vitis vinifera cell suspension cultures, were investigated. Of these, Co at all three used concentrations (5.0, 25, and 50 μM), Ag, and Cd at low concentration (5.0 μM) were most effective to stimulate the phenolic acid production, increasing the 3-O-glucosyl-resveratrol up to 1.6-fold of the control level (250.5 versus 152.4 μmol/g), 4 h after the treatments. Meanwhile, the elicitors at effective concentrations did not suppress cell growth, while the cell viability maintained. In contrast, Ag and Cd at high concentrations (25 and 50 μM) remarkably reduced the cell viability, decreasing the cell viability up to about 15 % of the control level, 24 h after the treatments. The heavy metal ions did not affect the anthocyanin production. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, peroxidase activity, medium pH value, and conductivity were only slightly elevated by the heavy metal ions. The results suggest that some of the secondary metabolites production was stimulated by the used elicitors, but there was not a stress response of the cells.

  15. Multiparametric flow cytometry allows rapid assessment and comparison of lactic acid bacteria viability after freezing and during frozen storage.

    PubMed

    Rault, Aline; Béal, Catherine; Ghorbal, Sarrah; Ogier, Jean-Claude; Bouix, Marielle

    2007-08-01

    Freezing is widely used for the long-term preservation of lactic acid bacteria, but often affects their viability and technological properties. Different methods are currently employed to determine bacterial cryotolerance, but they all require several hours or days before achieving results. The aim of this study was to establish the advantages of multiparametric flow cytometry by using two specific fluorescent probes to provide rapid assessment of the viability of four strains of Lactobacillus delbrueckii after freezing and during frozen storage. The relevance of carboxyfluorescein diacetate and propidium iodide to quantify bacterial viability was proven. When bacterial suspensions were simultaneously stained with these two fluorescent probes, three major subpopulations were identified: viable, dead and injured cells. The cryotolerance of four L. delbrueckii strains was evaluated by quantifying the relative percentages of each subpopulation before and after freezing, and throughout one month of storage at -80 degrees C. Results displayed significant differences in the resistance to freezing and frozen storage of the four strains when they were submitted to the same freezing and storage procedures. Whereas resistant strains displayed less than 10% of dead cells after one month of storage, one sensitive strain exhibited more than 50% of dead cells, together with 14% of stressed cells after freezing. Finally, this study proved that multiparametric flow cytometry was a convenient and rapid tool to evaluate the viability of lactic acid bacteria, and was well correlated with plate count results. Moreover, it made it possible to differentiate strains according to their susceptibility to freezing and frozen storage.

  16. Cellular interaction of different forms of aluminum nanoparticles in rat alveolar macrophages.

    PubMed

    Wagner, Andrew J; Bleckmann, Charles A; Murdock, Richard C; Schrand, Amanda M; Schlager, John J; Hussain, Saber M

    2007-06-28

    Nanomaterials, with dimensions in the 1-100 nm range, possess numerous potential benefits to society. However, there is little characterization of their effects on biological systems, either within the environment or on human health. The present study examines cellular interaction of aluminum oxide and aluminum nanomaterials, including their effect on cell viability and cell phagocytosis, with reference to particle size and the particle's chemical composition. Experiments were performed to characterize initial in vitro cellular effects of rat alveolar macrophages (NR8383) after exposure to aluminum oxide nanoparticles (Al2O3-NP at 30 and 40 nm) and aluminum metal nanoparticles containing a 2-3 nm oxide coat (Al-NP at 50, 80, and 120 nm). Characterization of the nanomaterials, both as received and in situ, was performed using transmission electron microscopy (TEM), dynamic light scattering (DLS), laser Doppler velocimetry (LDV), and/or CytoViva150 Ultra Resolution Imaging (URI)). Particles showed significant agglomeration in cell exposure media using DLS and the URI as compared to primary particle size in TEM. Cell viability assay results indicate a marginal effect on macrophage viability after exposure to Al2O3-NP at doses of 100 microg/mL for 24 h continuous exposure. Al-NP produced significantly reduced viability after 24 h of continuous exposure with doses from 100 to 250 microg/mL. Cell phagocytotic ability was significantly hindered by exposure to 50, 80, or 120 nm Al-NP at 25 microg/mL for 24 h, but the same concentration (25 microg/mL) had no significant effect on the cellular viability. However, no significant effect on phagocytosis was observed with Al2O3-NP. In summary, these results show that Al-NP exhibit greater toxicity and more significantly diminish the phagocytotic ability of macrophages after 24 h of exposure when compared to Al2O3-NP.

  17. Short communication: The effect of water temperature on the viability of silage inoculants.

    PubMed

    Mulrooney, C N; Kung, L

    2008-01-01

    The objective of this study was to determine if exposure to high temperatures in water affects the viability of various silage inoculants. Inoculants were enumerated on De Man, Rogosa, Sharpe agar to standardize a final count (colony-forming units) in water such that about 500 mL added to 1 tonne of wet forage would achieve a recommended application rate of about 100,000 cfu of lactic acid bacteria (LAB) per gram of wet forage. Testing was done in 4 sequences (SEQ). For each SEQ, inoculants were mixed in deionized water for 45 min at 30 degrees C followed by incubation for 6 h at 30 degrees C (SEQ 1), 35 degrees C (SEQ 2), 40 degrees C (SEQ 3), or 45 degrees C (SEQ 4) in duplicate 125-mL flasks rotating at 125 rpm. After 6 h, rotation was stopped and the temperature was lowered to 30 degrees C for the next 18 h for all SEQ. Numbers of LAB were enumerated at 0, 3, 6, and 24 h. Each sequence was repeated twice. Incubation at a moderate temperature (SEQ 1) did not affect the viability of the microbial inoculants. The viability of the inoculants declined with increasing temperature (SEQ 2 to 4) but the effect varied by inoculant. For some inoculants exposure to 35 degrees C resulted in substantial decreases in viable cells (loss of 0.5 to 1 log cfu/mL). Incubation at higher temperatures resulted in even greater losses in viability for some inoculants. Losses of more than 0.5 log cfu/mL would most likely make the application of these inoculants ineffective in the field. Lactobacillus plantarum MTD/1 was the most thermotolerant organism tested, because it was unaffected by all temperatures (30 to 45 degrees C) after 3 h of incubation. Lactobacillus plantarum MTD/1 and Lactobacillus buchneri 40788 also appeared to have better thermotolerance as their numbers substantially increased between 6 and 24 h in SEQ 4. These data show that some silage inoculants are more thermotolerant than others and that precautions should be taken to ensure that microbial inoculants that are

  18. Fetal viability as a threshold to personhood. A legal analysis.

    PubMed

    Peterfy, A

    1995-12-01

    This essay opens with an examination of US laws concerning fetal viability as they apply to induced abortion, to a mother's right to refuse medical treatment necessary to save the life of a fetus, and to the rights to file suit for the wrongful death of unborn children. The history of abortion policies in the US is traced from the common law period of the early 19th century to the restrictive post-Civil War laws and the decisions of the Supreme Court in Roe vs. Wade, which upheld the constitutionality of previability abortions; Webster vs. Reproductive Health Services, in which the Court assigned viability to the 20th week of pregnancy and acknowledged that States could have a compelling previability interest in the fetus; and the Casey decision, which provided tolerance for limits on the availability of abortion before viability as long as the limits did not create an "undue burden" on the woman seeking the abortion. Courts dealing with the issue of compelling a mother to undergo medical treatment to save her fetus have been inconsistent as they balanced the state's interest in the fetus against the mother's rights to privacy. Judges have tended to err on the side of forcing the medical interventions, but the most recent trend is against this sort of judgement. In these cases, fetal viability has also served as a dividing line. The inconsistency of the legal system is illustrated by the fact that, whereas the fetus now has a legal existence, wrongful death actions entered on behalf of a nonviable fetus have often been denied although courts have been more willing to extend protection to fetuses in wrongful death tort cases than in abortion or medical intervention cases. Criminal law has a unique set of rules for dealing with fetuses as some states have broadened their definitions of "homicide" to include fetuses, even nonviable fetuses. Courts, however, are reluctant to enlarge criminal statutes on their own. While the central position given to the role of

  19. Effect of botulinum neurotoxin type A (BoNTA) on the morphology and viability of 3T3 murine fibroblasts

    PubMed Central

    Bandala, Cindy; Terán-Melo, Juan Luis; Anaya-Ruiz, Maricruz; Mejía-Barradas, Cesar Miguel; Domínguez-Rubio, Rene; la Garza-Montano, Paloma De; Alfaro-Rodríguez, Alfonso; Lara-Padilla, Eleazar

    2015-01-01

    Aim: BoNTA is used in the treatment of ophthalmological disorders, muscular hyperactivity and pain. In recent years it has been described that BoNTA reduces cellular viability and induces apoptosis in prostate cells lines. Studies about the effect of BoNTA are no well known. There have been studies about the effect of BoNTA on the expression levels of collagenase in fibroblasts, but not on its morphological impact on these cells. The aim of this study was to determine the effect of BoNTA on the morphology and viability of the 3T3 fibroblast cell line. Material and methods: The 3T3 fibroblast cell line was cultured and the experimental group received 10 U BoNTA added to a 0.9% sterile saline solution in a reconstituted vial. The control group received saline solution only. Cultured cells were observed and photographed at 5, 10, 15 and 20 h. Cell viability was evaluated by means of the trypan blue test, and cell proliferation with the Proliferation Assay kit (PROMEGA). Results: The application of BoNTA to 3T3 fibroblast cells induced morphological changes, such as a loss of normal fibroblast morphology. Additionally, we observed the cytoplasmic retraction and spread phenomena. The nuclei showed other important changes with Giemsa staining. Conclusion: The results indicate that BoNTA induced a loss of spindle form, increase in cytoplasmic vesicles, and the presence of nuclear vesicles (compacted chromatin surrounded by a nuclear envelope). This suggests an apoptotic process and decreased cell viability. Further studies are needed to explore the mechanisms of these alterations. PMID:26464704

  20. Detection of viability of transplanted beta cells labeled with a novel contrast agent - polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles by magnetic resonance imaging.

    PubMed

    Zhang, Bo; Jiang, Biao; Chen, Ying; Huang, Hai; Xie, Qiuping; Kang, Muxing; Zhang, Hui; Zhai, Chuanxin; Wu, Yulian

    2012-01-01

    Islets can be visualized on MRI by labeling with superparamagnetic contrast agent during the transplantation procedure. However, whether the signal intensity reflects the cell number and cellular viability has not been determined. We used a self-synthesized novel superparamagnetic contrast agent -polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles (PVP-SPIO) - to label β-TC-6 cells (a mouse insulinoma cell line) or primary islets with commercial Feridex as a control. The labeling efficiency of two agents was compared by Prussian blue staining, intracellular iron content determination and MR scanning. Cells were exposed to hypoxia, high-glucose or exogenous H₂O₂ stimulation before/after PVP-SPIO labeling. Normal and injured cells were also transplanted into renal subcapsule. A clinically used 3.0 T MR scan was performed in vitro and 24 h post-transplantation to investigate the correlation between cellular viability and signal. Our PVP-SPIO displayed superior biocompatibility and magnetic properties. All of the cells could be labeled at 100 µg/ml iron concentration after 24 h incubation. At 100 µg/ml iron concentration, 1 × 10⁵ β cells labeled with PVP-SPIO could already be visualized in vitro by MRI, less than the detection threshold of Feridex. There existed a linear correlation between the number of labeled cells and R₂ value on the T₂ -weighted images. The signal intensity and the intracellular iron content declined along with the decreased viability of labeled cells. There was also a significant difference in signal intensity between injured and normal labeled cells after transplantation. From these results, we concluded that PVP-SPIO possessed superior cell labeling efficiency, and β cells could be labeled without compromising viability and function. The signal intensity on MRI might be a useful predictor to evaluate the number and the viability of PVP-SPIO-labeled cells.

  1. Effect of cellular mobility on immune response

    NASA Astrophysics Data System (ADS)

    Pandey, R. B.; Mannion, R.; Ruskin, H. J.

    2000-08-01

    Mobility of cell types in our HIV immune response model is subject to an intrinsic mobility and an explicit directed mobility, which is governed by Pmob. We investigate how restricting the explicit mobility, while maintaining the innate mobility of a viral-infected cell, affects the model's results. We find that increasing the explicit mobility of the immune system cells leads to viral dominance for certain levels of viral mutation. We conclude that increasing immune system cellular mobility indirectly increases the virus’ inherent mobility.

  2. Elliptical Particle Clustering in Cellular Flows

    NASA Astrophysics Data System (ADS)

    Atis, Severine; Sapsis, Themistoklis; Peacock, Thomas

    2015-11-01

    The transport of finite-sized objects by fluid flows is relevant to a wide variety of phenomena, such as debris transport on the ocean surface or bacteria advection in fluid environment. The shape of the advected objects can strongly alter their coupling with the surrounding flow field, and hence, greatly affecting their dispersion by the flow. We present the results of investigations of the behavior of neutrally buoyant, elliptical particles in two-dimensional cellular flows. We find that their trajectories, and overall organization, are markedly different than for spherical particles, with clear clustering for the elliptical particles associated with vortices.

  3. The Impact of 4-Nonylphenol on the Viability and Hormone Production of Mouse Leydig Cells.

    PubMed

    Jambor, T; Lukáčová, J; Tvrdá, E; Kňažická, Z; Forgács, Z; Lukáč, N

    2016-01-01

    Exogenous substances altering the function of the endocrine system and exhibiting adverse health effects on the organism are defined as endocrine disruptors. Nonylphenol is one of the most abundant alkylphenol ethoxylate derivatives, being detected in food products. Diverse studies have classified nonylphenol as hazardous to the health, especially to male reproduction. This in vitro study aimed to examine the effects of 4-nonylphenol on androstenedione