Science.gov

Sample records for affect cell viability

  1. Metformin selectively affects human glioblastoma tumor-initiating cell viability

    PubMed Central

    Würth, Roberto; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adirana; Corsaro, Alessandro; Parodi, Alessia; Sirito, Rodolfo; Massollo, Michela; Marini, Cecilia; Zona, Gianluigi; Fenoglio, Daniela; Sambuceti, Gianmario; Filaci, Gilberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

    2013-01-01

    Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect. PMID:23255107

  2. Oleuropein-Enriched Olive Leaf Extract Affects Calcium Dynamics and Impairs Viability of Malignant Mesothelioma Cells

    PubMed Central

    Marchetti, Carla; Clericuzio, Marco; Borghesi, Barbara; Cornara, Laura; Ribulla, Stefania; Gosetti, Fabio; Marengo, Emilio; Burlando, Bruno

    2015-01-01

    Malignant mesothelioma is a poor prognosis cancer in urgent need of alternative therapies. Oleuropein, the major phenolic of olive tree (Olea europaea L.), is believed to have therapeutic potentials for various diseases, including tumors. We obtained an oleuropein-enriched fraction, consisting of 60% w/w oleuropein, from olive leaves, and assessed its effects on intracellular Ca2+ and cell viability in mesothelioma cells. Effects of the oleuropein-enriched fraction on Ca2+ dynamics and cell viability were studied in the REN mesothelioma cell line, using fura-2 microspectrofluorimetry and MTT assay, respectively. Fura-2-loaded cells, transiently exposed to the oleuropein-enriched fraction, showed dose-dependent transient elevations of cytosolic Ca2+ concentration ([Ca2+]i). Application of standard oleuropein and hydroxytyrosol, and of the inhibitor of low-voltage T-type Ca2+ channels NNC-55-0396, suggested that the effect is mainly due to oleuropein acting through its hydroxytyrosol moiety on T-type Ca2+ channels. The oleuropein-enriched fraction and standard oleuropein displayed a significant antiproliferative effect, as measured on REN cells by MTT cell viability assay, with IC50 of 22 μg/mL oleuropein. Data suggest that our oleuropein-enriched fraction from olive leaf extract could have pharmacological application in malignant mesothelioma anticancer therapy, possibly by targeting T-type Ca2+ channels and thereby dysregulating intracellular Ca2+ dynamics. PMID:26693247

  3. 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

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

    PubMed

    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, p21(Waf1), 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

  5. Axitinib affects cell viability and migration of a primary foetal lung adenocarcinoma culture.

    PubMed

    Menna, Cecilia; De Falco, Elena; Pacini, Luca; Scafetta, Gaia; Ruggieri, Paola; Puca, Rosa; Petrozza, Vincenzo; Ciccone, Anna Maria; Rendina, Erino Angelo; Calogero, Antonella; Ibrahim, Mohsen

    2014-01-01

    Fetal lung adenocarcinoma (FLAC) is a rare variant of lung adenocarcinoma. Studies regarding FLAC have been based only on histopathological observations, thus representative in vitro models of FLAC cultures are unavailable. We have established and characterized a human primary FLAC cell culture, exploring its biology, chemosensitivity, and migration. FLAC cells and specimen showed significant upregulation of VEGF165 and HIF-1α mRNA levels. This observation was confirmed by in vitro chemosensitivity and migration assay, showing that only Axitinib was comparable to Cisplatin treatment. We provide a suitable in vitro model to further investigate the nature of this rare type of cancer. PMID:24380379

  6. α-Tocopheryl Succinate Affects Malignant Cell Viability, Proliferation, and Differentiation.

    PubMed

    Savitskaya, M A; Onischenko, G E

    2016-08-01

    The widespread occurrence of malignant tumors motivates great attention to finding and investigating effective new antitumor preparations. Such preparations include compounds of the vitamin E family. Among them, α-tocopheryl succinate (vitamin E succinate (VES)) has the most pronounced antitumor properties. In this review, various targets and mechanisms of the antitumor effect of vitamin E succinate are characterized. It has been shown that VES has multiple intracellular targets and effects, and as a result VES is able to induce apoptosis in tumor cells, inhibit their proliferation, induce differentiation, prevent metastasizing, and inhibit angiogenesis. However, VES has minimal effects on normal cells and tissues. Due to the variety of targets and selectivity of action, VES is a promising agent against malignant neoplasms. More detailed studies in this area can contribute to development of effective and safe chemotherapeutic preparations.

  7. α-Tocopheryl Succinate Affects Malignant Cell Viability, Proliferation, and Differentiation.

    PubMed

    Savitskaya, M A; Onischenko, G E

    2016-08-01

    The widespread occurrence of malignant tumors motivates great attention to finding and investigating effective new antitumor preparations. Such preparations include compounds of the vitamin E family. Among them, α-tocopheryl succinate (vitamin E succinate (VES)) has the most pronounced antitumor properties. In this review, various targets and mechanisms of the antitumor effect of vitamin E succinate are characterized. It has been shown that VES has multiple intracellular targets and effects, and as a result VES is able to induce apoptosis in tumor cells, inhibit their proliferation, induce differentiation, prevent metastasizing, and inhibit angiogenesis. However, VES has minimal effects on normal cells and tissues. Due to the variety of targets and selectivity of action, VES is a promising agent against malignant neoplasms. More detailed studies in this area can contribute to development of effective and safe chemotherapeutic preparations. PMID:27677550

  8. 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.

  9. 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

  10. Ureaplasma diversum infection in vitro alters prostaglandin E2 and prostaglandin F2a production by bovine endometrial cells without affecting cell viability.

    PubMed Central

    Kim, J J; Quinn, P A; Fortier, M A

    1994-01-01

    Bovine epithelial and stromal cells of the endometrium were inoculated with Ureaplasma diversum, pathogenic strain 2312, at 10(6) or 10(3) color-changing units (ccu)/ml in the presence of 1% fetal bovine serum (depleted of steroids by dextran-charcoal treatment) to assess the effect of infection on prostaglandin biosynthesis. When the inoculum of U. diversum was 10(6) ccu/ml, the concentration of U. diversum in the culture medium decreased with time. U. diversum was found on the epithelial and stromal cell monolayers, increasing in titer 100-fold, indicating that attachment and eventually growth occurred. When the inoculum was 10(3) ccu/ml, the titer of U. diversum remained the same or increased in the supernatant and increased on epithelial and stromal cells. The effect of infection was evaluated by measurement of the primary prostaglandin produced by each cell type, prostaglandin F2a for epithelial cells and prostaglandin E2 for stromal cells. Infection with U. diversum significantly decreased prostaglandin F2a accumulation, by 44.7% +/- 6.0% at 10(6) ccu/ml (P < or = 0.005) and 15.8% +/- 5.3% at 10(3) ccu/ml (P < or = 0.05) in epithelial cells. Prostaglandin E2 accumulation by stromal cells was decreased by 34.0% +/- 4.0% at 10(6) ccu/ml (P < or = 0.001) and by 13.5% +/- 2.7% at 10(3) ccu/ml (P < or = 0.005). Infection with 10(6) ccu/ml did not alter endometrial cell viability, as shown by protein measurement, trypan blue dye exclusion, and cell plating efficiency tests. Thus, alterations in prostaglandin production were not due to cell deterioration. These observations suggest that U. diversum can alter prostaglandin E2 and prostaglandin F2a patterns in primary cultures of bovine endometrial cells without affecting cell viability. PMID:8168914

  11. 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. PMID:27603212

  12. 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.

  13. Metformin selectively affects human glioblastoma tumor-initiating cell viability: A role for metformin-induced inhibition of Akt.

    PubMed

    Würth, Roberto; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adirano; Corsaro, Alessandro; Parodi, Alessia; Sirito, Rodolfo; Massollo, Michela; Marini, Cecilia; Zona, Gianluigi; Fenoglio, Daniela; Sambuceti, Gianmario; Filaci, Gilberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

    2013-01-01

    Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect.

  14. The conditioned medium from osteo-differentiating human mesenchymal stem cells affects the viability of triple negative MDA-MB231 breast cancer cells.

    PubMed

    Librizzi, Mariangela; Tobiasch, Edda; Luparello, Claudio

    2016-01-01

    This study aimed to investigate the effect of conditioned media (CM) from osteo-differentiating and adipo-differentiating human mesenchymal stem cells (MSCs) isolated from lipoaspirates of healthy female donors on the viability of triple-negative breast cancer cells MDA-MB231. The CM of undifferentiated and differentiating MSCs were collected after 7, 14, 21 and 28 days of culture. The effects of MSC CM on cell proliferation were assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 24 h. The effects of osteo-differentiating cell CM on apoptotic promotion, cell cycle impairment, mitochondrial transmembrane potential dissipation, production of reactive oxygen species and autophagosome accumulation were analysed by flow cytometry and Western blot. MTT assay showed that only CM collected from osteo-induced cells at day 28 (d28O-CM) reduced tumour cell viability. Treatment with d28O-CM restrained cell cycle progression through G2 phase, elicited a caspase-8-driven apoptotic effect already after 5 h of culture, and down-regulated autophagosome accumulation and beclin-1 expression. The finding that factor(s) secreted by osteo-differentiating MSCs shows properties of an apoptotic inducer and autophagy inhibitor on triple-negative breast cancer cells may have an important applicative potential that deserves further investigation.

  15. Partially Purified Extracts of Sea Anemone Anemonia viridis Affect the Growth and Viability of Selected Tumour Cell Lines

    PubMed Central

    Bulati, Matteo; Longo, Alessandra; Vlah, Sara; Bennici, Carmelo; Bonura, Angela; Tagliavia, Marcello; Mazzola, Salvatore

    2016-01-01

    In the last few years, marine species have been investigated for the presence of natural products with anticancer activity. Using reversed phase chromatography, low molecular weight proteins were fractionated from the sea anemone Anemonia viridis. Four different fractions were evaluated for their cytotoxic activity by means of erythrocyte haemolysis test, MTS, and LDH assays. Finally, the antiproliferative activities of three of these fractions were studied on PC3, PLC/PRF/5, and A375 human cancer cell lines. Our analysis revealed that the four fractions showed different protein contents and diverse patterns of activity towards human PBMC and cancer cell lines. Interestingly, fractions III and IV exerted cytotoxic effects on human cells. Conversely, fractions I and II displayed very low toxic effects associated with antiproliferative activities on cancer cell lines. PMID:27725939

  16. 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

  17. Viability of mesenchymal stem cells during electrospinning.

    PubMed

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

    2012-02-01

    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.

  18. 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. PMID:27681649

  19. 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.

  20. 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.

  1. Cholesterol level determines viability and mitogenicity, but it does not affect sodium butyrate-dependent sensitization of Colo 205 cells to TNF-α-induced apoptosis.

    PubMed

    Orzechowska, S; Pajak, B; Gajkowska, B; Orzechowski, A

    2011-02-01

    Transient treatment of human adenocarcinoma COLO 205 cells with lipit raft (LR) modulators (MßCD, NY, IMP) was followed by the challenge with metabolic inhibitors and selected anti-cancer drugs. To overturn cholesterol chelation, the MßCD, NY treatment was followed by cholesterol conjugates (CHOL-MßCD or CHOL-PEG). The TNF-α- and P(Ser473)-PKB/Akt1/2-mediated effects initiated at LR were evaluated with regard to cell viability and mitogenicity. Cholesterol chelators reversibly reduced cell survival, whereas some of the tested compounds had weak effects (CIS, CLA), stimulated (EGCG) or reduced (NaB) cell survival. Cellular localizations of LR-associated molecules (ceramides, Gαi-2 heterotrimeric protein, and TNF-R1) in different cellular compartments including the plasma membrane were observed in the respective photographs from TEM and SEM. Evidence from SEM also showed that TNF-R1 is clustered on the surface of COLO 205 cells without presence of cognate ligand but clustering is promoted by TNF-α, while it vanished after IMP treatment. COLO 205 cells remained immune to TNF-α-induced apoptosis unless NaB was added, in which case NaB-induced cell death was further potentiated by TNF-α. Combined NaB and TNF-α treatment was associated with marked changes in the expression of pro- and antiapoptotic proteins. In this study, we demonstrated that initial excess of prosurvival signals could be diminished by cholesterol chelators, whereas LR-independent cell survival could be targeted by NaB. Apparently, lipid rafts do not participate in NaB-dependent cell death.

  2. Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

    PubMed

    Dubińska-Magiera, Magda; Chmielewska, Magdalena; Kozioł, Katarzyna; Machowska, Magdalena; Hutchison, Christopher J; Goldberg, Martin W; Rzepecki, Ryszard

    2016-05-01

    Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

  3. 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.

  4. Metronidazole Decreases Viability of DLD-1 Colorectal Cancer Cell Line

    PubMed Central

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

    2013-01-01

    Abstract 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. PMID:23777253

  5. Nanoporous membrane-sealed microfluidic devices for improved cell viability.

    PubMed

    Masand, Shirley N; Mignone, Lindsay; Zahn, Jeffrey D; Shreiber, David I

    2011-12-01

    Cell-laden microfluidic devices have broad potential in various biomedical applications, including tissue engineering and drug discovery. However, multiple difficulties encountered while culturing cells within devices affecting cell viability, proliferation, and behavior has complicated their use. While active perfusion systems have been used to overcome the diffusive limitations associated with nutrient delivery into microchannels to support longer culture times, these systems can result in non-uniform oxygen and nutrient delivery and subject cells to shear stresses, which can affect cell behavior. Additionally, histological analysis of cell cultures within devices is generally laborious and yields inconsistent results due to difficulties in delivering labeling agents in microchannels. Herein, we describe a simple, cost-effective approach to preserve cell viability and simplify labeling within microfluidic networks without the need for active perfusion. Instead of bonding a microfluidic network to glass, PDMS, or other solid substrate, the network is bonded to a semi-permeable nanoporous membrane. The membrane-sealed devices allow free exchange of proteins, nutrients, buffers, and labeling reagents between the microfluidic channels and culture media in static culture plates under sterile conditions. The use of the semi-permeable membrane dramatically simplifies microniche cell culturing while avoiding many of the complications which arise from perfusion systems.

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

    PubMed

    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

  7. 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

  8. 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

  9. Early-age feed restriction affects viability and gene expression of satellite cells isolated from the gastrocnemius muscle of broiler chicks

    PubMed Central

    2012-01-01

    Background Muscle growth depends on the fusion of proliferate satellite cells to existing myofibers. We reported previously that 0–14 day intermittent feeding led to persistent retardation in myofiber hypertrophy. However, how satellite cells respond to such nutritional insult has not been adequately elucidated. Results One-day-old broiler chicks were allocated to control (Con, ad libitum feeding), intermittent feeding (IF, feed provided on alternate days) and re-feeding (RF, 2 days ad libitum feeding after 12 days of intermittent feeding) groups. Chickens were killed on Day 15 and satellite cells were isolated. When cultured, satellite cells from the IF group demonstrated significant retardation in proliferation and differentiation potential, while RF partly restored the proliferation rate and differentiation potential of the satellite cells. Significant up-regulation of insulin like growth factor I receptor (IGF-IR) (P<0.05) and thyroid hormone receptor α (TRα) (P<0.05), and down-regulation of growth hormone receptor (GHR) (P<0.01) and IGF-I (P<0.01) mRNA expression was observed in freshly isolated IF satellite cells when compared with Con cells. In RF cells, the mRNA expression of IGF-I was higher (P<0.05) and of TRα was lower (P<0.01) than in IF cells, suggesting that RF restored the mRNA expression of TRα and IGF-I, but not of GHR and IGF-IR. The Bax/Bcl-2 ratio tended to increase in the IF group, which was reversed in the RF group (P<0.05), indicating that RF reduced the pro-apoptotic influence of IF. Moreover, no significant effect of T3 was detected on cell survival in IF cells compared with Con (P<0.001) or RF (P<0.05) cells. Conclusions These data suggest that early-age feed restriction inhibits the proliferation and differentiation of satellite cells, induces changes in mRNA expression of the GH/IGF-I and thyroid hormone receptors in satellite cells, as well as blunted sensitivity of satellite cells to T3, and that RF partially reverses these

  10. Common European harmful algal blooms affect the viability and innate immune responses of Mytilus edulis larvae.

    PubMed

    De Rijcke, M; Vandegehuchte, M B; Vanden Bussche, J; Nevejan, N; Vanhaecke, L; De Schamphelaere, K A C; Janssen, C R

    2015-11-01

    Like marine diseases, harmful algal blooms (HABs) are globally increasing in frequency, severity and geographical scale. As a result, bivalves will have to face the combined threat of toxic algae and marine pathogens more frequently in the (near) future. These stressors combined may further affect the recruitment of ecologically and economically important bivalve species as HABs can affect the growth, viability and development of their larvae. To date, little is known on the specific effects of HABs on the innate immune system of bivalve larvae. This study therefore investigates whether two common harmful algae can influence the larval viability, development and immunological resilience of the blue mussel Mytilus edulis. Embryos of this model organism were exposed (48 h) to five densities of Pseudo-nitzschia multiseries or Prorocentrum lima cells. In addition, the effect of six concentrations of their respective toxins: domoic acid (DA) and okadaic acid (OA) were assessed. OA was found to significantly reduce larval protein phosphatase activity (p < 0.001) and larval viability (p < 0.01) at concentrations as low as 37.8 μg l(-1). P. multiseries (1400 cells ml(-1)), P. lima (150 cells ml(-1)) and DA (dosed five times higher than typical environmental conditions i.e. 623.2 μg l(-1)) increased the phenoloxidase (PO) innate immune activity of the mussel larvae. These results suggest that the innate immune response of even the earliest life stages of bivalves is susceptible to the presence of HABs.

  11. Common European harmful algal blooms affect the viability and innate immune responses of Mytilus edulis larvae.

    PubMed

    De Rijcke, M; Vandegehuchte, M B; Vanden Bussche, J; Nevejan, N; Vanhaecke, L; De Schamphelaere, K A C; Janssen, C R

    2015-11-01

    Like marine diseases, harmful algal blooms (HABs) are globally increasing in frequency, severity and geographical scale. As a result, bivalves will have to face the combined threat of toxic algae and marine pathogens more frequently in the (near) future. These stressors combined may further affect the recruitment of ecologically and economically important bivalve species as HABs can affect the growth, viability and development of their larvae. To date, little is known on the specific effects of HABs on the innate immune system of bivalve larvae. This study therefore investigates whether two common harmful algae can influence the larval viability, development and immunological resilience of the blue mussel Mytilus edulis. Embryos of this model organism were exposed (48 h) to five densities of Pseudo-nitzschia multiseries or Prorocentrum lima cells. In addition, the effect of six concentrations of their respective toxins: domoic acid (DA) and okadaic acid (OA) were assessed. OA was found to significantly reduce larval protein phosphatase activity (p < 0.001) and larval viability (p < 0.01) at concentrations as low as 37.8 μg l(-1). P. multiseries (1400 cells ml(-1)), P. lima (150 cells ml(-1)) and DA (dosed five times higher than typical environmental conditions i.e. 623.2 μg l(-1)) increased the phenoloxidase (PO) innate immune activity of the mussel larvae. These results suggest that the innate immune response of even the earliest life stages of bivalves is susceptible to the presence of HABs. PMID:26348409

  12. The Refsum disease marker phytanic acid, a branched chain fatty acid, affects Ca2+ homeostasis and mitochondria, and reduces cell viability in rat hippocampal astrocytes.

    PubMed

    Kahlert, Stefan; Schönfeld, Peter; Reiser, Georg

    2005-02-01

    The saturated branched chain fatty acid, phytanic acid, a degradation product of chlorophyll, accumulates in Refsum disease, an inherited peroxisomal disorder with neurological clinical features. To elucidate the pathogenic mechanism, we investigated the influence of phytanic acid on cellular physiology of rat hippocampal astrocytes. Phytanic acid (100 microM) induced an immediate transient increase in cytosolic Ca2+ concentration, followed by a plateau. The peak of this biphasic Ca2+ response was largely independent of extracellular Ca2+, indicating activation of cellular Ca2+ stores by phytanic acid. Phytanic acid depolarized mitochondria without causing in situ swelling of mitochondria. The slow decrease of mitochondrial potential is not consistent with fast and simultaneous opening of the mitochondrial permeability transition pore. However, phytanic acid induced substantial generation of reactive oxygen species. Phytanic acid caused astroglia cell death after a few hours of exposure. We suggest that the cytotoxic effect of phytanic acid seems to be due to a combined action on Ca2+ regulation, mitochondrial depolarization, and increased ROS generation in brain cells.

  13. Effect of glyceric acid calcium salt on the viability of ethanol-dosed gastric cells.

    PubMed

    Habe, Hiroshi; Sato, Shun; Fukuoka, Tokuma; Kitamoto, Dai; Sakaki, Keiji

    2011-01-01

    D-Glyceric acid (D-GA) calcium has been reported to accelerate ethanol oxidation in vivo in rats (Eriksson et al., Metabolism, 56, 895-898 (2007)). However, no other reports have shown that D-GA can reduce the harmful effects of ethanol. In this study, the effects of D-, L-, and DL-GA calcium on ethanol-dosed gastric cell viability were investigated using human gastric carcinoma cells (Kato III) and normal rat gastric mucosa cells (RGM1). Addition of 2% and 3 % ethanol to Kato III and RGM1 cells, respectively, decreased their cell viability by approximately 20-50 % after 24 or 72 h of cultivation. In 2 % ethanol-dosed Kato III cells cultivated for 24 h, addition of 0.002-20 µg/mL D- and L-GA calcium did not affect cell viability. Similarly, addition of less than 20 µg/mL DL-GA calcium did not affect cell viability. However, when 20 µg/mL DL-GA calcium was added, cell viability increased by 35.7 % after 72 h of incubation, compared to the viability of control cells without ethanol or GA. Addition of 20 µg/mL DL-GA calcium to 3 % ethanol-dosed RGM1 cells cultivated for 24 or 72 h also increased cell viability up to those observed in control cells. These results suggest that a racemic mixture of GA may have the strongest effect on enhancing the viability of ethanol-exposed cells. PMID:22027025

  14. 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.

  15. Motility, viability, and calcium in the sperm cells.

    PubMed

    Parodi, Jorge

    2014-04-01

    Sperm cells are complicated in vitro models. Their viability is limited, and physiology is complex. The study of their properties is of great application in the animal production as viable and functional gametes are essential. It has been shown that the decrease of sperm cell viability parallels an increase of the reactive oxygen species (ROS). Reactive oxygen species is secondary to normal metabolic processes of the cell-like flagellar movement. There is evidence of strategies that reduce ROS levels by using exogenous or endogenous antioxidants with the intention that seminal plasma protects the sperm cells and increases viability. Perhaps viability can increase by reducing that flagellar movement which is regulated by calcium. The phenomenon has not been fully characterized, but it is established that in certain mammalian models, the entrance of calcium via specific channels such as CATsper or voltage-dependent channels, signals flagellar movement. Previous reports have indicated that a change in the concentration of calcium or if the temperature is altered, the function of mammal sperm cells is reduced or blocked and viability prolonged. Fish sperm can remain immobile for several weeks but when activated the number of mobile and viable sperm is reduced at a faster rate. However, if the cells are not mobilized the semen can be preserved for longer periods. As presented in this paper, this supports the notion that by modulating calcium channels to reduce motility the viability of these cells can increase.

  16. 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

  17. In Vitro Periodontal Ligament Cell Viability in Different Storage Media.

    PubMed

    Sharma, Meenakshi

    2016-01-01

    The aim of this study was to evaluate the viability of periodontal ligament cells of avulsed teeth in three different storage media. Forty-five mature premolars extracted for orthodontic therapeutic purposes were randomly and equally divided into three groups according to the storage medium: milk (control), rice water and egg white. After placing extracted teeth for 30 min in storage media, the scrapings of the periodontal ligament (PDL) were collected in Falcon tubes containing collagenase in 2.5 mL of phosphate buffer saline and were incubated for 30 min and centrifuged for 5 min at 800 rpm. Cell viability was analyzed by Trypan blue exclusion. Rice water had a significantly higher number of viable cells compared to egg white and milk. There was no statistically significant difference between egg white and milk. Rice water may be able to maintain PDL cell viability of avulsed teeth better than egg white or milk. PMID:27652702

  18. Effect of Lanthanide Complex Structure on Cell Viability and Association

    PubMed Central

    2015-01-01

    A systematic study of the effect of hydrophobicity and charge on the cell viability and cell association of lanthanide metal complexes is presented. The terbium luminescent probes feature a macrocyclic polyaminocarboxylate ligand (DOTA) in which the hydrophobicity of the antenna and that of the carboxyamide pendant arms are independently varied. Three sensitizing antennas were investigated in terms of their function in vitro: 2-methoxyisophthalamide (IAM(OMe)), 2-hydroxyisophthalamide (IAM), and 6-methylphenanthridine (Phen). Of these complexes, Tb-DOTA-IAM exhibited the highest quantum yield, although the higher cell viability and more facile synthesis of the structurally related Tb-DOTA-IAM(OMe) platform renders it more attractive. Further modification of this latter core structure with carboxyamide arms featuring hydrophobic benzyl, hexyl, and trifluoro groups as well as hydrophilic amino acid based moieties generated a family of complexes that exhibit high cell viability (ED50 > 300 μM) regardless of the lipophilicity or the overall complex charge. Only the hexyl-substituted complex reduced cell viability to 60% in the presence of 100 μM complex. Additionally, cellular association was investigated by ICP-MS and fluorescence microscopy. Surprisingly, the hydrophobic moieties did not increase cell association in comparison to the hydrophilic amino acid derivatives. It is thus postulated that the hydrophilic nature of the 2-methoxyisophthalamide antenna (IAM(OMe)) disfavors the cellular association of these complexes. As such, responsive luminescent probes based on this scaffold would be appropriate for the detection of extracellular species. PMID:24901440

  19. In vitro electrochemical corrosion and cell viability studies on nickel-free stainless steel orthopedic implants.

    PubMed

    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.

  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. Influence of Waveform on Cell Viability during Ultrasound Exposure

    NASA Astrophysics Data System (ADS)

    Saliev, Timur; Feril, Loreto B.; McLean, Donald A.; Tachibana, Katsuro; Campbell, Paul A.

    2011-09-01

    We examined the role of ultrasound standing waves, and their travelling wave counterparts, on cell viability in an in-vitro insonation apparatus. Furthermore, the effect of distinct waveforms (sine and top-hat) was also explored, together with the role of microbubble presence. Measurements of cell viability in standing wave scenarios demonstrated a relatively higher rate of lysis (63.13±10.89% remaining viable) compared with the travelling wave data, where 96.22±4.0% remained viable. Significant differences were also seen as a function of waveform, where insonations employing top-hat wave shapes resulted in an average end stage viability of 30.31±5.71% compared with 61.94±14.28% in the sinusoidal counterparts.

  2. Inhibiting ice recrystallization and optimization of cell viability after cryopreservation.

    PubMed

    Chaytor, Jennifer L; Tokarew, Jacqueline M; Wu, Luke K; Leclère, Mathieu; Tam, Roger Y; Capicciotti, Chantelle J; Guolla, Louise; von Moos, Elisabeth; Findlay, C Scott; Allan, David S; Ben, Robert N

    2012-01-01

    The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220 mM solutions of disaccharides; however, the best cell viability was obtained when a 200 mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.

  3. Human periodontal ligament cell viability in milk and milk substitutes.

    PubMed

    Pearson, Robert M; Liewehr, Frederick R; West, Leslie A; Patton, William R; McPherson, James C; Runner, Royce R

    2003-03-01

    The purpose of this study was to determine the efficacy of several milk substitutes compared to whole milk in maintaining the viability of human periodontal ligament (PDL) cells on avulsed teeth. PDL cells were obtained from freshly extracted, healthy third molars and cultured in Eagle's minimal essential media (EMEM). The cells were plated onto 24-well culture plates and allowed to attach for 24 h. EMEM was replaced with refrigerated whole milk (positive control), reconstituted powdered milk, evaporated milk, or one of two baby formulas (Similac or Enfamil). Tap water served as the negative control. Tissue culture plates were incubated with the experimental media at 37 degrees C for 1, 2, 4, or 8 h. Cell viability was determined by a cell proliferation assay (CellTiter 96 AQ Assay), with absorbance read at 450 nM. A two-way ANOVA (p < 0.001) indicated that at 1 h there was no difference in the effect on PDL cell viability between any of the materials and whole milk. At 2 h, Enfamil and Similac performed significantly better than whole milk, whereas evaporated milk performed worse. At 4 h, Enfamil performed better than whole milk, whereas all other milk substitutes performed worse. At 8 h, all substitutes performed worse than whole milk. These results suggest that Enfamil, which is supplied in powder form that does not require special storage and has a shelf life of 18 months, is a more effective storage medium for avulsed teeth than pasteurized milk for at least 4 h.

  4. Thermal Pretreatment Improves Viability of Cryopreserved Human Endothelial Cells.

    PubMed

    Hofmann, Nicola; Sun, Huan; Chatterjee, Anamika; Saha, Debapriya; Glasmacher, Birgit

    2015-10-01

    A high survival rate of cryopreserved cells requires optimal cooling and thawing rates in the presence of a cryoprotective agent (CPA) or a combination of CPAs in adequate concentrations. One of the most widely used CPAs, dimethyl sulfoxide (Me2SO), however is toxic at high concentrations and has detrimental effects on cellular functions. Additional processing steps are necessary to remove the CPA after thawing, which make the process expensive and time consuming. Therefore it is of great interest to develop new cryoprotective strategies to replace the currently used CPAs or to reduce their concentration. The aim of this study was to investigate if thermal activation of human pulmonary microvascular endothelial cells (HPMEC ST-1.6R), prior to cryopreservation, could improve their post-thaw viability since the resulting heat shock protein expression acts as an intrinsic cellular protection mechanism. The results of this study suggest that both heat and cold shock pretreatments improve cryopreservation outcome of the HPMEC ST-1.6R cells. By re-cultivating cells after heat shock treatment before cryopreservation, a significant increase in cellular membrane integrity and adherence capacity could be achieved. However a combination of thermal activation and cryopreservation with alternative CPAs such as ectoine and L-proline could not further enhance the cell viability. The results of this study showed that pretreatment of endothelial cells with thermal activation could be used to reduce the Me2SO concentration required in order to preserve cell viability after cryopreservation. PMID:26419006

  5. Thermal Pretreatment Improves Viability of Cryopreserved Human Endothelial Cells.

    PubMed

    Hofmann, Nicola; Sun, Huan; Chatterjee, Anamika; Saha, Debapriya; Glasmacher, Birgit

    2015-10-01

    A high survival rate of cryopreserved cells requires optimal cooling and thawing rates in the presence of a cryoprotective agent (CPA) or a combination of CPAs in adequate concentrations. One of the most widely used CPAs, dimethyl sulfoxide (Me2SO), however is toxic at high concentrations and has detrimental effects on cellular functions. Additional processing steps are necessary to remove the CPA after thawing, which make the process expensive and time consuming. Therefore it is of great interest to develop new cryoprotective strategies to replace the currently used CPAs or to reduce their concentration. The aim of this study was to investigate if thermal activation of human pulmonary microvascular endothelial cells (HPMEC ST-1.6R), prior to cryopreservation, could improve their post-thaw viability since the resulting heat shock protein expression acts as an intrinsic cellular protection mechanism. The results of this study suggest that both heat and cold shock pretreatments improve cryopreservation outcome of the HPMEC ST-1.6R cells. By re-cultivating cells after heat shock treatment before cryopreservation, a significant increase in cellular membrane integrity and adherence capacity could be achieved. However a combination of thermal activation and cryopreservation with alternative CPAs such as ectoine and L-proline could not further enhance the cell viability. The results of this study showed that pretreatment of endothelial cells with thermal activation could be used to reduce the Me2SO concentration required in order to preserve cell viability after cryopreservation.

  6. Effect of dopamine on viability of BHK-21 cells.

    PubMed

    Moshkov, D A; Abramova, M B; Shubina, V S; Lavrovskaya, V P; Pavlik, L L; Lezhnev, E I

    2010-09-01

    We studied the effects of dopamine added to culture medium on survival of floating or adherent BHK-21 cells differing by organization of actin cytoskeleton. The viability of floating cells more drastically decreased with increasing dopamine concentration and duration of exposure than that of adherent cells. The cells worse adhered to the substrate and formed a monolayer. The formed monolayer degrades, cell borders become blurred, cells, polygonal in the control, are rounded. Preliminary blockade of dopamine receptors with haloperidol, inessential for cell survival and morphology, does not prevent the destructive effect of dopamine on the cells. Ultrastructural study revealed increased density of filamentous actin threads in deep compartments of cell cytoplasm after dopamine treatment, this increase being more pronounced in cells grown in suspension. Bearing in mind the polymerizing effect of dopamine on globular actin in vitro and the fact that the content of this protein in floating cells is higher than in adherent cells, we can conclude that the decrease in viability of BHK-21 cells is caused by interaction of dopamine with cytoplasmic globular actin. PMID:21246101

  7. Cell viability analysis using trypan blue: manual and automated methods.

    PubMed

    Louis, Kristine S; Siegel, Andre C

    2011-01-01

    One of the traditional methods of cell viability analysis is the use of trypan blue dye exclusion staining. This technique has been the standard methodology used in academic research laboratories and industrial biotechnology plants. Cells were routinely counted manually with a hemocytometer. In recent years, modern automated instrumentation has been introduced to supplement this traditional technique with the efficiency and reproducibility of computer control, advanced imaging, and automated sample handling.

  8. Zinc Restored the Decreased Vascular Smooth Muscle Cell Viability under Atherosclerotic Calcification Conditions

    PubMed Central

    Shin, Mee-Young; Kwun, In-Sook

    2014-01-01

    Zinc is considered to be involved in maintaining healthy vascular condition. Atherosclerotic calcification of vascular smooth muscle cells (VSMCs) occurs via the mechanism of cell death; therefore, cell viability is a critical factor for preventing VSMC calcification. In this study, we tested whether zinc affected VSMC viability under both normal physiological non-calcifying (0 mM P) and atherosclerotic calcifying conditions (3 and 5 mM P), since VSMC physiological characters change during the VSMC calcification process. The study results showed that an optimal zinc level (15 μM) restored the decreased VSMC viability which was induced under low zinc levels (0 and 1 μM) and calcifying conditions (3 and 5 mM P) at 9 and 15 days culture. This zinc-protecting effect for VSMC viability is more prominent under atherosclerotic calcifying condition (3 and 5 mM P) than normal condition (0 mM P). Also, the increased VSMC viability was consistent with the decreased Ca and P accumulation in VSMC cell layers. The results suggested that zinc could be an effective biomineral for preventing VSMC calcification under atherosclerotic calcifying conditions. PMID:25580404

  9. Cell Viability Assessment: Toward Content-Rich Platforms

    PubMed Central

    Ramirez, Christina Nicole; Antczak, Christophe; Djaballah, Hakim

    2013-01-01

    Importance of the field Monitoring cell viability in vitro is critical in many areas of biomedical research, and the ultimate goal in drug discovery is the ability to predict the in vivo toxicology of drug candidates based on their toxicity profile in vitro. Over the last decade, the contribution of high-throughput screening (HTS) toward this goal has been tremendous, providing the ability to screen compounds in parallel against multiple cell types. However, the toxic effects of drug candidates uncovered during clinical trials are by far the main reason for their failure. Over the same period, our understanding of programmed cell death has evolved dramatically with the identification of critical control points in the cell death pathways. As a result, cell viability should no longer be characterized solely on the basis of discrete endpoint measurements such as membrane permeability. Areas covered in this review/What the reader will gain This review summarizes the traditional viability assays currently commercially available, focusing on methods amenable to high density format. Assays categorized into the following classes are discussed: dye exclusion assays, DNA condensation-based assays and assays monitoring a metabolic function. We describe each approach, and using case studies, we emphasize their limitations. Take home message Current low-content methods based on single parameter readouts are prone to error due to the heterogeneity of cell populations and the multi-faceted nature of cell death. High-content approaches based on continuous, multiplexed readouts are becoming increasingly important for monitoring multiple markers of cell death induction simultaneously, on a cell by cell basis. The use of such content-rich platforms is a necessity to predict the toxicology of drug candidates accurately. PMID:22823019

  10. Strategies for periodontal ligament cell viability: An overview

    PubMed Central

    Goswami, Mousumi; Chaitra, TR; Chaudhary, Seema; Manuja, Naveen; Sinha, Ashish

    2011-01-01

    The long-term success of the reimplanted teeth is related to the maintenance of periodontal ligament (PDL) cell viability. Dental tissues are unique in comparison to most other tissues in the body due to their marked capacity for regeneration. Understanding the circumstances leading to repair and regeneration in oral tissues has been a formidable challenge. Numerous storage media have been introduced by many authors that help to maintain the PDL cell viability. To present an overview of the various available storage media. A literature search for the past 20 years was performed across the Internet database (Pubmed) and relevant citations using the keywords PDL cell viability, tooth avulsion, storage media, and the combination of all to retrieve around (n=225) citations. Articles that included follow-up of intervention for avulsed and re-implanted teeth were considered (n=44) and some literature review from well-known text books were considered. Literature supports that moist storage appears to be a more productive approach to optimize PDL cell survival. However, no medium is ideal and in vivo studies are inadequate. PMID:22025820

  11. Enhancement of cell viability after treatment with polyunsaturated fatty acids.

    PubMed

    Bartl, J; Walitza, S; Grünblatt, E

    2014-01-24

    Attention-deficit/hyperactivity disorder (ADHD) is highly prevalent in children and adolescents and both environmental and genetic factors play major roles. Polyunsaturated fatty acids (PUFAs) are postulated to contribute to the development of the infant brain and an imbalance in these may increase the risk of ADHD. In recent clinical studies, supplementation with PUFAs improved symptoms of ADHD in some cases. Similarly, some beneficial effects were observed with PUFA treatment in neuronal cell cultures. Therefore, in this study, we hypothesized that a specific PUFA combination (available on the market as Equazen™ [Vifor Pharma, Switzerland]) along with iron, zinc, or vitamin B5 (vitB5) would produce an additive beneficial effect on the viability of rat pheochromocytoma-12 dopaminergic cells. The specific PUFA combination alone, as well as added to each of the three nutrients, was tested in a dose-response manner. The specific PUFAs significantly improved cell viability, starting at very low doses (100pM) from 60h up to 90h; while the combined treatment with vitB5 and minerals did not provide additional benefit. Our results confirmed the beneficial effect of the specific PUFAs on neuronal cell viability; although supplementation with minerals and vitB5 did not enhance this effect.

  12. Decline of cell viability and mitochondrial activity in mouse skeletal muscle cell in a hypomagnetic field.

    PubMed

    Fu, Jing-Peng; Mo, Wei-Chuan; Liu, Ying; He, Rong-Qiao

    2016-05-01

    Hypomagnetic field (HMF), one of the key environmental risk factors for astronauts traveling in outer space, has previously been shown to repress locomotion of mammalians. However, underlying mechanisms of how HMF affects the motor system remains poorly understood. In this study, we created an HMF (<3 μT) by eliminating geomagnetic field (GMF, ∼50 μT) and exposed primary mouse skeletal muscle cells to this low magnetic field condition for a period of three days. HMF-exposed cells showed a decline in cell viability relative to GMF control, even though cells appeared normal in terms of morphology and survival rate. After a 3-day HMF-exposure, glucose consumption of skeletal muscle cells was significantly lower than GMF control, accompanied by less adenosine triphosphate (ATP) and adenosine diphosphate (ADP) content and higher ADP/ATP ratio. In agreement with these findings, mitochondrial membrane potential of HMF-exposed cells was also lower, whereas levels of cellular Reactive Oxygen Species were higher. Moreover, viability and membrane potential of isolated mitochondria were reduced after 1 h HMF-exposure in vitro. Our results indicate that mitochondria can directly respond to HMF at functional level, and suggest that HMF-induced decline in cell functionality results from a reduction in energy production and mitochondrial activity. PMID:27003876

  13. 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...

  14. 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.

  15. Progesterone promotes propagation and viability of mouse embryonic stem cells.

    PubMed

    Shen, Shan-Wei; Song, Hou-Yan

    2009-10-25

    It has been known that estrogen-17beta stimulates proliferation of mouse embryonic stem (mES) cells. To explore the function of another steroid hormone progesterone, we used MTT method and BrdU incorporation assay to obtain growth curves, clone forming assay to detect the propagation and viability of individual mES cells, Western blot to test the expression of ES cell marker gene Oct-4, fluorescence activated cell sorter (FACS) to test cell cycle, and real-time PCR to detect the expressions of cyclins, cyclin-dependent kinases and proto-oncogenes. The results showed that progesterone promoted proliferation of mES cells. The number of clones was more in progesterone-treated group than that in the control group. The expression of pluripotency-associated transcriptional factor Oct-4 changed little after progesterone treatment as shown by Western blot, indicating that most of mES cells were in undifferentiated state. The results of FACS proved that progesterone promoted DNA synthesis in mES cells. The proportion of mES cells in S+G(2)/M phase was higher in progesterone-treated group than that in the control group. Cyclins and cyclin-dependent kinases, as well as proto-oncogenes (c-myc, c-fos) were up-regulated when cells were treated with progesterone. The results obtained indicate that progesterone promotes propagation and viability of mES cells. The up-regulation of cell cycle-related factors might contribute to the function of progesterone.

  16. Comparison of ex vivo cultivated human limbal epithelial stem cell viability and proliferation on different substrates.

    PubMed

    Chakraborty, Anindita; Dutta, Jayanta; Das, Sumantra; Datta, Himadri

    2013-12-01

    Ocular surface injury causes serious vision-related problems especially when limbal stem cells are affected. Treatment lies in the transplantation of viable donor cells. Various substrates are used for the cultivation of limbal epithelial stem cells. In the present study, viability and proliferation of ex vivo cultured limbal epithelial stem cells were examined on a variety of substrates like collagen type IV, direct plastic Petri plate, intact amniotic membrane and denuded amniotic membrane. Viability and proliferation of cells were examined by colorimetric assay and [(3)H]-thymidine incorporation study. Furthermore, matrix metalloproteinase is known to be a key regulator in stem cell migration and proliferation. This enzyme activity was studied by gelatinolytic zymography. It was found from this study that although human limbal epithelial stem cells could be cultivated on different substrates such as collagen type IV, direct plastic Petri plate, intact amniotic membrane and denuded amniotic membrane, maximum growth and proliferation was observed when cultured on intact amniotic membrane. The number of patients suffering from limbal epithelial stem cell deficiency is large compared to donor tissues available for transplantation. Hence, increased cell viability and proliferation is required to serve more patients.

  17. Effect of microemulsions on cell viability of human dermal fibroblasts

    NASA Astrophysics Data System (ADS)

    Li, Juyi; Mironava, Tatsiana; Simon, Marcia; Rafailovich, Miriam; Garti, Nissim

    Microemulsions are optically clear, thermostable and isotropic mixture consisting of water, oil and surfactants. Their advantages of ease preparation, spontaneous formation, long-term stability and enhanced solubility of bioactive materials make them great potentials as vehicles in food and pharmaceutical applications. In this study, comparative in vitro cytotoxicity tests were performed to select a best formulation of microemulsion with the least toxicity for human dermal fibroblasts. Three different kinds of oils and six different kinds of surfactants were used to form microemulsions by different ratios. The effect of oil type and surfactant type as well as their proportions on cell proliferation and viability were tested.

  18. Rat visceral yolk sac cells: viability and expression of cell markers during maternal diabetes.

    PubMed

    Aires, M B; Santos, J R A; Souza, K S; Farias, P S; Santos, A C V; Fioretto, E T; Maria, D A

    2015-08-01

    The function of the visceral yolk sac (VYS) is critical for embryo organogenesis until final fetal development in rats, and can be affected by conditions such as diabetes. In view of the importance of diabetes during pregnancy for maternal and neonatal health, the objective of this study was to assess fetal weight, VYS cell markers, and viability in female Wistar rats (200-250 g) with induced diabetes (alloxan, 37 mg/kg) on the 8th gestational day (gd 8). At gd 15, rats from control (n=5) and diabetic (n=5) groups were anesthetized and laparotomized to remove the uterine horns for weighing of fetuses and collecting the VYS. Flow cytometry was used for characterizing VYS cells, and for determining mitochondrial activity, cell proliferation, DNA ploidy, cell cycle phases, and caspase-3 activity. Fetal weight was reduced in the diabetic group. Expression of the cell markers CD34, VEGFR1, CD115, CD117, CD14, CCR2, CD90, CD44, STRO-1, OCT3/4, and Nanog was detected in VYS cells in both groups. In the diabetic group, significantly decreased expression of CD34 (P<0.05), CCR2 (P<0.001), and OCT3/4 (P<0.01), and significantly increased expression of CD90 (P<0.05), CD117 (P<0.01), and CD14 (P<0.05) were observed. VYS cells with inactive mitochondria, activated caspase-3, and low proliferation were present in the rats with diabetes. Severe hyperglycemia caused by maternal diabetes had negative effects on pregnancy, VYS cell viability, and the expression of cell markers. PMID:26176314

  19. A bacteriophage detection tool for viability assessment of Salmonella cells.

    PubMed

    Fernandes, E; Martins, V C; Nóbrega, C; Carvalho, C M; Cardoso, F A; Cardoso, S; Dias, J; Deng, D; Kluskens, L D; Freitas, P P; Azeredo, J

    2014-02-15

    Salmonellosis, one of the most common food and water-borne diseases, has a major global health and economic impact. Salmonella cells present high infection rates, persistence over inauspicious conditions and the potential to preserve virulence in dormant states when cells are viable but non-culturable (VBNC). These facts are challenging for current detection methods. Culture methods lack the capacity to detect VBNC cells, while biomolecular methods (e.g. DNA- or protein-based) hardly distinguish between dead innocuous cells and their viable lethal counterparts. This work presents and validates a novel bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability. Salmonella Enteritidis cells in a VBNC physiological state were evaluated by cell culture, flow-cytometry and epifluorescence microscopy, and further assayed with a biosensor platform. Free PVP-SE1 phages in solution showed the ability to recognize VBNC cells, with no lysis induction, in contrast to the minor recognition of heat-killed cells. This ability was confirmed for immobilized phages on gold surfaces, where the phage detection signal follows the same trend of the concentration of viable plus VBNC cells in the sample. The phage probe was then tested in a magnetoresistive biosensor platform allowing the quantitative detection and discrimination of viable and VBNC cells from dead cells, with high sensitivity. Signals arising from 3 to 4 cells per sensor were recorded. In comparison to a polyclonal antibody that does not distinguish viable from dead cells, the phage selectivity in cell recognition minimizes false-negative and false-positive results often associated with most detection methods.

  20. Exogenous HGF Bypasses the Effects of ErbB Inhibition on Tumor Cell Viability in Medulloblastoma Cell Lines

    PubMed Central

    Zomerman, Walderik W.; Plasschaert, Sabine L. A.; Diks, Sander H.; Lourens, Harm-Jan; Meeuwsen-de Boer, Tiny; Hoving, Eelco W.; den Dunnen, Wilfred F. A.; de Bont, Eveline S. J. M.

    2015-01-01

    Recent clinical trials investigating receptor tyrosine kinase (RTK) inhibitors showed a limited clinical response in medulloblastoma. The present study investigated the role of micro-environmental growth factors expressed in the brain, such as HGF and EGF, in relation to the effects of hepatocyte growth factor receptor (MET) and epidermal growth factor receptor family (ErbB1-4) inhibition in medulloblastoma cell lines. Medulloblastoma cell lines were treated with tyrosine kinase inhibitors crizotinib or canertinib, targeting MET and ErbB1-4, respectively. Upon treatment, cells were stimulated with VEGF-A, PDGF-AB, HGF, FGF-2 or EGF. Subsequently, we measured cell viability and expression levels of growth factors and downstream signaling proteins. Addition of HGF or EGF phosphorylated MET or EGFR, respectively, and demonstrated phosphorylation of Akt and ERK1/2 as well as increased tumor cell viability. Crizotinib and canertinib both inhibited cell viability and phosphorylation of Akt and ERK1/2. Specifically targeting MET using shRNA’s resulted in decreased cell viability. Interestingly, addition of HGF to canertinib significantly enhanced cell viability as well as phosphorylation of Akt and ERK1/2. The HGF-induced bypass of canertinib was reversed by addition of crizotinib. HGF protein was hardly released by medulloblastoma cells itself. Addition of canertinib did not affect RTK cell surface or growth factor expression levels. This manuscript points to the bypassing capacity of exogenous HGF in medulloblastoma cell lines. It might be of great interest to anticipate on these results in developing novel clinical trials with a combination of MET and EGFR inhibitors in medulloblastoma. PMID:26496080

  1. A loss-of-function genetic screening identifies novel mediators of thyroid cancer cell viability.

    PubMed

    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

    2016-05-10

    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

  2. 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

  3. Development of a thermal sensor to probe cell viability and concentration in cell suspensions

    NASA Astrophysics Data System (ADS)

    Park, Byoung Kyoo; Yi, Namwoo; Park, Jaesung; Kim, Yonggoo; Kim, Dongsik

    2014-04-01

    This paper presents a novel biothermal sensor to probe cell viability and concentration of a cell suspension. The sensing technique exploits the thermophysical properties of the suspension, so no labeling of suspended cells is required. When the sensor is periodically heated, the amplitude and phase of the thermal signal are dependent on the thermal properties of the cell suspension, particularly the thermal conductivity k. We measured k of HeLa, hepatocyte, and NIH-3T3 J2 cell suspensions with various concentrations and viabilities. The results demonstrate that the k of a cell suspension has a strong correlation with its concentration and viability. Accordingly, k can be employed as an index of cell concentration and viability. Furthermore, without data processing to obtain k, the electric signal that reflects the thermal response of the sensor can be used as a tool to probe viability of a cell suspension in real time. The proposed thermal sensing technique offers label-free, non-invasive, long-term, and real-time means to probe the viability and concentration of cells in a suspension.

  4. Dendritic Cells Promote Pancreatic Viability in Mice with Acute Pancreatitis

    PubMed Central

    Bedrosian, Andrea S.; Nguyen, Andrew H.; Hackman, Michael; Connolly, Michael K.; Malhotra, Ashim; Ibrahim, Junaid; Cieza-Rubio, Napoleon E.; Henning, Justin R.; Barilla, Rocky; Rehman, Adeel; Pachter, H. Leon; Medina-Zea, Marco V.; Cohen, Steven M.; Frey, Alan B.; Acehan, Devrim; Miller, George

    2011-01-01

    Background & Aims Acute pancreatitis increases morbidity and mortality from organ necrosis by mechanisms that are incompletely understood. Dendritic cells (DCs) can promote or suppress inflammation, depending on their subtype and context. We investigated the roles of DC in development of acute pancreatitis. Methods Acute pancreatitis was induced in CD11c.DTR mice using caerulein or L-arginine; DCs were depleted by administration of diphtheria toxin. Survival was analyzed using Kaplan-Meier analysis. Results Numbers of MHC II+CD11c+DC increased 100-fold in pancreas of mice with acute pancreatitis, to account for nearly 15% of intra-pancreatic leukocytes. Intra-pancreatic DC acquired an immune phenotype in mice with acute pancreatitis; they expressed higher levels of MHC II and CD86 and increased production of interleukin-6, membrane cofactor protein (MCP)-1, and tumor necrosis factor (TNF)-α. However, rather than inducing an organ-destructive inflammatory process, DC were required for pancreatic viability; the exocrine pancreas died in mice that were depleted of DC and challenged with caerulein or L-arginine. All mice with pancreatitis that were depleted of DC died from acinar cell death within 4 days. Depletion of DC from mice with pancreatitis resulted in neutrophil infiltration and increased levels of systemic markers of inflammation. However, the organ necrosis associated with depletion of DC did not require infiltrating neutrophils, activation of NF-κB, or signaling by mitogen-activated protein kinase or TNF-α. Conclusions DC are required for pancreatic viability in mice with acute pancreatitis and might protect organs against cell stress. PMID:21801698

  5. Cell-Directed Assembly of Lipid-Silica Nanostructures Providing Extended Cell Viability

    NASA Astrophysics Data System (ADS)

    Baca, Helen K.; Ashley, Carlee; Carnes, Eric; Lopez, Deanna; Flemming, Jeb; Dunphy, Darren; Singh, Seema; Chen, Zhu; Liu, Nanguo; Fan, Hongyou; López, Gabriel P.; Brozik, Susan M.; Werner-Washburne, Margaret; Brinker, C. Jeffrey

    2006-07-01

    Amphiphilic phospholipids were used to direct the formation of biocompatible, uniform silica nanostructures in the presence of Saccharomyces cerevisiae and bacterial cell lines. The cell surfaces organize multilayered phospholipid vesicles that interface coherently with the silica host and help relieve drying stresses that develop with conventional templates. These host structures maintain cell accessibility, addressability, and viability in the absence of buffer or an external fluidic architecture. The cell surfaces are accessible and can be used to localize added proteins, plasmids, and nanocrystals. Prolonged cell viability combined with reporter protein expression enabled stand-alone cell-based sensing.

  6. Stem Cell Imaging: Tools to Improve Cell Delivery and Viability

    PubMed Central

    Wang, Junxin; Jokerst, Jesse V.

    2016-01-01

    Stem cell therapy (SCT) has shown very promising preclinical results in a variety of regenerative medicine applications. Nevertheless, the complete utility of this technology remains unrealized. Imaging is a potent tool used in multiple stages of SCT and this review describes the role that imaging plays in cell harvest, cell purification, and cell implantation, as well as a discussion of how imaging can be used to assess outcome in SCT. We close with some perspective on potential growth in the field. PMID:26880997

  7. Spatial Heterogeneity Analysis in Evaluation of Cell Viability and Apoptosis for Colorectal Cancer Cells

    PubMed Central

    Kucharavy, Herman; Hubbard, Karen; Uyar, Muharrem Ümit

    2016-01-01

    In evaluation of cell viability and apoptosis, spatial heterogeneity is quantified for cancerous cells cultured in 3-D in vitro cell-based assays under the impact of anti-cancer agents. In 48-h experiments using human colorectal cancer cell lines of HCT-116, SW-620, and SW-480, incubated cells are divided into control and drug administered groups, to be grown in matrigel and FOLFOX solution, respectively. Our 3-D cell tracking and data acquisition system guiding an inverted microscope with a digital camera is utilized to capture bright field and fluorescent images of colorectal cancer cells at multiple time points. Identifying the locations of live and dead cells in captured images, spatial point process and Voronoi tessellation methods are applied to extract morphological features of in vitro cell-based assays. For the former method, spatial heterogeneity is quantified with the second-order functions of Poisson point process, whereas the deviation in the area of Voronoi polygons is computed for the latter. With both techniques, the results indicate that the spatial heterogeneity of live cell locations increases as the viability of in in vitro cell cultures decreases. On the other hand, a decrease is observed for the heterogeneity of dead cell locations with the decrease in cell viability. This relationship between morphological features of in vitro cell-based assays and cell viability can be used for drug efficacy measurements and utilized as a biomarker for 3-D in vitro microenvironment assays. PMID:27574578

  8. Spatial Heterogeneity Analysis in Evaluation of Cell Viability and Apoptosis for Colorectal Cancer Cells.

    PubMed

    Saribudak, Aydin; Kucharavy, Herman; Hubbard, Karen; Uyar, Muharrem Umit

    2016-01-01

    In evaluation of cell viability and apoptosis, spatial heterogeneity is quantified for cancerous cells cultured in 3-D in vitro cell-based assays under the impact of anti-cancer agents. In 48-h experiments using human colorectal cancer cell lines of HCT-116, SW-620, and SW-480, incubated cells are divided into control and drug administered groups, to be grown in matrigel and FOLFOX solution, respectively. Our 3-D cell tracking and data acquisition system guiding an inverted microscope with a digital camera is utilized to capture bright field and fluorescent images of colorectal cancer cells at multiple time points. Identifying the locations of live and dead cells in captured images, spatial point process and Voronoi tessellation methods are applied to extract morphological features of in vitro cell-based assays. For the former method, spatial heterogeneity is quantified with the second-order functions of Poisson point process, whereas the deviation in the area of Voronoi polygons is computed for the latter. With both techniques, the results indicate that the spatial heterogeneity of live cell locations increases as the viability of in in vitro cell cultures decreases. On the other hand, a decrease is observed for the heterogeneity of dead cell locations with the decrease in cell viability. This relationship between morphological features of in vitro cell-based assays and cell viability can be used for drug efficacy measurements and utilized as a biomarker for 3-D in vitro microenvironment assays. PMID:27574578

  9. Improving Viability of Stem Cells During Syringe Needle Flow Through the Design of Hydrogel Cell Carriers

    PubMed Central

    Aguado, Brian A.; Mulyasasmita, Widya; Su, James; Lampe, Kyle J.

    2012-01-01

    Cell transplantation is a promising therapy for a myriad of debilitating diseases; however, current delivery protocols using direct injection result in poor cell viability. We demonstrate that during the actual cell injection process, mechanical membrane disruption results in significant acute loss of viability at clinically relevant injection rates. As a strategy to protect cells from these damaging forces, we hypothesize that cell encapsulation within hydrogels of specific mechanical properties will significantly improve viability. We use a controlled in vitro model of cell injection to demonstrate success of this acute protection strategy for a wide range of cell types including human umbilical vein endothelial cells (HUVEC), human adipose stem cells, rat mesenchymal stem cells, and mouse neural progenitor cells. Specifically, alginate hydrogels with plateau storage moduli (G′) ranging from 0.33 to 58.1 Pa were studied. A compliant crosslinked alginate hydrogel (G′=29.6 Pa) yielded the highest HUVEC viability, 88.9%±5.0%, while Newtonian solutions (i.e., buffer only) resulted in 58.7%±8.1% viability. Either increasing or decreasing the hydrogel storage modulus reduced this protective effect. Further, cells within noncrosslinked alginate solutions had viabilities lower than media alone, demonstrating that the protective effects are specifically a result of mechanical gelation and not the biochemistry of alginate. Experimental and theoretical data suggest that extensional flow at the entrance of the syringe needle is the main cause of acute cell death. These results provide mechanistic insight into the role of mechanical forces during cell delivery and support the use of protective hydrogels in future clinical stem cell injection studies. PMID:22011213

  10. Effects of biosurfactants on the viability and proliferation of human breast cancer cells

    PubMed Central

    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. PMID:24949273

  11. Imatinib alters cell viability but not growth factors levels in TM4 Sertoli cells

    PubMed Central

    Hashemnia, Seyyed Mohammad Reza; Atari-Hajipirloo, Somayeh; Roshan-Milani, Shiva; Valizadeh, Nasim; Mahabadi, Sonya; Kheradmand, Fatemeh

    2016-01-01

    Background: The anticancer agent imatinib (IM) is a small molecular analog of ATP that inhibits tyrosine kinase activity of platelet derived growth factors (PDGFs) and stem cell factor (SCF) receptor in cancer cells. However these factors have a key role in regulating growth and development of normal Sertoli, Leydig and germ cells. Objective: The aim of this study was to determine cell viability, PDGF and SCF levels in mouse normal Sertoli cells exposed to IM. Materials and Methods: In this experimental study, the mouse TM4 Sertoli cells were treated with 0, 2.5, 5, 10 and 20 μM IM for 2, 4 or 6 days. The cell viability and growth factors levels were assessed by MTT and ELISA methods, respectively. For statistical analysis, One-Way ANOVA was performed. Results: IM showed significant decrease in Sertoli cell viability compared to control group (p=0.001). However, IM increased PDGF and SCF level insignificantly (p>0.05). Conclusion: Results suggested that IM treatment induced a dose dependent reduction of cell viability in Sertoli cells. It seems that treatment with this anticancer drug is involved in the fertility process. Further studies are needed to evaluate the role of PDGF and SCF in this cell. PMID:27738659

  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. 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. PMID:26886589

  14. Effect of laser fluence on yeast cell viability in laser-assisted cell transfer

    NASA Astrophysics Data System (ADS)

    Lin, Yafu; Huang, Yong; Wang, Gaoyan; Tzeng, Tzeng-Rong J.; Chrisey, Douglas B.

    2009-08-01

    Matrix-assisted pulsed-laser evaporation direct-write (MAPLE DW) has been emerging as a promising biological construct fabrication technique. The post-transfer cell viability in MAPLE DW depends on various operation conditions such as the applied laser fluence; unfortunately, the effect of laser fluence on the post-transfer cell viability has not been well elucidated. This work aims to study the effect of laser fluence on the post-transfer cell viability and the cell recovery ability in MAPLE DW of yeast cells (Saccharomyces cerevisiae). It has been observed that (1) yeast cell viability decreases as the laser fluence increases from 85 to around 1500mJ/cm2 and (2) some of the MAPLE DW process-induced cell damage is reversible. The post-transfer yeast cell recovery is a function of laser fluence; however, this dependence relationship is not monotonic. Future work is needed to better understand the physical and chemical mechanisms of the above observations.

  15. Effect of methoxychlor on Ca(2+) movement and viability in MDCK renal tubular cells.

    PubMed

    Cheng, He-Hsiung; Lu, Yi-Chau; Lu, Ti; Cheng, Jin-Shiung; Mar, Guang-Yuan; Fang, Yi-Chien; Chai, Kuo-Liang; Jan, Chung-Ren

    2012-10-01

    The effect of the insecticide methoxychlor on the physiology of renal tubular cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca(2+) concentrations ([Ca(2+) ](i) ) in MDCK renal tubular cells using the Ca(2+) -sensitive fluorescent dye fura-2. Methoxychlor at 5-20 μM increased [Ca(2+) ](i) in a concentration-dependent manner. The signal was reduced by 80% by removing extracellular Ca(2+) . Methoxychlor-induced Ca(2+) entry was not affected by nifedipine and SK&F96365 but was inhibited by econazole and protein kinase C modulators. In Ca(2+) -free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) partly inhibited methoxychlor-induced [Ca(2+) ](i) rise. Incubation with methoxychlor also inhibited thapsigargin- or BHQ-induced [Ca(2+) ](i) rise. Inhibition of phospholipase C with U73122 nearly abolished methoxychlor-induced [Ca(2+) ](i) rise. At 5-15 μM, methoxychlor slightly increased cell viability, whereas at 20 μM, it decreased viability. The cytotoxic effect of methoxychlor was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid/AM (BAPTA/AM). Annexin V-FITC data suggest that 10 μM methoxychlor inhibited apoptosis, while 20 μM methoxychlor enhanced apoptosis. Methoxychlor (10 and 20 μM) increased the production of reactive oxygen species. Together, in renal tubular cells, methoxychlor induced [Ca(2+) ](i) rise by inducing phospholipase C-dependent Ca(2+) release from multiple stores and Ca(2+) entry via protein kinase C- and econazole-sensitive channels. Methoxychlor slightly enhanced or inhibited cell viability in a concentration-dependent, Ca(2+) -independent manner. Methoxychlor induced cell death that may involve apoptosis via mitochondrial pathways.

  16. 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.

  17. Do trichothecenes reduce viability of circulating blood cells and modify haemostasis parameters?

    PubMed

    Froquet, R; Arnold, F; Batina, P; Parent-Massin, D

    2003-01-01

    This manuscript describes the results of experiments conducted using human blood cells to determine the ability of T-2 toxin and DON to cause changes in clotting time, platelet aggregation, red blood cell haemolysis, RBC glucose content, lactate release, glutathione depletion, as well as white blood cell viability. In vitro results showed that haemostasis parameters and erythrocytes were not affected at concentrations able to induce inhibition of haematopoietic progenitor proliferation. In the presence of 10(-8) M and 10(-6) M T-2, the leucocyte number decreased at 24 h by 30% and 50% respectively. A 50% decrease in leucocyte number was observed for 10(-5) M DON. Results were compared with haematopoietic progenitor sensitivities. Due to the differences in sensitivities between mature blood cells and haematopoietic progenitors, haematological problems associated with trichothecene intoxication could be attributed to haematopoiesis inhibition.

  18. Loss of GLUT4 induces metabolic reprogramming and impairs viability of breast cancer cells.

    PubMed

    Garrido, Pablo; Osorio, Fernando G; Morán, Javier; Cabello, Estefanía; Alonso, Ana; Freije, José M P; González, Celestino

    2015-01-01

    Metabolic reprogramming strategies focus on the normalization of metabolism of cancer cells and constitute promising targets for cancer treatment. Here, we demonstrate that the glucose transporter 4 (GLUT4) has a prominent role in basal glucose uptake in MCF7 and MDA-MB-231 breast cancer cells. We show that shRNA-mediated down-regulation of GLUT4 diminishes glucose uptake and induces metabolic reprogramming by reallocating metabolic flux to oxidative phosphorylation. This reallocation is reflected on an increased activity of the mitochondrial oxidation of pyruvate and lower lactate release. Altogether, GLUT4 inhibition compromises cell proliferation and critically affects cell viability under hypoxic conditions, providing proof-of-principle for the feasibility of using pharmacological approaches to inhibit GLUT4 in order to induce metabolic reprogramming in vivo in breast cancer models.

  19. Cytoplasmic myosin-exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability.

    PubMed

    Cui, X; Zhang, L; Magli, A R; Catera, R; Yan, X-J; Griffin, D O; Rothstein, T L; Barrientos, J; Kolitz, J E; Allen, S L; Rai, K R; Chiorazzi, N; Chu, C C

    2016-01-01

    The degree of chronic lymphocytic leukemia (CLL) B-cell antigen receptor (BCR) binding to myosin-exposed apoptotic cells (MEACs) correlates with worse patient outcomes, suggesting a link to disease activity. Therefore, we studied MEAC formation and the effects of MEAC binding on CLL cells. In cell line studies, both intrinsic (spontaneous or camptothecin-induced) and extrinsic (FasL- or anti-Fas-induced) apoptosis created a high percent of MEACs over time in a process associated with caspase-3 activation, leading to cytoplasmic myosin cleavage and trafficking to cell membranes. The involvement of common apoptosis pathways suggests that most cells can produce MEACs and indeed CLL cells themselves form MEACs. Consistent with the idea that MEAC formation may be a signal to remove dying cells, we found that natural IgM antibodies bind to MEACs. Functionally, co-culture of MEACs with CLL cells, regardless of immunoglobulin heavy-chain variable region gene mutation status, improved leukemic cell viability. Based on inhibitor studies, this improved viability involved BCR signaling molecules. These results support the hypothesis that stimulation of CLL cells with antigen, such as those on MEACs, promotes CLL cell viability, which in turn could lead to progression to worse disease. PMID:26220042

  20. 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 and testosterone production as well as on the viability of Leydig cells of NMRI mice. The cells were cultured for 44 h with addition of 0.04; 0.2; 1.0; 2.5 and 5.0 μg/ml of 4-nonylphenol and compared to the control. Quantification of testosterone and androstenedione directly from aliquots of the medium was performed by enzyme-linked immunosorbent assay. Cell viability was measured by the metabolic activity assay for mitochondrial functional activity. Androstenedione production significantly (P < 0.001) increased with 1.0; 2.5 and 5.0 μg/ml 4-nonylphenol. Although cAMP-stimulated testosterone production was not significantly affected by 4-nonylphenol, a tendency to attenuate the level of testosterone in the Leydig cells treated with 2.5 and 5.0 μg/ml 4-nonylphenol was observed. The viability of mouse Leydig cells was slightly increased at the lowest doses of 4-nonylphenol (0.04 and 0.2 μg/ml). We also observed an increase at higher concentrations of the substance (1.0; 2.5 and 5.0 μg/ml), but this increase was not significant. Further investigations are required to establish the biological significance and possible reproductive implications. PMID:27085008

  1. 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.

  2. Effect of Cold Plasma on Cell Viability and Collagen Synthesis in Cultured Murine Fibroblasts

    NASA Astrophysics Data System (ADS)

    Shi, Xingmin; Cai, Jingfen; Xu, Guimin; Ren, Hongbin; Chen, Sile; Chang, Zhengshi; Liu, Jinren; Huang, Chongya; Zhang, Guanjun; Wu, Xili

    2016-04-01

    An argon atmospheric pressure plasma jet was employed to treat L929 murine fibroblasts cultured in vitro. Experimental results showed that, compared with the control cells, the treatment of fibroblasts with 15 s of plasma led to a significant increase of cell viability and collagen synthesis, while the treatment of 25 s plasma resulted in a remarkable decrease. Exploration of related mechanisms suggested that cold plasma could up-regulate CyclinD1 gene expression and down-regulate p27 gene expression at a low dose, while it could down-regulate CyclinD1 expression and up-regulate p27 expression at a higher dose, thus altering the cell cycle progression, and then affecting cell viability and collagen synthesis of fibroblasts. supported partly by National Natural Science Foundation of China (Nos. 81372076, 51307133 and 51221005), China National Funds for Distinguished Young Scientists (No. 51125029), the Sci-Tech Project of Shaanxi Province of China (No. 2010K16-04), and the Fundamental Research Funds for the Central Universities of China (No. xkjc2013004)

  3. Emerging role of calcium-activated potassium channel in the regulation of cell viability following potassium ions challenge in HEK293 cells and pharmacological modulation.

    PubMed

    Tricarico, Domenico; Mele, Antonietta; Calzolaro, Sara; Cannone, Gianluigi; Camerino, Giulia Maria; Dinardo, Maria Maddalena; Latorre, Ramon; Conte Camerino, Diana

    2013-01-01

    Emerging evidences suggest that Ca(2+)activated-K(+)-(BK) channel is involved in the regulation of cell viability. The changes of the cell viability observed under hyperkalemia (15 mEq/L) or hypokalemia (0.55 mEq/L) conditions were investigated in HEK293 cells expressing the hslo subunit (hslo-HEK293) in the presence or absence of BK channel modulators. The BK channel openers(10(-11)-10(-3)M) were: acetazolamide(ACTZ), Dichlorphenamide(DCP), methazolamide(MTZ), bendroflumethiazide(BFT), ethoxzolamide(ETX), hydrochlorthiazide(HCT), quercetin(QUERC), resveratrol(RESV) and NS1619; and the BK channel blockers(2 x 10(-7)M-5 x 10(-3)M) were: tetraethylammonium(TEA), iberiotoxin(IbTx) and charybdotoxin(ChTX). Experiments on cell viability and channel currents were performed using cell counting kit-8 and patch-clamp techniques, respectively. Hslo whole-cell current was potentiated by BK channel openers with different potency and efficacy in hslo-HEK293. The efficacy ranking of the openers at -60 mV(Vm) was BFT> ACTZ >DCP ≥RESV≥ ETX> NS1619> MTZ≥ QUERC; HCT was not effective. Cell viability after 24 h of incubation under hyperkalemia was enhanced by 82+6% and 33+7% in hslo-HEK293 cells and HEK293 cells, respectively. IbTx, ChTX and TEA enhanced cell viability in hslo-HEK293. BK openers prevented the enhancement of the cell viability induced by hyperkalemia or IbTx in hslo-HEK293 showing an efficacy which was comparable with that observed as BK openers. BK channel modulators failed to affect cell currents and viability under hyperkalemia conditions in the absence of hslo subunit. In contrast, under hypokalemia cell viability was reduced by -22+4% and -23+6% in hslo-HEK293 and HEK293 cells, respectively; the BK channel modulators failed to affect this parameter in these cells. In conclusion, BK channel regulates cell viability under hyperkalemia but not hypokalemia conditions. BFT and ACTZ were the most potent drugs either in activating the BK current and in preventing

  4. 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.

  5. Production of intracellular reactive oxygen species and change of cell viability induced by atmospheric pressure plasma in normal and cancer cells

    NASA Astrophysics Data System (ADS)

    Ja Kim, Sun; Min Joh, Hea; Chung, T. H.

    2013-10-01

    The effects of atmospheric pressure plasma jet on cancer cells (human lung carcinoma cells) and normal cells (embryonic kidney cells and bronchial epithelial cells) were investigated. Using a detection dye, the production of intracellular reactive oxygen species (ROS) was found to be increased in plasma-treated cells compared to non-treated and gas flow-treated cells. A significant overproduction of ROS and a reduction in cell viability were induced by plasma exposure on cancer cells. Normal cells were observed to be less affected by the plasma-mediated ROS, and cell viability was less changed. The selective effect on cancer and normal cells provides a promising prospect of cold plasma as a cancer therapy.

  6. 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.

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

    PubMed Central

    Tremblay, Michel G.; Moss, Tom

    2016-01-01

    ABSTRACT 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. PMID:27399837

  8. The Cytotoxic Role of Intermittent High Glucose on Apoptosis and Cell Viability in Pancreatic Beta Cells

    PubMed Central

    Zhang, Zhen; Li, Jing; Yang, Lei; Chen, Rongping; Yang, Rui; Zhang, Hua; Cai, Dehong; Chen, Hong

    2014-01-01

    Objectives. Glucose fluctuations are both strong predictor of diabetic complications and crucial factor for beta cell damages. Here we investigated the effect of intermittent high glucose (IHG) on both cell apoptosis and proliferation activity in INS-1 cells and the potential mechanisms. Methods. Cells were treated with normal glucose (5.5 mmol/L), constant high glucose (CHG) (25 mmol/L), and IHG (rotation per 24 h in 11.1 or 25 mmol/L) for 7 days. Reactive oxygen species (ROS), xanthine oxidase (XOD) level, apoptosis, cell viability, cell cycle, and expression of cyclinD1, p21, p27, and Skp2 were determined. Results. We found that IHG induced more significant apoptosis than CHG and normal glucose; intracellular ROS and XOD levels were more markedly increased in cells exposed to IHG. Cells treated with IHG showed significant decreased cell viability and increased cell proportion in G0/G1 phase. Cell cycle related proteins such as cyclinD1 and Skp2 were decreased significantly, but expressions of p27 and p21 were increased markedly. Conclusions. This study suggested that IHG plays a more toxic effect including both apoptosis-inducing and antiproliferative effects on INS-1 cells. Excessive activation of cellular stress and regulation of cyclins might be potential mechanism of impairment in INS-1 cells induced by IHG. PMID:24772447

  9. Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

    PubMed Central

    Johnson, M. Brittany; Criss, Alison K.

    2013-01-01

    Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells. PMID:24056524

  10. Real-time cell viability assays using a new anthracycline derivative DRAQ7®

    PubMed Central

    Akagi, Jin; Kordon, Magdalena; Zhao, Hong; Matuszek, Anna; Dobrucki, Jurek; Errington, Rachel; Smith, Paul J; Takeda, Kazuo; Darzynkiewicz, Zbigniew; Wlodkowic, Donald

    2012-01-01

    The exclusion of charged fluorescent dyes by intact cells has become a well-established assay for determining viability of cells. In search for a non-invasive fluorescent probe capable of long-term monitoring of cell death in real-time, we evaluated a new anthracycline derivative DRAQ7. The novel probe does not penetrate the plasma membrane of living cells but when the membrane integrity is compromised, it enters and binds readily to nuclear DNA to report cell death. It proved to be non-toxic to a panel of cancer cell lines grown continuously for up to 72 hours and did not induce any detectable DNA damage signaling when analyzed using laser scanning microscopy and flow cytometry. DRAQ7 provided a sensitive, real-time readout of cell death induced by a variety of stressors such as hypoxia, starvation and drug-induced cytotoxicity. The overall responses to anti-cancer agents and resulting pharmacological dose-response profiles were not affected by the growth of tumor cells in the presence DRAQ7. Moreover, we for the first time introduced a near real-time microflow cytometric assay based on combination of DRAQ7 and mitochondrial inner membrane potential (ΔΨm) sensitive probe TMRM. We provide evidence that this low-dosage, real-time labeling procedure provides multi-parameter and kinetic fingerprint of anti-cancer drug action. PMID:23165976

  11. Changes of cytokine production and cell viability of peripheral blood mononuclear cells from silicosis patients: effect of in vitro treatment with acetylsalicylic acid.

    PubMed

    Dobreva, Zlatka Georgieva; Prakova, Gospodinka Radeva; Slavov, Emil Slavov; Stanilova, Spaska Angelova

    2010-02-01

    In this study, IL-6 and IL-12p40 production and cell viability of peripheral blood mononuclear cells from silicosis patients after in vitro stimulation were investigated. Furthermore, the effects of introducing acetylsalicylic acid to stimulated patients' peripheral blood mononuclear cells on cytokine production and cell viability were determined. Nine patients with moderate silicosis, 11 with severe silicosis and 14 healthy subjects were recruited for this study. The level of IL-6 produced by patients peripheral blood mononuclear cells decreased depending on the stage of the disease. The addition of acetylsalicylic acid had significantly suppressive effect on the IL-6 production by lipopolysaccharide-stimulated patients' peripheral blood mononuclear cells. Acetylsalicylic acid treatment of C3 binding glycoprotein-stimulated patients' peripheral blood mononuclear cells led to significant upregulation of IL-12p40 production. Results showed a stage-dependent decrease of cell viability of peripheral blood mononuclear cells from silicosis patients. Acetylsalicylic acid significantly decreased cell viability entirely in stimulated peripheral blood mononuclear cells from patients with severe silicosis. In conclusion, this study showed that the disease progression affects peripheral blood mononuclear cells in patients with silicosis and causes functional changes that became apparent after stimulation. Our study demonstrated that in severe silicosis the treatment with acetylsalicylic acid, as an anti-inflammatory agent, might not be beneficial for patients.

  12. Assessment of planctomycetes cell viability after pollutants exposure.

    PubMed

    Flores, Carlos; Catita, José A M; Lage, Olga Maria

    2014-08-01

    In this study, the growth of six different planctomycetes, a particular ubiquitous bacterial phylum, was assessed after exposure to pollutants. In addition and for comparative purposes, Pseudomonas putida, Escherichia coli and Vibrio anguillarum were tested. Each microorganism was exposed to several concentrations of 21 different pollutants. After exposure, bacteria were cultivated using the drop plate method. In general, the strains exhibited a great variation of sensitivity to pollutants in the order: V. anguillarum > planctomycetes > P. putida > E. coli. E. coli showed resistance to all pollutants tested, with the exception of phenol and sodium azide. Copper, Ridomil® (fungicide), hydrazine and phenol were the most toxic pollutants. Planctomycetes were resistant to extremely high concentrations of nitrate, nitrite and ammonium but they were the only bacteria sensitive to Previcur N® (fungicide). Sodium azide affected the growth on plates of E. coli, P. putida and V. anguillarum, but not of planctomycetes. However, this compound affected planctomycetes cell respiration but with less impact than in the aforementioned bacteria. Our results provide evidence for a diverse response of bacteria towards pollutants, which may influence the structuring of microbial communities in ecosystems under stress, and provide new insights on the ecophysiology of planctomycetes.

  13. Ice-Binding Protein Derived from Glaciozyma Can Improve the Viability of Cryopreserved Mammalian Cells.

    PubMed

    Kim, Hak Jun; Shim, Hye Eun; Lee, Jun Hyuck; Kang, Yong-Cheol; Hur, Young Baek

    2015-12-28

    Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1°C/min in a -80°C freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.

  14. Tick-Borne Encephalitis Virus Infects Rat Astrocytes but Does Not Affect Their Viability

    PubMed Central

    Potokar, Maja; Korva, Miša; Jorgačevski, Jernej; Avšič-Županc, Tatjana; Zorec, Robert

    2014-01-01

    Tick-borne encephalitis virus (TBEV) causes one of the most dangerous human neuroinfections in Europe and Asia. To infect neurons it must cross the blood-brain-barrier (BBB), and presumably also cells adjacent to the BBB, such as astrocytes, the most abundant glial cell type. However, the knowledge about the viral infection of glial cells is fragmental. Here we studied whether TBEV infects rat astrocytes. Rats belong to an animal group serving as a TBEV amplifying host. We employed high resolution quantitative fluorescence microscopy to investigate cell entry and cytoplasmic mobility of TBEV particles along with the effect on the cell cytoskeleton and cell survival. We report that infection of astrocytes with TBEV increases with time of exposure to TBEV and that with post-infection time TBEV particles gained higher mobility. After several days of infection actin cytoskeleton was affected, but cell survival was unchanged, indicating that rat astrocytes resist TBEV-mediated cell death, as reported for other mammalian cells. Therefore, astrocytes may present an important pool of dormant TBEV infections and a new target for therapeutic intervention. PMID:24465969

  15. MAML1 regulates cell viability via the NF-{kappa}B pathway in cervical cancer cell lines

    SciTech Connect

    Kuncharin, Yanin; Sangphech, Naunpun; Kueanjinda, Patipark; Bhattarakosol, Parvapan; Palaga, Tanapat

    2011-08-01

    The Notch signaling pathway plays important roles in tumorigenesis in a context-dependent manner. In human cervical cancer, alterations in Notch signaling have been reported, and both tumor-suppressing and tumor-promoting roles of Notch signaling have been proposed; however, the precise molecular mechanisms governing these roles in cervical cancer remain controversial. MAML is a transcriptional co-activator originally identified by its role in Notch signaling. Recent evidence suggests that it also plays a role in other signaling pathways, such as the p53 and {beta}-catenin pathways. MAML is required for stable formation of Notch transcriptional complexes at the promoters of Notch target genes. Chromosomal translocations affecting MAML have been shown to promote tumorigenesis. In this study, we used a truncated dominant-negative MAML1 (DN-MAML) to investigate the role of MAML in HPV-positive cervical cancer cell lines. Three human cervical cancer cell lines (HeLa, SiHa and CaSki) expressed all Notch receptors and the Notch target genes Hes1 and MAML1. Among these 3 cell lines, constitutive appearance of cleaved Notch1 was found only in CaSki cells, which suggests that Notch1 is constitutively activated in this cell line. Gamma secretase inhibitor (GSI) treatment, which suppresses Notch receptor activation, completely abrogated this form of Notch1 but had no effect on cell viability. Overexpression of DN-MAML by retroviral transduction in CaSki cells resulted in significant decreases in the mRNA levels of Hes1 and Notch1 but had no effects on the levels of MAML1, p53 or HPV E6/E7. DN-MAML expression induced increased viability of CaSki cells without any effect on cell cycle progression or cell proliferation. In addition, clonogenic assay experiments revealed that overexpression of DN-MAML resulted in increased colony formation compared to the overexpression of the control vector. When the status of the NF-{kappa}B pathway was investigated, CaSki cells overexpressing

  16. Gallic acid reduces cell viability, proliferation, invasion and angiogenesis in human cervical cancer cells

    PubMed Central

    ZHAO, BING; HU, MENGCAI

    2013-01-01

    Gallic acid is a trihydroxybenzoic acid, also known as 3,4,5-trihydroxybenzoic acid, which is present in plants worldwide, including Chinese medicinal herbs. Gallic acid has been shown to have cytotoxic effects in certain cancer cells, without damaging normal cells. The objective of the present study was to determine whether gallic acid is able to inhibit human cervical cancer cell viability, proliferation and invasion and suppress cervical cancer cell-mediated angiogenesis. Treatment of HeLa and HTB-35 human cancer cells with gallic acid decreased cell viability in a dose-dependent manner. BrdU proliferation and tube formation assays indicated that gallic acid significantly decreased human cervical cancer cell proliferation and tube formation in human umbilical vein endothelial cells, respectively. Additionally, gallic acid decreased HeLa and HTB-35 cell invasion in vitro. Western blot analysis demonstrated that the expression of ADAM17, EGFR, p-Akt and p-Erk was suppressed by gallic acid in the HeLa and HTB-35 cell lines. These data indicate that the suppression of ADAM17 and the downregulation of the EGFR, Akt/p-Akt and Erk/p-Erk signaling pathways may contribute to the suppression of cancer progression by Gallic acid. Gallic acid may be a valuable candidate for the treatment of cervical cancer. PMID:24843386

  17. Gallic acid suppresses cell viability, proliferation, invasion and angiogenesis in human glioma cells

    PubMed Central

    Lu, Yong; Jiang, Feng; Jiang, Hao; Wu, Kalina; Zheng, Xuguang; Cai, Yizhong; Katakowski, Mark; Chopp, Michael; To, Shing-Shun Tony

    2010-01-01

    Gallic acid, an organic acid, also known as 3,4,5-trihydroxybenzoic acid, is cytotoxic against certain cancer cells, without harming normal cells. The objective of this study is to evaluate whether gallic acid can inhibit glioma cell viability, proliferation, invasion and reduce glioma cell mediated angiogenesis. Treatment of U87 and U251n glioma cells with gallic acid inhibited cell viability in a dose- and time-dependent manner. BrdU and tube formation assays indicated that gallic acid significantly decreased glioma cell proliferation and tube formation in mouse brain endothelial cells, respectively. In addition, gallic acid decreased U87 cell invasion in vitro. Western blot analysis showed that expression of ADAM17, p-Akt and p-Erk was suppressed by gallic acid in both U87 and U251n cell lines. These data suggest that suppression of ADAM17 and downregulation of PI3K/Akt and Ras/MAPK signaling pathways may contribute to gallic acid-induced decrease of invasiveness. Gallic acid may be a valuable candidate for treatment of brain tumor. PMID:20553913

  18. Effect of epithermal neutrons on viability of glioblastoma tumor cells in vitro.

    PubMed

    Mostovich, L A; Gubanova, N V; Kutsenko, O S; Aleinik, V I; Kuznetsov, A S; Makarov, A N; Sorokin, I N; Taskaev, S Yu; Nepomnyashchikh, G I; Grigor'eva, E V

    2011-06-01

    We studied in vitro effect of epithermal neutrons in various doses on viability of glioblastoma U87 tumor cells. Increasing the dose from 1.9 to 4.1 Sv promoted cell death. Cytofluorimetric analysis revealed no activation of apoptosis in the irradiated cells, which attested to necrotic death of the tumor cells exposed to epithermal neutron radiation.

  19. Soy milk as a storage medium to preserve human fibroblast cell viability: an in vitro study.

    PubMed

    Moura, Camilla Christian Gomes; Soares, Priscilla Barbosa Ferreira; Reis, Manuella Verdinelli de Paula; Fernandes Neto, Alfredo Júlio; Soares, Carlos José

    2012-01-01

    Soy milk (SM) is widely consumed worldwide as a substitute for cow milk. It is a source of vitamins, carbohydrates and sugars, but its capacity to preserve cell viability has not been evaluated. The purpose of the present study was to investigate the efficacy of SM to maintain the viability of human fibroblasts at short periods compared with different cow milks. Human mouth fibroblasts were cultured and stored in the following media at room temperature: 10% Dulbecco's Modified Eagle Medium (DMEM) (positive control group); long shelf-life ultra-high temperature whole cow milk (WM); long shelf-life ultra-high temperature skim cow milk (SKM); powdered cow milk (PM); and soy milk (SM). After 5, 15, 30 and 45 min, cell viability was analyzed using the MTT assay. Data were analyzed statistically by the Kruskal-Wallis test with post-analysis using the Dunn's method (α=0.05). SKM showed the lowest capacity to maintain cell viability in all analyzed times (p<0.05). At 30 and 45 min, the absorbance levels in control group (DMEM) and SM were significantly higher than in SKM (p<0.05). Cell viability decreased along the time (5-45 min). The results indicate that SM can be used as a more adequate storage medium for avulsed teeth. SKM was not as effective in preserving cell viability as the cell culture medium and SM.

  20. 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

  1. 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.

  2. 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. PMID:26406457

  3. Comparison of methods used for assessing the viability and vitality of yeast cells.

    PubMed

    Kwolek-Mirek, Magdalena; Zadrag-Tecza, Renata

    2014-11-01

    Determination of cell viability is the most commonly used method for assessing the impact of various types of stressors in toxicity research and in industrial microbiology studies. Viability is defined as a percentage of live cells in a whole population. Although cell death is one of the consequences of toxicity, chemical or physical factors may exert their toxic effects through a number of cellular alterations that may compromise cell ability to divide without necessarily leading to cell death. This aspect represents the term 'cell vitality' defined as physiological capabilities of cells. It is important to note that cell viability and cell vitality represent two different aspects of cell functions, and both are required for the estimation of the physiological state of a cell after exposure to various types of stressors and chemical or physical factors. In this paper, we introduced a classification of available methods for estimating both viability and vitality in Saccharomyces cerevisiae yeast cells (wild-type and Δsod1 mutant) in which the effects of selected oxidants causing oxidative stress is evaluated. We present the advantages as well as disadvantages of the selected methods and assess their usefulness in different types of research.

  4. 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.

  5. 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. PMID:25070929

  6. Is cell viability always directly related to corrosion resistance of stainless steels?

    PubMed

    Salahinejad, E; Ghaffari, M; Vashaee, D; Tayebi, L

    2016-05-01

    It has been frequently reported that cell viability on stainless steels is improved by increasing their corrosion resistance. The question that arises is whether human cell viability is always directly related to corrosion resistance in these biostable alloys. In this work, the microstructure and in vitro corrosion behavior of a new class of medical-grade stainless steels were correlated with adult human mesenchymal stem cell viability. The samples were produced by a powder metallurgy route, consisting of mechanical alloying and liquid-phase sintering with a sintering aid of a eutectic Mn-Si alloy at 1050 °C for 30 and 60 min, leading to nanostructures. In accordance with transmission electron microscopic studies, the additive particles for the sintering time of 30 min were not completely melted. Electrochemical impedance spectroscopic experiments suggested the higher corrosion resistance for the sample sintered for 60 min; however, a better cell viability on the surface of the less corrosion-resistant sample was unexpectedly found. This behavior is explained by considering the higher ion release rate of the Mn-Si additive material, as preferred sites to corrosion attack based on scanning electron microscopic observations, which is advantageous to the cells in vitro. In conclusion, cell viability is not always directly related to corrosion resistance in stainless steels. Typically, the introduction of biodegradable and biocompatible phases to biostable alloys, which are conventionally anticipated to be corrosion-resistant, can be advantageous to human cell responses similar to biodegradable metals. PMID:26952444

  7. Effects of a novel pesticide-particle conjugate on viability and reactive oxygen species generation in neuronal (PC12) cells.

    PubMed

    Sooresh, Aishwarya; Sayes, Christie M; Pine, Michelle

    2015-04-01

    Development of new methods and compounds to eradicate insect vectors are desperately needed. To that end, our team has previously described the synthesis and characterization of a conjugate comprised of a silver nanoparticle core encapsulated by the pyrethroid pesticide, deltamethrin (pesticide encapsulated silver nanoparticle termed "PENS"). For this current work, the PENS conjugate was tested in neuronal cultured cells to compare the cytotoxic responses to the unconjugated pesticide deltamethrin - a known neurotoxic agent and pristine silver nanoparticles. The PC12 (pheochromocytoma of the rat adrenal medulla) cell line was chosen as a model neuronal culture system. Cells were exposed to known concentrations of PENS, deltamethrin or silver nanoparticle suspensions to assess the degree of toxicity in vitro. After 24 hours of incubation, cell viability and intracellular reactive oxygen species (ROS) were measured. Bright field images of high dose exposures to dosing solutions were also acquired to evaluate cell morphology. Exposure to PENS resulted in a 17% decline in viability at the highest concentration of 45 µM while exposure to deltamethrin caused a 47% decrease. These results suggest that cellular viability was less adversely affected by PENS than by the deltamethrin. Also, ROS production following PENS exposure indicated that the newly developed conjugate was responding in a similar manner as that of cells treated with deltamethrin only.

  8. Metabolic activity of sperm cells: correlation with sperm cell concentration, viability and motility in the rabbit.

    PubMed

    Sabés-Alsina, Maria; Planell, Núria; Gil, Sílvia; Tallo-Parra, Oriol; Maya-Soriano, Maria José; Taberner, Ester; Piles, Miriam; Sabés, Manel; Lopez-Bejar, Manel

    2016-10-01

    The resazurin reduction test (RRT) is a useful technique to assess the metabolic rate of sperm cells. RRT depends on the ability of metabolically active cells to reduce the non-fluorescent dye resazurin to the fluorescent resorufin. The aim of this study was to develop a vital fluorometric method to evaluate metabolic activity of rabbit sperm cells. Twenty-five rabbit males were included in the study. Viability and morphology, motility and metabolic activity were evaluated using an eosin-nigrosin staining, a computer-assisted semen analysis (CASA) and the RRT, respectively. Spearman rank correlation analysis was used to determine the correlation between RRT and semen parameters. After evaluation, a concentration of 10 × 106 sperm cells/ml was selected for further experiments with RRT. No significant correlation was found between the RRT results and the motility parameters. However, after RRT a significant positive correlation between relative fluorescence units and the percentage of alive spermatozoa (r = 0.62; P = 0.001) and a negative one with the percentage of sperm cells with acrosomic abnormalities (r = -0.45; P < 0.05) were detected. The vital assessment of metabolic rate of sperm cells by RRT could provide more information about semen quality than other routine semen analysis, correlating with sperm viability and acrosome status information.

  9. The effect of TRAIL molecule on cell viability in in vitro beta cell culture.

    PubMed

    Tekmen, I; Ozyurt, D; Pekçetin, C; Buldan, Z

    2007-06-01

    Insulin-dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disorder triggered by autoreactive T cells directed to pancreas beta-cell antigens. In this disorder, more than 90% of beta cells are destroyed. Cell death may be mediated via soluble or membrane-bound cell death ligands. One of these ligands may be tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF-alpha superfamily. In the present study, we examined whether TRAIL had cytotoxic effects on adult rat pancreas beta cell cultures and INS1-E rat insulinoma cell line cultures or not. In this study, cell destruction models were built with TRAIL concentrations of 10, 100 and 1000 ng. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for evaluating cell viability. It was detected that cell cultures with TRAIL added showed no differences statistically when compared with control cultures containing no toxic additions. These results showed that TRAIL did not have significant cytotoxic effects on pancreas beta cell culture and INS-1E rat insulinoma cell line cultures. Detection of the expression of TRAIL receptors and natural apoptosis inhibitor proteins will be favourable to investigate the resistance mechanisms to TRAIL-induced cell death in this cell culture system.

  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. N-Acetyl cysteine restores viability and function of rat odontoblast-like cells impaired by polymethylmethacrylate dental resin extract.

    PubMed

    Yamada, Masahiro; Kojima, Norinaga; Att, Wael; Hori, Norio; Suzuki, Takeo; Ogawa, Takahiro

    2009-01-01

    There is concern that dental-resin materials directly loaded on a prepared tooth adversely affect dental pulp tissue by releasing the resin chemicals through dentinal tubes. This study determined whether self-curing polymethyl methacrylate (PMMA)-based dental resin extract adversely affected the viability and function of odontoblast-like cells and whether the cytotoxicity of this resin, if any, could be eliminated by N-acetyl cysteine, an antioxidant amino acid derivative. Odontoblast-like cells isolated from rat maxillary incisor dental pulp tissue were exposed to a PMMA resin extract with or without N-acetyl cysteine for 1 h and then cultured in osteoblastic media. The percentage of viable cells 24 h after seeding was 20% in cells exposed to the resin extract without N-acetyl cysteine, whereas 45% of cells were viable after exposure to the N-acetyl cysteine-supplemented extract. The cells that had been exposed to the extract showed a strong tendency for apoptosis associated with the increased reactive oxygen species production and decreased intracellular glutathione level, which was improved by the addition of N-acetyl cysteine. N-Acetyl cysteine supplementation almost completely restored the significantly reduced alkaline phosphatase activity and matrix mineralization by the resin extract. These results conclusively demonstrated that exposure of odontoblast-like cells to the resin extract impaired the cell viability and function and, more intriguingly, N-acetyl cysteine supplementation to the extract significantly prevented these toxic effects.

  12. Exposure to Music Alters Cell Viability and Cell Motility of Human Nonauditory Cells in Culture.

    PubMed

    Lestard, Nathalia R; Capella, Marcia A M

    2016-01-01

    Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells. PMID:27478480

  13. Exposure to Music Alters Cell Viability and Cell Motility of Human Nonauditory Cells in Culture

    PubMed Central

    Lestard, Nathalia R.

    2016-01-01

    Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells. PMID:27478480

  14. Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

    SciTech Connect

    Matveeva, V. G. Antonova, L. V. Velikanova, E. A.; Sergeeva, E. A.; Krivkina, E. O.; Glushkova, T. V.; Kudryavtseva, Yu. A.; Barbarash, O. L.; Barbarash, L. S.

    2015-10-27

    We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwoven scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.

  15. Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

    NASA Astrophysics Data System (ADS)

    Matveeva, V. G.; Antonova, L. V.; Velikanova, E. A.; Sergeeva, E. A.; Krivkina, E. O.; Glushkova, T. V.; Kudryavtseva, Yu. A.; Barbarash, O. L.; Barbarash, L. S.

    2015-10-01

    We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwoven scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.

  16. [Water activity and food stability. I. Effects on viability of Saccharomyces cerevisiae cells (author's transl)].

    PubMed

    Guerzoni, M E; Suzzi, G; Lerici, C R; Bartolini, R; Testa, G

    1976-01-01

    Biological activity of microorganism is related to water activity (aw). In this paper the effect of glicerol as humectant on Saccharomyces cerevisiae viability was considered. The irreversible loss of viability was observed only for values inferior than 0,75. The K+ presence promoted an increasing of cell viability and growth. We have evaluated the changes of the most important components of cell poll; the increasing of glicerol amount of the system induced a drastic fall of aminoacids, purines and K ions content, but it increased the Na ions content. The exposure of cells to increasing glicerol concentrations, caused an aminoacids and purines excretion related to contact time; after a few hours this material was readsorbed by cells. PMID:799835

  17. Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanisms

    PubMed Central

    Ochoa-Alvarez, Jhon A.; Krishnan, Harini; Pastorino, John G.; Nevel, Evan; Kephart, David; Lee, Joseph J.; Retzbach, Edward P.; Shen, Yongquan; Fatahzadeh, Mahnaz; Baredes, Soly; Kalyoussef, Evelyne; Honma, Masaru; Adelson, Martin E.; Kaneko, Mika K.; Kato, Yukinari; Young, Mary Ann; Deluca-Rapone, Lisa; Shienbaum, Alan J.; Yin, Kingsley; Jensen, Lasse D.; Goldberg, Gary S.

    2015-01-01

    Podoplanin (PDPN) is a unique transmembrane receptor that promotes tumor cell motility. Indeed, PDPN may serve as a chemotherapeutic target for primary and metastatic cancer cells, particularly oral squamous cell carcinoma (OSCC) cells that cause most oral cancers. Here, we studied how a monoclonal antibody (NZ-1) and lectin (MASL) that target PDPN affect human OSCC cell motility and viability. Both reagents inhibited the migration of PDPN expressing OSCC cells at nanomolar concentrations before inhibiting cell viability at micromolar concentrations. In addition, both reagents induced mitochondrial membrane permeability transition to kill OSCC cells that express PDPN by caspase independent nonapoptotic necrosis. Furthermore, MASL displayed a surprisingly robust ability to target PDPN on OSCC cells within minutes of exposure, and significantly inhibited human OSCC dissemination in zebrafish embryos. Moreover, we report that human OSCC cells formed tumors that expressed PDPN in mice, and induced PDPN expression in infiltrating host murine cancer associated fibroblasts. Taken together, these data suggest that antibodies and lectins may be utilized to combat OSCC and other cancers that express PDPN. PMID:25826087

  18. Activity of partially inhibited serine palmitoyltransferase is sufficient for normal sphingolipid metabolism and viability of HSN1 patient cells.

    PubMed

    Dedov, Vadim N; Dedova, Irina V; Merrill, Alfred H; Nicholson, Garth A

    2004-03-01

    Hereditary sensory neuropathy type I (HSN1) is a common degenerative disorder of peripheral sensory neurons. HSN1 is caused by mutations in the gene, encoding the long chain base 1 of serine palmitoyltransferase (SPT) [Nat. Genet. 27 (2001) 309]. Here, we show a 44% reduction of SPT activity in transformed lymphocytes from HSN1 patients with mutation T399G in the SPTLC1 gene. However, the decrease in SPT activity had no effect on de novo sphingolipid biosynthesis, cellular sphingolipid content, cell proliferation and death (apoptosis and necrosis). The removal of extracellular sphingolipids did not affect viability of HSN1 cells. We also found no significant difference in whole blood counts, viability, and permeability to Triton X-100 of primary lymphocytes from HSN1 patients. These results suggest that, despite the inhibition of mutant allele, the activity of nonmutant allele of STP may be sufficient for adequate sphingolipid biosynthesis and cell viability. Therefore, the neurodegeneration in HSN1 is likely to be caused by subtler and rather long-term effect(s) of these mutations such as loss of a cell-type selective facet of sphingolipid metabolism and/or function, or perhaps accumulation of toxic species, including abnormal protein(s) as in other neurodegenerations. PMID:14990347

  19. Effect of temozolomide on the viability of musculoskeletal sarcoma cells

    PubMed Central

    KUSABE, YUTA; KAWASHIMA, HIROYUKI; OGOSE, AKIRA; SASAKI, TARO; ARIIZUMI, TAKASHI; HOTTA, TETSUO; ENDO, NAOTO

    2015-01-01

    Musculoskeletal sarcomas (MSS) are a heterogeneous group of malignancies with relatively high mortality rates. The prognosis for patients with MSS is poor, with few drugs inducing measurable activity. Alkylating agents, namely ifosfamide and dacarbazine, which act nonspecifically on proliferating cells, are the typical therapy prescribed for advanced MSS. A novel alkylating agent, temozolomide (TMZ), has several advantages over existing alkylating agents. TMZ induces the formation of O6-methylguanine in DNA, thereby inducing mismatches during DNA replication and the subsequent activation of apoptotic pathways. However, due to conflicting data in the literature, the mechanism of TMZ action has remained elusive. Therefore, the present study aimed to evaluate apoptosis in MSS cells treated with TMZ, and to evaluate the correlation between TMZ action and survival pathways, including the phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 mitogen activated protein kinase (MAPK) pathways. Cell proliferation was evaluated by performing an XTT (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay. Apoptotic morphological changes, for example chromatin condensation, were evaluated by fluorescence confocal microscopy. The expression of the apoptosis-associated proteins caspase-3, poly adenosine diphosphate ribose polymerase (PARP), Akt and ERK1/2, was determined by western blotting. The results of the present study indicated that, in certain MSS cells, the IC50 value was lower than that in TMZ-sensitive U-87 MG cells. Furthermore, TMZ treatment was associated with apoptotic morphological changes and the expression levels of pro-apoptotic cleaved caspase-3 and PARP were also increased in TMZ-treated MSS cells. In addition, the results indicated that PI3K/Akt and ERK1/2 MAPK were constitutively phosphorylated in MSS cells, and phosphorylation of PI3K/Akt was suppressed in certain

  20. Mathematical analysis of mis-estimation of cell subsets in flow cytometry: viability staining revisited.

    PubMed

    Petrunkina, A M; Harrison, R A P

    2011-05-31

    Many research projects in cell biology now use flow cytometry for analysis or for isolation of specific cell types. In such studies, cell viability is obviously a crucial issue. However, many studies appear to rely upon light-scattering characteristics to identify and gate out non-viable cells, despite the fact that reliable identification of such cells can only be achieved through staining with impermeable fluorescent nuclear dyes such as propidium iodide or 7-amino actinomycin. In this paper we apply mathematical analysis to the theoretical problem of quantifying cell sub-populations labeled with two or more fluorescent markers, comparing situations in which dead cells have been identified with those in which cell viability has not been assessed. We demonstrate that in all cases in which dead cells are present within the population, percentages of live sub-populations in different subsets are mis-estimated. In cases where the pattern of marker expression differs greatly between live and dead cells, or where the proportion of dead cells is high, this mis-estimation will be aggravated; the subsets pattern will therefore be biased in a population selected only on the basis of light-scatter behavior. The importance of accurately detecting and gating out dead cells is illustrated by an experimental example accompanying the mathematical analysis. To conclude, identification of dead cells by means of viability stains should be an absolute routine in practical flow cytometry, so as to avoid mis-estimation in sorting or analysis.

  1. Cell biological analyses of anther morphogenesis and pollen viability in Arabidopsis and rice.

    PubMed

    Chang, Fang; Zhang, Zaibao; Jin, Yue; Ma, Hong

    2014-01-01

    Major advances have been made in recent years in our understanding of anther development through a combination of genetic studies, cell biological technologies, biochemical analysis, microarray and high-throughput sequencing-based approaches. In this chapter, we summarize the widely used protocols for pollen viability staining; the investigation of anther morphogenesis by light microscopy of semi-thin sections; TUNEL assay for programmed tapetum cell death; and laser microdissection procedures to obtain specialized cells or cell layers for carrying out transcriptomics.

  2. A Protocol for a High-Throughput Multiplex Cell Viability Assay.

    PubMed

    Gilbert, Daniel F; Boutros, Michael

    2016-01-01

    High-throughput cell viability assays are broadly used in RNAi and small molecule screening experiments to identify compounds that selectively kill cancer cells or as counter screens to exclude the compounds that have a generic effect on cell growth. While there are several assaying techniques available, cellular fitness is often assessed on the basis of one single and often rather indirect physiological indicator. This can lead to inconsistencies and poor correspondence between cell viability screening experiments, conducted under comparable conditions but with different viability indicators. Multiplexing, i.e., the combination of different individual assaying techniques in one experiment and subsequent comparative analysis of multiparametric data can decrease inter-assay variability and increase dataset concordance. Here, we describe a protocol for a multiplexing approach for high-throughput cell viability screening to address the issues encountered in the classical strategy using a single fitness indicator described above. The method combines a biochemical, luminescence-based approach and two fluorescence-based assay types. The biochemical method assesses cellular fitness by quantifying intracellular ATP concentration. Calcein labeling reflects cell fitness through membrane integrity and indirect measurement of ATP-dependent enzymatic esterase activity. Hoechst DNA stain correlates cell fitness with cellular DNA content. The presented multiplexing approach is suitable for low, medium and high-throughput screening and has the potential to decrease inter-assay variability and increase dataset concordance as well as reproducibility of experimental results. PMID:27581285

  3. 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. PMID:26888289

  4. 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.

  5. The JAMM motif of human deubiquitinase Poh1 is essential for cell viability.

    PubMed

    Gallery, Melissa; Blank, Jonathan L; Lin, Yinghui; Gutierrez, Juan A; Pulido, Jacqueline C; Rappoli, David; Badola, Sunita; Rolfe, Mark; Macbeth, Kyle J

    2007-01-01

    Poh1 deubiquitinase activity is required for proteolytic processing of polyubiquitinated substrates by the 26S proteasome, linking deubiquitination to complete substrate degradation. Poh1 RNA interference (RNAi) in HeLa cells resulted in a reduction in cell viability and an increase in polyubiquitinated protein levels, supporting the link between Poh1 and the ubiquitin proteasome pathway. To more specifically test for any requirement of the zinc metalloproteinase motif of Poh1 to support cell viability and proteasome function, we developed a RNAi complementation strategy. Effects on cell viability and proteasome activity were assessed in cells with RNAi of endogenous Poh1 and induced expression of wild-type Poh1 or a mutant form of Poh1, in which two conserved histidines of the proposed catalytic site were replaced with alanines. We show that an intact zinc metalloproteinase motif is essential for cell viability and 26S proteasome function. As a required enzymatic component of the proteasome, Poh1 is an intriguing therapeutic drug target for cancer.

  6. Cooling rate dependent biophysical and viability response shift with attachment state in human dermal fibroblast cells.

    PubMed

    Choi, Jeunghwan; Bischof, John C

    2011-12-01

    While studies on the freezing of cells in suspension have been carried out extensively, corresponding studies with cells in the attached state and in tissue or tissue-equivalents are less developed. As attachment is a hallmark of the tissue state it is important to understand its impact on biophysics and viability to better apply freezing towards tissue preservation. The current study reports on observed biophysical response changes observed during freezing human dermal fibroblasts in suspension, attached cell, and fibrin tissue-equivalent models. Specifically, intracellular ice formation is shown to increase and dehydration is inferred to increase from suspension to attached systems. Biophysical model parameters fit to these experimental observations reflect the higher kinetics in the attached state. Post-thaw viability values from fast cooling rates were higher for suspension systems, and correlated well with the amount of IIF observed. On the other hand, viability values from slow cooling rates were higher for attached systems, although the degree of dehydration was predicted to be comparable to suspension cells. This disconnect between biophysics and viability predictions at slow rates clearly requires further investigation as it runs counter to our current understanding of dehydration injury in cells. This may suggest a possible protective effect of the attachment state on cell systems.

  7. Interferon-gamma produced by microglia and the neuropeptide PACAP have opposite effects on the viability of neural progenitor cells.

    PubMed

    Mäkelä, Johanna; Koivuniemi, Raili; Korhonen, Laura; Lindholm, Dan

    2010-01-01

    Inflammation is part of many neurological disorders and immune reactions may influence neuronal progenitor cells (NPCs) contributing to the disease process. Our knowledge about the interplay between different cell types in brain inflammation are not fully understood. It is important to know the mechanisms and factors involved in order to enhance regeneration and brain repair. We show here that NPCs express receptors for interferon-gamma (IFNgamma), and IFNgamma activates the signal transducer and activator of transcription (STAT) protein-1. IFNgamma reduced cell proliferation in NPCs by upregulation of the cell cycle protein p21 as well as induced cell death of NPCs by activating caspase-3. Studies of putative factors for rescue showed that the neuropeptide, Pituitary adenylate cyclase-activating polypeptide (PACAP) increased cell viability, the levels of p-Bad and reduced caspase-3 activation in the NPCs. Medium from cultured microglia contained IFNgamma and decreased the viability of NPCs, whilst blocking with anti-IFNgamma antibodies counteracted this effect. The results show that NPCs are negatively influenced by IFNgamma whereas PACAP is able to modulate its action. The interplay between IFNgamma released from immune cells and PACAP is of importance in brain inflammation and may affect the regeneration and recruitment of NPCs in immune diseases. The observed effects of IFNgamma on NPCs deserve to be taken into account in human anti-viral therapies particularly in children with higher rates of brain stem cell proliferation.

  8. Effect of Varying Fluid Shear Stress on Cancer Stem Cell Viability & Protein Expression

    NASA Astrophysics Data System (ADS)

    Domier, Ria; Kim, Yonghyun; Dozier, David; Triantafillu, Ursula

    2013-11-01

    Cancer stem cells cultured in vitro in stirred bioreactors are exposed to shear stress. By observing the effect of shear stress on cancer stem cell viability, laboratory cell growth could be optimized. In addition, metastasized cancer stem cells in vivo are naturally exposed to shear stress, a factor influencing stem cell differentiation, while circulating in the bloodstream. Changes in protein expression after exposure to shear stress could allow for identification and targeting of circulating cancer cells. In this study, blood flow through capillaries was simulated by using a syringe pump to inject suspensions of Kasumi-1 leukemia stem cells into model blood vessels composed of PEEK tubing 125 microns in diameter. The Hagen-Poisseuille equation was used to solve for operating flow rates based on specified amounts of shear stress. After exposure, cell counts and viabilities were observed using an optical microscope and proteins were analyzed using Western blotting. It was observed that at a one minute exposure to stress, cell viability increased as the amount of shear was increased from 10 to 60 dynes per square centimeter. Results from this research are applicable to optimization of large-scale stem cell growth in bioreactors as well as to the design of targeted cancer therapies. Funding from NSF REU grant #1062611 is gratefully acknowledged.

  9. Human hepatocyte isolation and relationship of cell viability to early graft function.

    PubMed

    Mitry, Ragai R; Hughes, Robin D; Aw, Marion M; Terry, Claire; Mieli-Vergani, Giorgina; Girlanda, Raffaele; Muiesan, Paolo; Rela, Mohamed; Heaton, Nigel D; Dhawan, Anil

    2003-01-01

    Hepatocyte transplantation is emerging as an additional modality of treatment for patients with acute liver failure or liver-based metabolic disorders. The procedure requires isolation of high-quality hepatocytes from unused donor livers. Hepatocytes were isolated from 20 donor livers (11 right lobes, 3 left lateral segments, 6 whole livers) using a collagenase perfusion technique. Cell viability (median 56%, range 13-95%) and yield (median 1.4 x 10(9) cells, range 2.0 x 10(6)-1.8 x 10(10) cells) varied according to the tissue available. Fatty livers rejected for transplantation gave lower cell viability (median 45%, range 25-59%). There was a significant correlation between age of donor (median 21 years, range 7-66 years) and viability of isolated hepatocytes in vitro (r = -0.683, p = 0.001). The 13 segments of livers were from reduced/split grafts used for clinical transplantation in 9 children and 4 adults. There was no significant correlation between in vitro cell viability and clinical parameters including intensive care stay, serum aspartate aminotransferase,and international normalized ratio (in the first 7 days), and allograft rejection or other early posttransplant complications, in patients transplanted with the corresponding tissue. PMID:12693666

  10. Effects of ethylene glycol ethers on cell viability in the human neuroblastoma SH-SY5Y cell line.

    PubMed

    Regulska, Magdalena; Pomierny, Bartosz; Basta-Kaim, Agnieszka; Starek, Andrzej; Filip, Małgorzata; Lasoń, Władysław; Budziszewska, Bogusława

    2010-01-01

    Ethylene glycol ethers (EGEs) are a class of chemicals used extensively in the manufacture of a wide range of domestic and industrial products, which may result in human exposure and toxicity. Hematologic and reproductive toxicity of EGEs are well known whereas their action on neuronal cell viability has not been studied so far. In the present study, we investigated the effects of some EGEs on cell viability and on the hydrogen peroxide-induced damage in the human neuroblastoma (SH-SY5Y) cells. It has been found that 2-phenoxyethanol in a concentration-dependent manner (5-25 mM, 24 h) increased the basal and H(2)O(2)-induced lactate dehydrogenase (LDH) release and 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT) reduction. 2-Butoxyethanol given alone did not affect LDH release and MTT reduction but concentration-dependently enhanced the cytotoxic effect of H(2)O(2). 2-Isopropoxyethanol significantly and concentration-dependently (1-25 mM) increased the basal LDH release and attenuated MTT reduction, but did not potentiate the cytotoxic effect of H(2)O(2). Contrary to this, 2-methoxyethanol did not show a cytotoxic effect while 2-ethoxyethanol at high concentrations intensified the hydrogen peroxide action. This study demonstrated that among the EGEs studied, 2-phenoxyethanol showed the most consistent cytotoxic effect on neurons in in vitro conditions and enhanced the hydrogen peroxide action. 2-Isopropoxyethanol had also a potent cytotoxic effect, but it did not enhance the hydrogen peroxide action, whereas 2-butoxyethanol only potentiated cytotoxic effect of H(2)O(2). It is concluded that the results of the present study should be confirmed in in vivo conditions and that some EGEs, especially 2-phenoxyethanol, 2-butoxyethanol and 2-isopropoxyethanol, may be responsible for initiation or exacerbation of neuronal cell damage.

  11. Oxygen-Purged Microfluidic Device to Enhance Cell Viability in Photopolymerized PEG Hydrogel Microparticles.

    PubMed

    Xia, Bingzhao; Krutkramelis, Kaspars; Oakey, John

    2016-07-11

    Encapsulating cells within biocompatible materials is a widely used strategy for cell delivery and tissue engineering. While cells are commonly suspended within bulk hydrogel-forming solutions during gelation, substantial interest in the microfluidic fabrication of miniaturized cell encapsulation vehicles has more recently emerged. Here, we utilize multiphase microfluidics to encapsulate cells within photopolymerized picoliter-volume water-in-oil droplets at high production rates. The photoinitiated polymerization of polyethylene glycol diacrylate (PEGDA) is used to continuously produce solid particles from aqueous liquid drops containing cells and hydrogel forming solution. It is well understood that this photoinitiated addition reaction is inhibited by oxygen. In contrast to bulk polymerization in which ambient oxygen is rapidly and harmlessly consumed, allowing the polymerization reaction to proceed, photopolymerization within air permeable polydimethylsiloxane (PDMS) microfluidic devices allows oxygen to be replenished by diffusion as it is depleted. This sustained presence of oxygen and the consequential accumulation of peroxy radicals produce a dramatic effect upon both droplet polymerization and post-encapsulation cell viability. In this work we employ a nitrogen microjacketed microfluidic device to purge oxygen from flowing fluids during photopolymerization. By increasing the purging nitrogen pressure, oxygen concentration was attenuated, and increased post-encapsulation cell viability was achieved. A reaction-diffusion model was used to predict the cumulative intradroplet concentration of peroxy radicals, which corresponded directly to post-encapsulation cell viability. The nitrogen-jacketed microfluidic device presented here allows the droplet oxygen concentration to be finely tuned during cell encapsulation, leading to high post-encapsulation cell viability. PMID:27285343

  12. Effect of ZnO nanoparticles on nasopharyngeal cancer cells viability and respiration

    NASA Astrophysics Data System (ADS)

    Prasanth, R.; Gopinath, D.

    2013-03-01

    Development of a therapeutic drugs based on nanoparticles requires a better understanding of the mechanism of selective cyto-toxic effects of nanopaticles over cancer cells. Scanning electrochemical microscopy provides opportunity to measure the real time chemical process at cell proximity in the presence of nanoparticle. Herein, the respiration process in nasopharyngeal cancer cells is investigated with the help of scanning electrochemical microscopy. The cell viability has been tested with MTT assay. The results show that ZnO nanoparticles have time and dose dependent effect in nasopharyngeal cancer cells and the cell respiration rate decreases with time.

  13. Influence of different buffers (HEPES/MOPS) on keratinocyte cell viability and microbial growth.

    PubMed

    Dias, Kássia de Carvalho; Barbugli, Paula Aboud; Vergani, Carlos Eduardo

    2016-06-01

    This study assessed the effect of the buffers 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 3-(N-morpholino) propanesulfonic acid (MOPS) on keratinocyte cell viability and microbial growth. It was observed that RPMI buffered with HEPES, supplemented with l-glutamine and sodium bicarbonate, can be used as a more suitable medium to promote co-culture. PMID:27060444

  14. 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.

  15. Bacterial biofilm formation on the bladder epithelium of spinal cord injured patients. II. Toxic outcome on cell viability.

    PubMed

    Reid, G; Kang, Y S; Lacerte, M; Tieszer, C; Hayes, K C

    1993-08-01

    As a follow up to our first study of 10 spinal cord injured patients, a further 8 patients were investigated over 2 months for biofilm formation on their bladder epithelial cells and for evidence that these uropathogens damage the host bladder. All the patients were found to be colonized with uropathogens, regardless of whether or not they were receiving antibiotics. Using vital staining, it was discovered that there was a significant reduction (33%) in bladder cell viability in the presence of bacterial biofilms compared to controls. This was not associated with cell turnover rates. In vitro tests showed a similar reduction in cell viability when uropathogens were incubated with bladder cells. In addition, white cell counts were significantly elevated in the patients' urine, indicative of an infectious and/or inflamed state. There was no difference between symptomatic and asymptomatic patients in their mean bacterial adhesion counts. Patients were just as likely to be symptomatic as asymptomatic when on antibiotics. In summary, the presence of virulent organisms in the bladder does adversely affect the host, even when the patient has insignificant signs and symptoms of infection, thereby raising concerns over the decision not to treat the patient. Unless specific antibiotics are used which eradicate adherent biofilms from the bladder, the treatment of symptomatic patients will only impact upon the signs and symptoms in some patients, and not alter their susceptibility to reinfection.

  16. A combined approach for the assessment of cell viability and cell functionality of human fibrochondrocytes for use in tissue engineering.

    PubMed

    Garzón, Ingrid; Carriel, Victor; Marín-Fernández, Ana Belén; Oliveira, Ana Celeste; Garrido-Gómez, Juan; Campos, Antonio; Sánchez-Quevedo, María Del Carmen; Alaminos, Miguel

    2012-01-01

    Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5-P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5-P6 for cell therapy protocols.

  17. A Combined Approach for the Assessment of Cell Viability and Cell Functionality of Human Fibrochondrocytes for Use in Tissue Engineering

    PubMed Central

    Garzón, Ingrid; Carriel, Victor; Marín-Fernández, Ana Belén; Oliveira, Ana Celeste; Garrido-Gómez, Juan; Campos, Antonio; Sánchez-Quevedo, María del Carmen; Alaminos, Miguel

    2012-01-01

    Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5–P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5–P6 for cell therapy protocols. PMID:23272194

  18. Digitoxin and a synthetic monosaccharide analog inhibit cell viability in lung cancer cells

    SciTech Connect

    Elbaz, Hosam A.; Stueckle, Todd A.; Wang, Hua-Yu Leo; O'Doherty, George A.; Lowry, David T.; Sargent, Linda M.; Wang, Liying; Dinu, Cerasela Zoica; Rojanasakul, Yon

    2012-01-01

    Mechanisms of digitoxin-inhibited cell growth and induced apoptosis in human non-small cell lung cancer (NCI-H460) cells remain unclear. Understanding how digitoxin or derivate analogs induce their cytotoxic effect below therapeutically relevant concentrations will help in designing and developing novel, safer and more effective anti-cancer drugs. In this study, NCI-H460 cells were treated with digitoxin and a synthetic analog D6-MA to determine their anti-cancer activity. Different concentrations of digitoxin and D6-MA were used and the subsequent changes in cell morphology, viability, cell cycle, and protein expressions were determined. Digitoxin and D6-MA induced dose-dependent apoptotic morphologic changes in NCI-H460 cells via caspase-9 cleavage, with D6-MA possessing 5-fold greater potency than digitoxin. In comparison, non-tumorigenic immortalized bronchial and small airway epithelial cells displayed significantly less apoptotic sensitivity compared to NCI-H460 cells suggesting that both digitoxin and D6-MA were selective for NSCLC. Furthermore, NCI-H460 cells arrested in G(2)/M phase following digitoxin and D6-MA treatment. Post-treatment evaluation of key G2/M checkpoint regulatory proteins identified down-regulation of cyclin B1/cdc2 complex and survivin. Additionally, Chk1/2 and p53 related proteins experienced down-regulation suggesting a p53-independent cell cycle arrest mechanism. In summary, digitoxin and D6-MA exert anti-cancer effects on NCI-H460 cells through apoptosis or cell cycle arrest, with D6-MA showing at least 5-fold greater potency relative to digitoxin. -- Highlights: ► Digitoxin and synthetic analog D6-MA induced apoptotic morphologic changes in NCI-H460 cells in a dose-dependent manner. ► Apoptotic cell death induced by analog was 5-fold more potent when compared to digitoxin. ► NCI-H460 cells arrested in G(2)/M phase following digitoxin (≥ 5 nM) and analog (≥ 1 nM) treatment. ► Digitoxin inhibited the expression of cyclin

  19. Effects of four rice paddy herbicides on algal cell viability and the relationship with population recovery.

    PubMed

    Nagai, Takashi; Ishihara, Satoru; Yokoyama, Atsushi; Iwafune, Takashi

    2011-08-01

    Paddy herbicides are a high-risk concern for aquatic plants, including algae, because they easily flow out from paddy fields into rivers, with toxic effects. The effect on algal population dynamics, including population recovery after timed exposure, must be assessed. Therefore, we demonstrated concentration-response relationships of four paddy herbicides for algal growth inhibition and mortality, and the relationship between the effect on algal cell viability and population recovery following exposure. We used SYTOX Green dye assay and flow cytometry to assess cell viability of the alga Pseudokirchneriella subcapitata. Live cells could be clearly distinguished from dead cells during herbicide exposure. Our results showed that pretilachlor and quinoclamine had both algicidal and algistatic effects, whereas bensulfuron-methyl only had an algistatic effect, and pentoxazone only had an algicidal effect. Then, a population recovery test following a 72-h exposure was conducted. The algal population recovered in all tests, but the periods required for recovery differed among exposure concentrations and herbicides. The periods required for recovery were inconsistent with the dead cell ratio at the beginning of the recovery test; that is, population recovery could not be described only by cell viability. Consequently, the temporal effect of herbicides and subsequent recovery of the algal population could be described not only by the toxicity characteristics but also by toxicokinetics, such as rate of uptake, transport to the target site, and elimination of the substance from algal cells. PMID:21590715

  20. Ursolic acid and oleanolic acid from Eriobotrya fragrans inhibited the viability of A549 cells.

    PubMed

    Yuan, Yuan; Gao, Yongshun; Song, Gang; Lin, Shunquan

    2015-02-01

    Loquat {Eriobotrya japonica (Lindl.)}, a kind of Chinese herb, has many efficacies such as anti-inflammatory, antimicrobial and curing chronic bronchitis. However, reports on the pharmacological action of wild loquat extract are limited. In this work, the A549 cell line was selected to study the inhibitory effect of ursolic acid and oleanolic acid (UA, OA) from the leaves of E. fragrans. Results showed that UA/OA inhibited A549 cell viability and induced apoptosis in a dose and time dependent manner. The cell fraction in the G0/G1 phase dramatically increased under treatment with UA/OA. Data showed that UA activated the expression of PARP. UA and OA down-regulated MMP-2 and Bcl-2; on the contrary, they up-regulated Bid. This work demonstrated that UA/OA extracted from wild loquat leaves can significantly inhibit the viability of A549 cells.

  1. Comparison of viability and antioxidant capacity between canine adipose-derived mesenchymal stem cells and heme oxygenase-1-overexpressed cells after freeze-thawing

    PubMed Central

    KIM, Mijung; KIM, Yongsun; LEE, Seunghoon; KUK, Minyoung; KIM, Ah Young; KIM, Wanhee; KWEON, Oh-Kyeong

    2015-01-01

    Allogenic adipose-derived mesenchymal stem cells (Ad-MSCs) are an alternative source for cytotherapy owing to their antioxidant and anti-inflammatory effects. Frozen-thawed allogenic Ad-MSCs can be used instantly for this purpose. However, the viability and function of frozen-thawed Ad-MSCs have not been clearly evaluated. The purpose of this study was to compare the viability and function of Ad-MSCs and heme oxygenase-1 (HO-1)-overexpressed Ad-MSCs in vitro after freeze-thawing. The viability, proliferation, antioxidant capacity and mRNA gene expression of growth factors were evaluated. Frozen-thawed cells showed significantly lower viability than fresh cells (77% for Ad-MSCs and 71% for HO-1 Ad-MSCs, P<0.01). However, the proliferation rate of frozen-thawed Ad-MSCs increased and did not differ from that of fresh Ad-MSCs after 3 days of culture. In contrast, the proliferation rate of HO-1-overexpressed Ad-MSCs was lower than that of Ad-MSCs. The mRNA expression levels of TGF-β, HGF and VEGF did not differ between fresh and frozen-thawed Ad-MSCs, but COX-2 and IL-6 had significantly higher mRNA expression in frozen cells than fresh cells (P<0.05). Fresh Ad-MSCs exhibited higher HO-1 mRNA expression than frozen-thawed Ad-MSCs, and fresh HO-1-overexpressed Ad-MSCs exhibited higher than fresh Ad-MSCs (P<0.05). However, there was no significant difference between fresh and frozen HO-1-overexpressed Ad-MSCs. The antioxidant capacity of HO-1-overexpressed Ad-MSCs was significantly higher than that of Ad-MSCs. Cryopreservation of Ad-MSCs negatively affects viability and antioxidant capacity, and HO-1-overexpressed Ad-MSCs might be useful to maximize the effect of Ad-MSCs for cytotherapy. PMID:26725542

  2. Viability selection affects black but not yellow plumage colour in greenfinches.

    PubMed

    Hõrak, Peeter; Männiste, Marju

    2016-01-01

    Much of the debate surrounding the selective forces responsible for the expression of conspicuous plumage colouration is centred on the question of precisely which individual qualities are signalled by carotenoid- and melanin-based pigments. To examine this and other related issues, we performed viability selection analyses in wild-caught captive male greenfinches (Carduelis chloris) in Estonia during winters between 2003 and 2014. Based on our measurements, birds with a darker black eumelanin-based colouration of tail feathers survived better than those whose tail feathers had a paler black colouration. The carotenoid-based yellow colouration of the same feathers was not associated with mortality in captivity and showed much less between-year variation in the field than the black colouration. Between year-variation in the black (but not yellow) colouration of feathers was parallel in wild-grown feathers (on birds in the wild) and laboratory-grown ones (on birds held temporarily in captivity). Taken together, these findings imply that eumelanotic colouration in greenfinches is currently under selection and suggest the presence of sufficient genetic variation for a rapid response to selection. In particular, tail feathers have become darker black since the emergence of avian trichomonosis, which is known to selectively kill paler individuals. PMID:26386701

  3. Dehydroepiandrosterone inhibits cell proliferation and improves viability by regulating S phase and mitochondrial permeability in primary rat Leydig cells.

    PubMed

    Liu, Lin; Wang, Dian; Li, Longlong; Ding, Xiao; Ma, Haitian

    2016-07-01

    Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement and exhibits putative anti‑aging properties. However, the molecular basis of the actions of DHEA, particularly on the biological characteristics of target cells, remain unclear. The aim of the current study was to investigate the effects of DHEA on cell viability, cell proliferation, cell cycle and mitochondrial function in primary rat Leydig cells. Adult Leydig cells were purified by Percoll gradient centrifugation, and cell proliferation was detected using a Click-iT® EdU Assay kit and cell cycle assessment performed using flow cytometry. Mitochondrial membrane potential was detected using JC-1 staining assay. The results of the current study demonstrate that DHEA decreased cell proliferation in a dose‑dependent manner, whereas it improved cell viability in a time‑dependent and dose‑dependent manner. Flow cytometry analysis demonstrated that DHEA treatment increased the S phase cell population and decreased the G2/M cell population. Cyclin A and CDK2 mRNA levels were decreased in primary rat Leydig cells following DHEA treatment. DHEA treatment decreased the transmembrane electrical gradient in primary Leydig cells, whereas treatment significantly increased succinate dehydrogenase activity. These results indicated that DHEA inhibits primary rat Leydig cell proliferation by decreasing cyclin mRNA level, whereas it improves cells viability by modulating the permeability of the mitochondrial membrane and succinate dehydrogenase activity. These findings may demonstrate an important molecular mechanism by which DHEA activity is mediated. PMID:27220727

  4. Dehydroepiandrosterone inhibits cell proliferation and improves viability by regulating S phase and mitochondrial permeability in primary rat Leydig cells

    PubMed Central

    LIU, LIN; WANG, DIAN; LI, LONGLONG; DING, XIAO; MA, HAITIAN

    2016-01-01

    Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement and exhibits putative anti-aging properties. However, the molecular basis of the actions of DHEA, particularly on the biological characteristics of target cells, remain unclear. The aim of the current study was to investigate the effects of DHEA on cell viability, cell proliferation, cell cycle and mitochondrial function in primary rat Leydig cells. Adult Leydig cells were purified by Percoll gradient centrifugation, and cell proliferation was detected using a Click-iT® EdU Assay kit and cell cycle assessment performed using flow cytometry. Mitochondrial membrane potential was detected using JC-1 staining assay. The results of the current study demonstrate that DHEA decreased cell proliferation in a dose-dependent manner, whereas it improved cell viability in a time-dependent and dose-dependent manner. Flow cytometry analysis demonstrated that DHEA treatment increased the S phase cell population and decreased the G2/M cell population. Cyclin A and CDK2 mRNA levels were decreased in primary rat Leydig cells following DHEA treatment. DHEA treatment decreased the transmembrane electrical gradient in primary Leydig cells, whereas treatment significantly increased succinate dehydrogenase activity. These results indicated that DHEA inhibits primary rat Leydig cell proliferation by decreasing cyclin mRNA level, whereas it improves cells viability by modulating the permeability of the mitochondrial membrane and succinate dehydrogenase activity. These findings may demonstrate an important molecular mechanism by which DHEA activity is mediated. PMID:27220727

  5. Differential Redox Regulation of Ca2+ Signaling and Viability in Normal and Malignant Prostate Cells

    PubMed Central

    Holzmann, Christian; Kilch, Tatiana; Kappel, Sven; Dörr, Kathrin; Jung, Volker; Stöckle, Michael; Bogeski, Ivan; Peinelt, Christine

    2015-01-01

    In prostate cancer, reactive oxygen species (ROS) are elevated and Ca2+ signaling is impaired. Thus, several novel therapeutic strategies have been developed to target altered ROS and Ca2+ signaling pathways in prostate cancer. Here, we investigate alterations of intracellular Ca2+ and inhibition of cell viability caused by ROS in primary human prostate epithelial cells (hPECs) from healthy tissue and prostate cancer cell lines (LNCaP, DU145, and PC3). In hPECs, LNCaP and DU145 H2O2 induces an initial Ca2+ increase, which in prostate cancer cells is blocked at high concentrations of H2O2. Upon depletion of intracellular Ca2+ stores, store-operated Ca2+ entry (SOCE) is activated. SOCE channels can be formed by hexameric Orai1 channels; however, Orai1 can form heteromultimers with its homolog, Orai3. Since the redox sensor of Orai1 (Cys-195) is absent in Orai3, the Orai1/Orai3 ratio in T cells determines the redox sensitivity of SOCE and cell viability. In prostate cancer cells, SOCE is blocked at lower concentrations of H2O2 compared with hPECs. An analysis of data from hPECs, LNCaP, DU145, and PC3, as well as previously published data from naive and effector TH cells, demonstrates a strong correlation between the Orai1/Orai3 ratio and the SOCE redox sensitivity and cell viability. Therefore, our data support the concept that store-operated Ca2+ channels in hPECs and prostate cancer cells are heteromeric Orai1/Orai3 channels with an increased Orai1/Orai3 ratio in cells derived from prostate cancer tumors. In addition, ROS-induced alterations in Ca2+ signaling in prostate cancer cells may contribute to the higher sensitivity of these cells to ROS. PMID:26445441

  6. 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. PMID:27419079

  7. 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.

  8. An Optimized Injectable Hydrogel Scaffold Supports Human Dental Pulp Stem Cell Viability and Spreading

    PubMed Central

    Jones, T. D.; Kefi, A.; Sun, S.; Cho, M.; Alapati, S. B.

    2016-01-01

    Introduction. HyStem-C™ is a commercially available injectable hydrogel composed of polyethylene glycol diacrylate (PEGDA), hyaluronan (HA), and gelatin (Gn). These components can be mechanically tuned to enhance cell viability and spreading. Methods. The concentration of PEGDA with an added disulfide bond (PEGSSDA) was varied from 0.5 to 8.0% (w/v) to determine the optimal concentration for injectable clinical application. We evaluated the cell viability of human dental pulp stem cells (hDPSCs) embedded in 2% (w/v) PEGSSDA-HA-Gn hydrogels. Volume ratios of HA : Gn from 100 : 0 to 25 : 75 were varied to encourage hDPSC spreading. Fibronectin (Fn) was added to our model to determine the effect of extracellular matrix protein concentration on hDPSC behavior. Results. Our preliminary data suggests that the hydrogel gelation time decreased as the PEGSSDA cross-linker concentration increased. The PEGSSDA-HA-Gn was biocompatible with hDPSCs, and increased ratios of HA : Gn enhanced cell viability for 14 days. Additionally, cell proliferation with added fibronectin increased significantly over time at concentrations of 1.0 and 10.0 μg/mL in PEGDA-HA-Gn hydrogels, while cell spreading significantly increased at Fn concentrations of 0.1 μg/mL. Conclusions. This study demonstrates that PEG-based injectable hydrogels maintain hDPSC viability and facilitate cell spreading, mainly in the presence of extracellular matrix (ECM) proteins. PMID:27294191

  9. 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

    2016-01-01

    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

  10. 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

    2016-01-01

    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

  11. Quantitative study of electroporation-mediated molecular uptake and cell viability.

    PubMed Central

    Canatella, P J; Karr, J F; Petros, J A; Prausnitz, M R

    2001-01-01

    Electroporation's use for laboratory transfection and clinical chemotherapy is limited by an incomplete understanding of the effects of electroporation parameters on molecular uptake and cell viability. To address this need, uptake of calcein and viability of DU 145 prostate cancer cells were quantified using flow cytometry for more than 200 different combinations of experimental conditions. The experimental parameters included field strength (0.1-3.3 kV/cm), pulse length (0.05-20 ms), number of pulses (1-10), calcein concentration (10-100 microM), and cell concentration (0.6-23% by volume). These data indicate that neither electrical charge nor energy was a good predictor of electroporation's effects. Instead, both uptake and viability showed a complex dependence on field strength, pulse length, and number of pulses. The effect of cell concentration was explained quantitatively by electric field perturbations caused by neighboring cells. Uptake was shown to vary linearly with external calcein concentration. This large quantitative data set may be used to optimize electroporation protocols, test theoretical models, and guide mechanistic interpretations. PMID:11159443

  12. Cell viability of acute myeloid leukaemia blasts in culture correlates with treatment outcome.

    PubMed

    Maha, Abdullah; Cheong, Soon-Keng; Leong, Chooi-Fun; Seow, Heng-Fong

    2008-02-01

    Despite the advances in understanding the pathophysiology of acute myeloid leukaemia (AML), the cure rate for acute myeloid leukaemia patients remains low. Cytogenetic abnormalities and age are the prognostic factors that guide treatment decisions. However, many AML patients still die. The biological factors that influence treatment outcome are largely unknown. Thus, the objective of our study was to use the in vitro viability test to correlate with treatment outcome. Acute myeloid leukaemia blasts demonstrated differing ability to survive in culture. Our examination of blast phenotype at various days in culture showed two possible growth directions. First, cells underwent maturation by increased expression of CD16 and down-regulated CD34 (a haemopoietic stem cell marker). These cells also appeared to have undergone apoptosis. Alternatively, cells continued to survive in culture and maintained high expression of CD34. An MTT assay was carried out to determine viability after three days of culture. Lower optical density values were obtained for samples that underwent apoptosis and higher values were obtained for samples that survived in culture. Apoptosis was measured by Annexin V/propidium iodide staining. A comparison between results of MTT assay and duration of disease free survival revealed that a higher viability in vitro correlated significantly with shorter survival duration in the patient (R -0.761, p=0.002, n=13). Thus, this study further supports the hypothesis that AML patients with poor survival may be related to having blasts with a biologically more immature or stem cell-like nature.

  13. Influence of carvacrol and 1,8-cineole on cell viability, membrane integrity, and morphology of Aeromonas hydrophila cultivated in a vegetable-based broth.

    PubMed

    de Sousa, Jossana Pereira; de Oliveira, Kataryne Árabe Rimá; de Figueiredo, Regina Celia Bressan Queiroz; de Souza, Evandro Leite

    2015-02-01

    This study investigated the effects of carvacrol (CAR) and 1,8-cineole (CIN) alone (at the MIC) or in combination at subinhibitory amounts (both at 1/8 MIC) on the cell viability, membrane permeability, and morphology of Aeromonas hydrophila INCQS 7966 (A. hydrophila) cultivated in a vegetable-based broth. CAR and CIN alone or in combination severely affected the viability of the bacteria and caused dramatic changes in the cell membrane permeability, leading to cell death, as observed by confocal laser microscopy. Scanning and transmission electron microscopy images of bacterial cells exposed to CAR or CIN or the mixture of both compounds revealed severe changes in cell wall structure, rupture of the plasma membrane, shrinking of cells, condensation of cytoplasmic content, leakage of intracellular material, and cell collapse. These findings suggest that CAR and CIN alone or in combination at subinhibitory amounts could be applied to inhibit the growth of A. hydrophila in foods, particularly as sanitizing agents in vegetables.

  14. 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.

  15. Evaporation-induced stimulation of bacterial osmoregulation for electrical assessment of cell viability.

    PubMed

    Ebrahimi, Aida; Alam, Muhammad Ashraful

    2016-06-28

    Bacteria cells use osmoregulatory proteins as emergency valves to respond to changes in the osmotic pressure of their external environment. The existence of these emergency valves has been known since the 1960s, but they have never been used as the basis of a viability assay to tell dead bacteria cells apart from live ones. In this paper, we show that osmoregulation provides a much faster, label-free assessment of cell viability compared with traditional approaches that rely on cell multiplication (growth) to reach a detectable threshold. The cells are confined in an evaporating droplet that serves as a dynamic microenvironment. Evaporation-induced increase in ionic concentration is reflected in a proportional increase of the droplet's osmotic pressure, which in turn, stimulates the osmoregulatory response from the cells. By monitoring the time-varying electrical conductance of evaporating droplets, bacterial cells are identified within a few minutes compared with several hours in growth-based methods. To show the versatility of the proposed method, we show detection of WT and genetically modified nonhalotolerant cells (Salmonella typhimurium) and dead vs. live differentiation of nonhalotolerant (such as Escherichia coli DH5α) and halotolerant cells (such as Staphylococcus epidermidis). Unlike the growth-based techniques, the assay time of the proposed method is independent of cell concentration or the bacteria type. The proposed label-free approach paves the road toward realization of a new class of real time, array-formatted electrical sensors compatible with droplet microfluidics for laboratory on a chip applications.

  16. The Effect of Tuning Cold Plasma Composition on Glioblastoma Cell Viability

    PubMed Central

    Cheng, Xiaoqian; Sherman, Jonathan; Murphy, William; Ratovitski, Edward; Canady, Jerome; Keidar, Michael

    2014-01-01

    Previous research in cold atmospheric plasma (CAP) and cancer cell interaction has repeatedly proven that the cold plasma induced cell death. It is postulated that the reactive oxygen species (ROS) and reactive nitrogen species (RNS) play a major role in the CAP cancer therapy. In this paper, we seek to determine a mechanism of CAP therapy on glioblastoma cells (U87) through an understanding of the composition of the plasma, including treatment time, voltage, flow-rate and plasma-gas composition. In order to determine the threshold of plasma treatment on U87, normal human astrocytes (E6/E7) were used as the comparison cell line. Our data showed that the 30 sec plasma treatment caused 3-fold cell death in the U87 cells compared to the E6/E7 cells. All the other compositions of cold plasma were performed based on this result: plasma treatment time was maintained at 30 s per well while other plasma characteristics such as voltage, flow rate of source gas, and composition of source gas were changed one at a time to vary the intensity of the reactive species composition in the plasma jet, which may finally have various effect on cells reflected by cell viability. We defined a term “plasma dosage” to summarize the relationship of all the characteristics and cell viability. PMID:24878760

  17. Impact of thermal effects induced by ultrasound on viability of rat C6 glioma cells.

    PubMed

    Kujawska, T; Secomski, W; Bilmin, K; Nowicki, A; Grieb, P

    2014-07-01

    In order to have consistent and repeatable effects of sonodynamic therapy (SDT) on various cancer cells or tissue lesions we should be able to control a delivered ultrasound energy and thermal effects induced. The objective of this study was to investigate viability of rat C6 glioma cells in vitro depending on the intensity of ultrasound in the region of cells and to determine the exposure time inducing temperature rise above 43 °C, which is known to be toxic for cells. For measurements a planar piezoelectric transducer with a diameter of 20 mm and a resonance frequency of 1.06 MHz was used. The transducer generated tone bursts with 94 μs duration, 0.4 duty-cycle and initial intensity ISATA (spatial averaged, temporal averaged) varied from 0.33 W/cm(2) to 8 W/cm(2) (average acoustic power varied from 1 W to 24 W). The rat C6 glioma cells were cultured on a bottom of wells in 12-well plates, incubated for 24h and then exposed to ultrasound with measured acoustic properties, inducing or causing no thermal effects leading to cell death. Cell viability rate was determined by MTT assay (a standard colorimetric assay for assessing cell viability) as the ratio of the optical densities of the group treated by ultrasound to the control group. Structural cellular changes and apoptosis estimation were observed under a microscope. Quantitative analysis of the obtained results allowed to determine the maximal exposure time that does not lead to the thermal effects above 43 °C in the region of cells for each initial intensity of the tone bursts used as well as the threshold intensity causing cell death after 3 min exposure to ultrasound due to thermal effects. The averaged threshold intensity was found to be about 5.7 W/cm(2). PMID:24589258

  18. Histone deacetylase inhibitors reduce WB-F344 oval cell viability and migration capability by suppressing AKT/mTOR signaling in vitro.

    PubMed

    Zhang, Peng; Zhu, Xiaofeng; Wu, Ying; Hu, Ronglin; Li, Dongming; Du, Jun; Jiao, Xingyuan; He, Xiaoshun

    2016-01-15

    Histone deacetylase (HDAC) can blockDNA replication and transcription and altered HDAC expression was associated with tumorigenesis. This study investigated the effects of HDAC inhibitors on hepatic oval cells and aimed to delineate the underlying molecular events. Hepatic oval cells were treated with two different HDAC inhibitors, suberoylanilidehydroxamic acid (SAHA) and trichostatin-A (TSA). Cells were subjected to cell morphology, cell viability, cell cycle, and wound healing assays. The expression of proteins related to both apoptosis and the cell cycle, and proteins of the AKT/mammalian target of rapamycin (mTOR) signaling pathway were analyzed by Western blot. The data showed that HDAC inhibitors reduced oval cell viability and migration capability, and arrested oval cells at the G0/G1 and S phases of the cell cycle, in a dose- and time-dependent manner. HDAC inhibitors altered cell morphology and reduced oval cell viability, and downregulated the expression of PCNA, cyclinD1, c-Myc and Bmi1 proteins, while also suppressing AKT/mTOR and its downstream target activity. In conclusion, this study demonstrates that HDAC inhibitors affect oval cells by suppressing AKT/mTOR signaling. PMID:26558695

  19. Effect of a novel Omegacoeur®/Doluperine® nutritional combination on human embryonic kidney cell viability.

    PubMed

    Sottejeau, Y; Patel, A M; Gerber, G; Pierre, S V; Maixent, J M

    2010-01-01

    Holistica Laboratories (Eguilles, France) developed the nutritional supplements Omegacoeur® and Doluperine® based on two of the most ancient and unique dietary health traditions. Omegacoeur® is formulated to supply key active components of Mediterranean diet (omega 3,6,9 fatty acids, garlic, and basil) and the formulation of Doluperine® was based on the Ayurvedic tradition (curcuma, pepper, ginger extracts). Interestingly, recent studies suggest that an combination of the ingredients supplied by these two supplements could provide additional and previously unanticipated benefit through synergistic actions of some of their key components. However, the effect of such combination on human cell viability has not been investigated. In this present article, a review of the various effects of the individual compounds of the new combination and the reported active doses, and the result of a study of an combination of Omegacoeur® / Dolupérine® on Human Embryonic Kidney (HEK 293) cells. Incremental doses of 4 Omegacoeur® / Dolupérine® combinations prepared so that the molar ratio DHA (Docosahexaenoic acid) in Omegacoeur® / curcumin in Dolupérine® was kept constant, at 2.5 DHA / 1 curcumin, were added to the culture media. After 24h of incubation, cell viability was assessed by the trypan blue exclusion method. The data suggest that the combination of Omegacoeur® with Dolupérine® does not affect HEK 293 cells viability in the range of doses that have demonstrated beneficial effects in earlier studies. PMID:21062574

  20. Effect of a novel Omegacoeur®/Doluperine® nutritional combination on human embryonic kidney cell viability.

    PubMed

    Sottejeau, Y; Patel, A M; Gerber, G; Pierre, S V; Maixent, J M

    2010-10-05

    Holistica Laboratories (Eguilles, France) developed the nutritional supplements Omegacoeur® and Doluperine® based on two of the most ancient and unique dietary health traditions. Omegacoeur® is formulated to supply key active components of Mediterranean diet (omega 3,6,9 fatty acids, garlic, and basil) and the formulation of Doluperine® was based on the Ayurvedic tradition (curcuma, pepper, ginger extracts). Interestingly, recent studies suggest that an combination of the ingredients supplied by these two supplements could provide additional and previously unanticipated benefit through synergistic actions of some of their key components. However, the effect of such combination on human cell viability has not been investigated. In this present article, a review of the various effects of the individual compounds of the new combination and the reported active doses, and the result of a study of an combination of Omegacoeur® / Dolupérine® on Human Embryonic Kidney (HEK 293) cells. Incremental doses of 4 Omegacoeur® / Dolupérine® combinations prepared so that the molar ratio DHA (Docosahexaenoic acid) in Omegacoeur® / curcumin in Dolupérine® was kept constant, at 2.5 DHA / 1 curcumin, were added to the culture media. After 24h of incubation, cell viability was assessed by the trypan blue exclusion method. The data suggest that the combination of Omegacoeur® with Dolupérine® does not affect HEK 293 cells viability in the range of doses that have demonstrated beneficial effects in earlier studies.

  1. Effects of risedronate on the morphology and viability of gingiva-derived mesenchymal stem cells

    PubMed Central

    KIM, BO-BAE; KO, YOUNGKYUNG; PARK, JUN-BEOM

    2015-01-01

    Risedronate has been used for the prevention and treatment of postmenopausal and corticosteroid-induced osteoporosis. The present study was performed to evaluate the effects of risedronate on the morphology and viability of human stem cells derived from the gingiva. Stem cells derived from the gingiva were grown in the presence of risedronate at concentrations that ranged from 1 to 10 µM. The morphology of the cells was viewed under an inverted microscope, and cell proliferation was analyzed with a cell counting kit-8 (CCK-8) on days 2, 4 and 7. The untreated control group showed a spindle-shaped, fibroblast-like morphology. The shapes of the cells treated with 1 and 5 µM risedronate were similar to that of the control group on day 2. However, morphology of the 10 µM group markedly differed from that of the control group. The shapes of the cells in the 1, 5 and 10 µM groups were rounder, and pronounced alterations when compared with the untreated control group were noted in all groups on day 7. The cultures growing in the presence of risedronate showed decreased CCK-8 values on day 7. In conclusion, risedronate produced notable alterations in the morphology of the cells and reduced the viability of gingival mesenchymal stem cells. PMID:26623028

  2. Triptolide reduces the viability of osteosarcoma cells by reducing MKP-1 and Hsp70 expression

    PubMed Central

    ZHAO, LEI; JIANG, BO; WANG, DONG; LIU, WEI; ZHANG, HUAWU; LIU, WEISHENG; QIU, ZHEN

    2016-01-01

    Osteosarcoma is the most common type of malignant bone tumor found in adolescents and young adults. The aim of the present study was to determine whether triptolide, a diterpene epoxide extracted from the Tripterygium plant, was able effectively decrease the viability of osteosarcoma cells. The underlying molecular mechanisms are also investigated. The human osteosarcoma cell lines U-2 OS and MG-63 were used in this study. The U-2 OS and MG-63 cells were treated with 0, 5, 10, 25 or 50 nM triptolide. Cells treated with dimethyl sulfoxide only were used as the no drug treatment control. A commercial MTT kit was used to determine the effects of triptolide on cells. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is frequently overexpressed in tumor tissues, possibly related to the failure of a number of chemotherapeutics. Heat shock protein 70 (Hsp70) is a chaperone molecule that is able to increase drug resistance. The protein expression levels of MKP-1 and Hsp70 were determined using western blot analysis. The results indicate that triptolide effectively reduced the viability of the osteosarcoma cells. Furthermore, triptolide was found to effectively reduce MKP-1 expression and Hsp70 levels. Further analysis showed that triptolide reduced MKP-1 mRNA expression in the U-2 OS and MG-63 cells. Triptolide reduced Hsp70 mRNA expression levels in U-2 OS and MG-63 cells. These results suggest that triptolide effectively decreases the viability of osteosarcoma cells. These effects may be associated with the decreased expression of MKP-1 and Hsp70 levels. These results suggest that triptolide may be used in the treatments of osteosarcoma. PMID:27168842

  3. In situ viability detection assays induce heat-shock protein 70 expression in spermatozoa without affecting the chromatin integrity.

    PubMed

    Asokan, Y; Honguntikar, S D; Uppangala, S; Salian, S R; Kumar, D; Kalthur, G; Adiga, S K

    2015-10-01

    To differentiate dead spermatozoa from viable but immotile spermatozoa, several techniques are being used during ICSI. As processed spermatozoa from poor-quality ejaculate are confronted with a higher risk of experiencing stress on exposure to altered osmotic conditions or chemicals, this study was undertaken to determine the expression of stress response gene Hsp70 and chromatin integrity in spermatozoa subjected to in situ viability assays such as hypo-osmotic swelling (HOS) test, modified hypo-osmotic swelling (M-HOS) test and pentoxifylline in 25 fresh and frozen-thawed asthenozoospermic ejaculates. RT-PCR and immunofluorescence detection of Hsp70 were performed to elucidate the expression and localisation of Hsp70 in spermatozoa, whereas DNA fragmentation analysis was performed by sperm chromatin dispersion assay. Exposure of fresh and frozen-thawed asthenozoospermic spermatozoa to M-HOS and pentoxifylline significantly increased Hsp70 expression as evidenced by increased RNA expression and immunolocalisation of Hsp70 protein in sperm head (P < 0.05-0.001). However, chromatin integrity was not significantly affected in any groups until 6 h of post-exposure time period. Our results suggest that conventional HOS may be preferred for the in situ detection of the viability as there was no immediate stress response and chromatin instability in the exposed spermatozoa.

  4. Chromium oxide nano-particles induce stress in bacteria: probing cell viability.

    PubMed

    Singh, Gulshan; Vajpayee, Poornima; Khatoon, Imrana; Jyoti, Anurag; Dhawan, Alok; Gupta, K C; Shanker, Rishi

    2011-02-01

    In this study, viability of an environmentally relevant bacterium, Escherichia coli exposed to 0-100 microg/mL chromium oxide nanoparticles (Cr2O3, Nps) for 120 min in Luria Bertani broth was evaluated by Propidium monoazide (PMA) assisted Q-PCR and standard plate count (SPC) method. Viable count for E. coli grown in Cr2O3, Nps amended medium was more by PMA assisted Q-PCR than SPC. Thus, the observations made in this study suggest that the inclusion of PMA assisted Q-PCR for viability assessment in Nps toxicity studies will provide the real count for the viable cells comprising of both viable and viable but not culturable (VBNC) cells. PMID:21485854

  5. The effect of mitotane on viability, steroidogenesis and gene expression in NCI‑H295R adrenocortical cells.

    PubMed

    Lehmann, Tomasz P; Wrzesiński, Tomasz; Jagodziński, Paweł P

    2013-03-01

    Mitotane, also known as o,p'‑DDD or (RS)‑1‑chl-oro‑2‑[2,2‑dichloro‑1‑(4‑chlorophenyl)‑ethyl]‑benzene, is an adrenal cortex-specific cytotoxic drug used in the therapy of adrenocortical carcinoma (ACC). The drug also inhibits steroidogenesis, however, the mechanisms of its anticancer and antisteroidogenic effects remain unknown. At present, data on the impact of mitotane on cell viability and the regulation of genes encoding proteins associated with steroids synthesis in the adrenal cortex, including cortisol and dehydroepiandrosterone sulfate (DHEAS), are limited and contradictory. In the present study, the effect of 24‑h mitotane treatment on viability of the ACC cell line, NCI‑H295R, was analyzed, identifying a decrease in cell viability and an increase in caspase‑3 and ‑7 activities. Mitotane treatment also led to decreased cortisol and DHEAS concentration in the culture media. Concomitantly, mitotane resulted in decreased mRNA levels of two cytochromes P450 (CYP11A1 and CYP17A1), mRNAs encoding proteins involved in the synthesis of cortisol and DHEAS. Mitotane did not affect mRNA levels of cyclin dependent kinase inhibitor 1A (encoding p21) and MYC (encoding cMyc). cMyc and p21 are key transcription factors associated with cell cycle regulation. However, mitotane inhibited expression of transforming growth factor β1 gene, encoding a potent inhibitor of cell proliferation and steroidogenesis. PRKAR1A, a protein kinase A regulatory subunit, is involved in the activation of steroidogenesis. PRKAR1A mRNA levels were reduced following 24‑h treatment with mitotane. Results indicate that mitotane markedly inhibited expression of genes involved in steroidogenesis, secretion of cortisol and DHEAS. Reduced expression of TGFB1 cannot account fully for the effect of mitotane on CYP11A1 and CYP17A1. We hypothesized that reduced viability of NCI‑H295R cells in the presence of mitotane may be a result of apoptosis triggered by increased

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

    PubMed Central

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

    2016-01-01

    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. PMID:27255403

  7. 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.

  8. The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability

    PubMed Central

    Arigony, Ana Lúcia Vargas; de Oliveira, Iuri Marques; Bordin, Diana Lilian; Prá, Daniel; Pêgas Henriques, João Antonio

    2013-01-01

    Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed. PMID:23781504

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

    PubMed

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

    2016-01-01

    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/cm(2)), 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. PMID:27255403

  10. DJ-1 Expression in Cervical Carcinoma and its Effects on Cell Viability and Apoptosis.

    PubMed

    Wang, Han; Gao, Weiwei

    2016-01-01

    BACKGROUND This study aimed to investigate the expression of DJ-1 in cervical carcinoma and its effects on cell viability and apoptosis. MATERIAL AND METHODS Cervical carcinoma cell line Hela and 85 tissue samples, including 45 primary tumor biopsies, 30 para-carcinoma tissues, and 10 normal cervical tissues samples were used in this study. The expressions of DJ-1 in cervical carcinoma tissue, para-carcinoma tissue, and normal tissue samples were investigated by immunohistochemistry. DJ-1 expression in Hela cells was also investigated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. DJ-1 was interfered and transfected with siRNA, then cell viability and apoptosis were assayed by MTT and flow cytometry, respectively. Additionally, the expressions of phosphatase and tensin homolog (PTEN), AKT, and phospho-AKT (P-AKT) were detected. RESULTS Immunohistochemistry results showed that DJ-1 was highly expressed in cervical carcinoma tissues. In Hela cells, the expression of DJ-1 was significantly higher than that in normal controls (P<0.05). When cells were treated with DJ-1 siRNA, the cell viability decreased significantly (P<0.05), and the percentage of apoptosis cells increased significantly (P<0.05). In addition, the expressions of PTEN and AKT were significantly higher in the DJ-1 siRNA treatment group than those in the control group (P<0.05). The expression of p-AKT was significantly lower in the DJ-1 siRNA treatment group than in the control group and the DJ-1 over-expression group (P<0.05). CONCLUSIONS The aberrant up-regulation of DJ-1 expression might be an important step in the pathogenesis of cervical carcinoma. PMID:27544688

  11. DJ-1 Expression in Cervical Carcinoma and its Effects on Cell Viability and Apoptosis

    PubMed Central

    Wang, Han; Gao, Weiwei

    2016-01-01

    Background This study aimed to investigate the expression of DJ-1 in cervical carcinoma and its effects on cell viability and apoptosis. Material/Methods Cervical carcinoma cell line Hela and 85 tissue samples, including 45 primary tumor biopsies, 30 para-carcinoma tissues, and 10 normal cervical tissues samples were used in this study. The expressions of DJ-1 in cervical carcinoma tissue, para-carcinoma tissue, and normal tissue samples were investigated by immunohistochemistry. DJ-1 expression in Hela cells was also investigated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. DJ-1 was interfered and transfected with siRNA, then cell viability and apoptosis were assayed by MTT and flow cytometry, respectively. Additionally, the expressions of phosphatase and tensin homolog (PTEN), AKT, and phospho-AKT (P-AKT) were detected. Results Immunohistochemistry results showed that DJ-1 was highly expressed in cervical carcinoma tissues. In Hela cells, the expression of DJ-1 was significantly higher than that in normal controls (P<0.05). When cells were treated with DJ-1 siRNA, the cell viability decreased significantly (P<0.05), and the percentage of apoptosis cells increased significantly (P<0.05). In addition, the expressions of PTEN and AKT were significantly higher in the DJ-1 siRNA treatment group than those in the control group (P<0.05). The expression of p-AKT was significantly lower in the DJ-1 siRNA treatment group than in the control group and the DJ-1 over-expression group (P<0.05). Conclusions The aberrant up-regulation of DJ-1 expression might be an important step in the pathogenesis of cervical carcinoma. PMID:27544688

  12. Cationic Pillararenes Potently Inhibit Biofilm Formation without Affecting Bacterial Growth and Viability.

    PubMed

    Joseph, Roymon; Naugolny, Alissa; Feldman, Mark; Herzog, Ido M; Fridman, Micha; Cohen, Yoram

    2016-01-27

    It is estimated that up to 80% of bacterial infections are accompanied by biofilm formation. Since bacteria in biofilms are less susceptible to antibiotics than are bacteria in the planktonic state, biofilm-associated infections pose a major health threat, and there is a pressing need for antibiofilm agents. Here we report that water-soluble cationic pillararenes differing in the quaternary ammonium groups efficiently inhibited the formation of biofilms by clinically important Gram-positive pathogens. Biofilm inhibition did not result from antimicrobial activity; thus, the compounds should not inhibit growth of natural bacterial flora. Moreover, none of the cationic pillararenes caused detectable membrane damage to red blood cells or toxicity to human cells in culture. The results indicate that cationic pillararenes have potential for use in medical applications in which biofilm formation is a problem. PMID:26745311

  13. Effect of antibiotics against Mycoplasma sp. on human embryonic stem cells undifferentiated status, pluripotency, cell viability and growth.

    PubMed

    Romorini, Leonardo; Riva, Diego Ariel; Blüguermann, Carolina; Videla Richardson, Guillermo Agustin; Scassa, Maria Elida; Sevlever, Gustavo Emilio; Miriuka, Santiago Gabriel

    2013-01-01

    Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate into specialized cells and hold great promise as models for human development and disease studies, cell-replacement therapies, drug discovery and in vitro cytotoxicity tests. The culture and differentiation of these cells are both complex and expensive, so it is essential to extreme aseptic conditions. hESCs are susceptible to Mycoplasma sp. infection, which is hard to detect and alters stem cell-associated properties. The purpose of this work was to evaluate the efficacy and cytotoxic effect of Plasmocin(TM) and ciprofloxacin (specific antibiotics used for Mycoplasma sp. eradication) on hESCs. Mycoplasma sp. infected HUES-5 884 (H5 884, stable hESCs H5-brachyury promoter-GFP line) cells were effectively cured with a 14 days Plasmocin(TM) 25 µg/ml treatment (curative treatment) while maintaining stemness characteristic features. Furthermore, cured H5 884 cells exhibit the same karyotype as the parental H5 line and expressed GFP, through up-regulation of brachyury promoter, at day 4 of differentiation onset. Moreover, H5 cells treated with ciprofloxacin 10 µg/ml for 14 days (mimic of curative treatment) and H5 and WA09 (H9) hESCs treated with Plasmocin(TM) 5 µg/ml (prophylactic treatment) for 5 passages retained hESCs features, as judged by the expression of stemness-related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers (brachyury, Nkx2.5 and cTnT: mesoderm; AFP: endoderm; nestin and Pax-6: ectoderm) was verified in in vitro differentiated antibiotic-treated hESCs. In conclusion, we found that Plasmocin(TM) and ciprofloxacin do not affect hESCs stemness and pluripotency nor cell viability. However, curative treatments slightly diminished cell growth rate. This cytotoxic effect was reversible as cells regained normal growth rate upon antibiotic withdrawal.

  14. A colorimetric assay for determination of cell viability in algal cultures.

    PubMed

    Capasso, Juan M; Cossío, Belén R; Berl, Tomás; Rivard, Christopher J; Jiménez, Carlos

    2003-07-01

    In this work, we propose the determination of cell viability in algal cultures by using a colorimetric assay widely used for estimation of cell proliferation in animal cell cultures. The method is based on in vivo reduction by metabolically active cells of a tetrazolium compound (MTS=3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt) to a colored formazan, with maximal absorbance at 490 nm, that is released to the culture medium. For this purpose, we have tested two microalgae with high commercial value (Dunaliella and Spirulina) and two seaweeds with different morphology (Ulva and Gracilaria). Color development in this assay is directly proportional to the number of viable cells, to the incubation time in the presence of the assay solution, and to the incubation temperature. A direct significant correlation was found between algal photosynthesis rate and color development in all species used through this work. Moreover, the intensity of absorbance at 490 nm was significantly lower in stressed cells (e.g. in nutrient-limited cultures, in the presence of toxic substances, and in osmotically-stressed cultures). We conclude that cell viability of algal cultures can be rapidly and easily estimated through colorimetric determination of the reduction of MTS to formazan.

  15. Factors Influencing Protoplast Viability of Suspension-Cultured Rice Cells during Isolation Process.

    PubMed

    Ishii, S

    1988-09-01

    Callus cells of rice (Oryza sativa L.) that were actively dividing in suspension culture had lost the ability to divide during the isolation process of protoplasts. Factors influencing the protoplast viability were examined using highly purified preparations of cellulase C(1), xylanase, and pectin lyase, which were essential enzymes for the isolation of protoplasts from the rice cells. The treatment of the cells with xylanase and pectin lyase, both of which are macerating enzymes, caused cellular damage. Xylanase treatment was more detrimental to the cells. Osmotic stress, cell wall fragments solubilized by xylanase, and disassembly of cortical microtubules were not the primary factors which damaged the rice cells and protoplasts. The addition of AgNO(3), an inhibitor of ethylene action, to the protoplast isolation medium increased the number of colonies formed from the cultured protoplasts, although the yield of protoplasts was reduced by the addition. Superoxide radical (O(2)-) was generated from the cells treated with xylanase or pectin lyase. The addition of superoxide dismutase and catalase to the protoplast isolation medium resulted in a marked improvement in protoplast viability especially when the non-additive control protoplasts formed colonies with a low frequency. The addition of glutathione peroxidase and phospholipase A(2), which have been known to reduce and detoxify lipid hydroperoxides in membranes, to the protoplast culture medium significantly increased the frequency of colony formation. These results suggested that some of the damage to rice protoplasts may be caused by oxygen toxicity.

  16. Magnetically induced electrostimulation of human osteoblasts results in enhanced cell viability and osteogenic differentiation

    PubMed Central

    HIEMER, BETTINA; ZIEBART, JOSEFIN; JONITZ-HEINCKE, ANIKA; GRUNERT, PHILIP CHRISTIAN; SU, YUKUN; HANSMANN, DORIS; BADER, RAINER

    2016-01-01

    The application of electromagnetic fields to support the bone-healing processes is a therapeutic approach for patients with musculoskeletal disorders. The ASNIS-III s-series screw is a bone stimulation system providing electromagnetic stimulation; however, its influence on human osteoblasts (hOBs) has not been extensively investigated. Therefore, in the present study, the impact of this system on the viability and differentiation of hOBs was examined. We used the ASNIS-III s screw system in terms of a specific experimental test set-up. The ASNIS-III s screw system was used for the application of electromagnetic fields (EMF, 3 mT, 20 Hz) and electromagnetic fields combined with an additional alternating electric field (EMF + EF) (3 mT, 20 Hz, 700 mV). The stimulation of primary hOBs was conducted 3 times per day for 45 min over a period of 72 h. Unstimulated cells served as the controls. Subsequently, the viability, the gene expression of differentiation markers and pro-collagen type 1 synthesis of the stimulated osteoblasts and corresponding controls were investigated. The application of both EMF and EMF + EF using the ASNIS-III s screw system revealed a positive influence on bone cell viability and moderately increased the synthesis of pro-collagen type 1 compared to the unstimulated controls. Stimulation with EMF resulted in a slightly enhanced gene expression of type 1 collagen and osteocalcin; however, stimulation with EMF + EF resulted in a significant increase in alkaline phosphatase (1.4-fold) and osteocalcin (1.6-fold) levels, and a notable increase in the levels of runt-related transcription factor 2 (RUNX-2; 1.54-fold). Our findings demonstrate that stimulation with electromagnetic fields and an additional alternating electric field has a positive influence on hOBs as regards cell viability and the expression of osteoblastic differentiation markers. PMID:27220915

  17. Histopathology of growth anomaly affecting the coral, Montipora capitata: implications on biological functions and population viability.

    PubMed

    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

  18. Secondary metabolites from nonhost plants affect the motility and viability of phytopathogenic Aphanomyces cochlioides zoospores.

    PubMed

    Islam, Md Tofazzal

    2008-01-01

    The motile zoospores of the damping-off pathogen Aphanomyces cochlioides aggregate on host plants (e.g., sugar beet, spinach) guided by the host-specific plant signal cochliophilin A before infection. To assess the potential role of secondary metabolites in nonhost resistance, acetone extracts of 200 nonhost traditional medicinal plants from Chinese and Bangladeshi origins were tested for the motility behaviour of A. cochlioides zoospores using a particle bioassay method. Nearly one third of the tested plant extracts exhibited diverse deleterious activities such as repellent, stimulant, motility halting and lysis against A. cochlioides zoospores. Among these active plants, an extract of the Chinese medicinal plant Dalbergia odorifera displayed potent repellent activity toward zoospores. Chromatographic separation of D. odorifera constituents revealed that the repellent activity was regulated by the cumulative effect of three motility-affecting isoflavonoids, viz. (+/-)-medicarpin (repellent at 150 microg/ml), (-)-claussequinone (stimulant at 100 microg/ml) and formononetin (stimulant and attractant at 50 microg/ml). A mixture (1:1:1, w/w/w) of these three compounds exhibited only repellent activity toward zoospores at a concentration lower than 50 microg/ml. These results suggest that nonhost plants might possess potential bioactive secondary metabolites to ward off zoosporic phytopathogens. PMID:18533468

  19. Automated cell viability assessment using a microfluidics based portable imaging flow analyzer

    PubMed Central

    Jagannadh, Veerendra Kalyan; Adhikari, Jayesh Vasudeva; Gorthi, Sai Siva

    2015-01-01

    In this work, we report a system-level integration of portable microscopy and microfluidics for the realization of optofluidic imaging flow analyzer with a throughput of 450 cells/s. With the use of a cellphone augmented with off-the-shelf optical components and custom designed microfluidics, we demonstrate a portable optofluidic imaging flow analyzer. A multiple microfluidic channel geometry was employed to demonstrate the enhancement of throughput in the context of low frame-rate imaging systems. Using the cell-phone based digital imaging flow analyzer, we have imaged yeast cells present in a suspension. By digitally processing the recorded videos of the flow stream on the cellphone, we demonstrated an automated cell viability assessment of the yeast cell population. In addition, we also demonstrate the suitability of the system for blood cell counting. PMID:26015835

  20. Automated cell viability assessment using a microfluidics based portable imaging flow analyzer.

    PubMed

    Jagannadh, Veerendra Kalyan; Adhikari, Jayesh Vasudeva; Gorthi, Sai Siva

    2015-03-01

    In this work, we report a system-level integration of portable microscopy and microfluidics for the realization of optofluidic imaging flow analyzer with a throughput of 450 cells/s. With the use of a cellphone augmented with off-the-shelf optical components and custom designed microfluidics, we demonstrate a portable optofluidic imaging flow analyzer. A multiple microfluidic channel geometry was employed to demonstrate the enhancement of throughput in the context of low frame-rate imaging systems. Using the cell-phone based digital imaging flow analyzer, we have imaged yeast cells present in a suspension. By digitally processing the recorded videos of the flow stream on the cellphone, we demonstrated an automated cell viability assessment of the yeast cell population. In addition, we also demonstrate the suitability of the system for blood cell counting. PMID:26015835

  1. 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.

  2. 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

  3. Effect of laser treatment on the attachment and viability of mesenchymal stem cell responses on shape memory NiTi alloy.

    PubMed

    Chan, C W; Hussain, I; Waugh, D G; Lawrence, J; Man, H C

    2014-09-01

    The objectives of this study were to investigate the effect of laser-induced surface features on the morphology, attachment and viability of mesenchymal stem cells (MSCs) at different periods of time, and to evaluate the biocompatibility of different zones: laser-melted zone (MZ), heat-affected zone (HAZ) and base metal (BM) in laser-treated NiTi alloy. The surface morphology and composition were studied by scanning electron microscope (SEM) and X-ray photoemission spectroscopy (XPS), respectively. The cell morphology was examined by SEM while the cell counting and viability measurements were done by hemocytometer and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The results indicated that the laser-induced surface features, such as surface roughening, presence of anisotropic dendritic pattern and complete surface Ni oxidation were beneficial to improve the biocompatibility of NiTi as evidenced by the highest cell attachment (4 days of culture) and viability (7 days of culture) found in the MZ. The biocompatibility of the MZ was the best, followed by the BM with the HAZ being the worst. The defective and porous oxide layer as well as the coarse grained structure might attribute to the inferior cell attachment (4 days of culture) and viability (7 days of culture) on the HAZ compared with the BM which has similar surface morphology.

  4. Evaluation of Periodontal Ligament Cell Viability in Three Different Storage Media: An in Vitro Study

    PubMed Central

    Sharma, Sanjay; Reddy, Y. G.; Mittal, Rakesh; Agarwal, Vishal; Singh, Chanchal; Singh, Amandeep

    2015-01-01

    Objectives: This study was undertaken to evaluate the viability of periodontal ligament (PDL) cells of avulsed teeth in three different storage media. Materials and Methods: Forty-five premolars extracted for orthodontic therapeutic purposes were randomly and equally divided into three groups based on storage media used [Group I: milk (control); Group II: aloe vera (experimental); Group III: egg white (experimental)]. Following extractions, the teeth were placed in one of the three different storage media for 30 minutes, following which the scrapings of the PDL from these teeth were collected in Falcon tubes containing collagenase enzyme in 2.5 mL of phosphate buffered saline. The tubes were subsequently incubated for 30 minutes and centrifuged for five minutes at 800 rpm. The obtained PDL cells were stained with Trypan Blue and were observed under optical microscope. The percentage of viable cells was calculated. Results: Aloe vera showed the highest percentage of viable cells (114.3±8.0), followed by egg white (100.9±6.3) and milk (101.1±7.3). Conclusion: Within the limitations of this study, it appears that aloe vera maintains PDL cell viability better than egg white or milk. PMID:26877742

  5. Oxygen delivery from hyperbarically loaded microtanks extends cell viability in anoxic environments.

    PubMed

    Cook, Colin A; Hahn, Kathryn C; Morrissette-McAlmon, Justin B F; Grayson, Warren L

    2015-06-01

    Oxygen diffusion limitations within nascent tissue engineered (TE) grafts lead to the development of hypoxic regions, cell death, and graft failure. Previous efforts have been made to deliver oxygen within TE scaffolds, including peroxide-doping, perfluorocarbons, and hyperbaric oxygen therapy, to mitigate these effects and help maintain post transplantation cell viability, but these have suffered from significant drawbacks. Here we present a novel approach utilizing polymeric hollow-core microspheres that can be hyperbarically loaded with oxygen and subsequently provide prolonged oxygen delivery. These oxygen carriers are termed, microtanks. With an interest in orthopedic applications, we combined microtanks within polycaprolactone to form solid phase constructs with oxygen delivery capabilities. The mathematical laws governing oxygen delivery from microtank-loaded constructs are developed along with empirical validation. Constructs achieved periods of oxygen delivery out to 6 days, which was shown to prolong the survival of human adipose derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs) as well as to enhance their cellular morphology under anoxic conditions. The results of this study suggest the microtank approach may be a feasible means of maintaining cell viability in TE scaffolds during the critical period of vascularization in vivo.

  6. Oxygen Delivery from Hyperbarically Loaded Microtanks Extends Cell Viability in Anoxic Environments

    PubMed Central

    Cook, Colin A.; Hahn, Kathryn C.; Morrissette-McAlmon, Justin B.F.; Grayson, Warren L.

    2016-01-01

    Oxygen diffusion limitations within nascent tissue engineered (TE) grafts lead to the development of hypoxic regions, cell death, and graft failure. Previous efforts have been made to deliver oxygen within TE scaffolds, including peroxide-doping, perfluorocarbons, and hyperbaric oxygen therapy, to mitigate these effects and help maintain post transplantation cell viability, but these have suffered from significant drawbacks. Here we present a novel approach utilizing polymeric hollow-core microspheres that can be hyperbarically loaded with oxygen and subsequently provide prolonged oxygen delivery. These oxygen carriers are termed, microtanks. With an interest in orthopedic applications, we combined microtanks within polycaprolactone to form solid phase constructs with oxygen delivery capabilities. The mathematical laws governing oxygen delivery from microtank-loaded constructs are developed along with empirical validation. Constructs achieved periods of oxygen delivery out to 6 days, which was shown to prolong the survival of human adipose derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs) as well as to enhance their cellular morphology under anoxic conditions. The results of this study suggest the microtank approach may be a feasible means of maintaining cell viability in TE scaffolds during the critical period of vascularization in vivo. PMID:25818444

  7. Evaluation of the effects of Cimicifugae Rhizoma on the morphology and viability of mesenchymal stem cells

    PubMed Central

    JEONG, SU-HYEON; LEE, JI-EUN; KIM, BO-BAE; KO, YOUNGKYUNG; PARK, JUN-BEOM

    2015-01-01

    Cimicifugae Rhizoma is a traditional herbal medicine used to treat various diseases in Korea, China and Japan. Cimicifugae Rhizoma is primarily derived from Cimicifuga heracleifolia Komarov or Cimicifuga foetida Linnaeus. Cimicifugae Rhizoma has been used as an anti-inflammatory, analgesic and antipyretic remedy. The present study was performed to evaluate the extracts of Cimicifugae Rhizoma on the morphology and viability of human stem cells derived from gingiva. Stem cells derived from gingiva were grown in the presence of Cimicifugae Rhizoma at final concentrations that ranged from 0.001 to 1,000 µg/ml. The morphology of the cells was viewed under an inverted microscope and the analysis of cell proliferation was performed using a Cell Counting kit-8 (CCK-8) assay on days 1, 3, 5 and 7. Under an optical microscope, the control cells exhibited a spindle-shaped, fibroblast-like morphology. The shapes of the cells in the groups treated with 0.001, 0.01, 0.1, 1 and 10 µg/ml Cimicifugae Rhizoma were similar to the shapes in the control group. Significant alterations in morphology were noted in the 100 and 1,000 µg/ml groups when compared with the control group. The cells in the 100 and 1,000 µg/ml groups were rounder, and fewer cells were present. The cultures that were grown in the presence of Cimicifugae Rhizoma at a concentration of 0.001 µg/ml on day 1 had an increased CCK-8 value. The cultures grown in the presence of Cimicifugae Rhizoma at a concentration of 10 µg/ml on day 7 had a reduced CCK-8 value. Within the limits of this study, Cimicifugae Rhizoma influenced the viability of stem cells derived from the gingiva, and its direct application onto oral tissues may have adverse effects at high concentrations. The concentration and application time of Cimicifugae Rhizoma should be meticulously controlled to obtain optimal results. PMID:26622366

  8. Flow Cytometry Approach to Quantify the Viability of Milk Somatic Cell Counts after Various Physico-Chemical Treatments

    PubMed Central

    Li, Na; Richoux, Romain; Perruchot, Marie-Hélène; Boutinaud, Marion; Mayol, Jean-François; Gagnaire, Valérie

    2015-01-01

    Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better

  9. A MULTIPLEXED ASSAY FOR DETERMINATION OF NEUROTOXICANT EFFECTS ON SPONTANEOUS NETWORK ACTIVITY AND CELL VIABILITY FROM MICROELECTRODE ARRAYS

    EPA Science Inventory

    AbstractTITLE: A MULTIPLEXED ASSAY FOR DETERMINATION OF NEUROTOXICANT EFFECTS ON SPONTANEOUS NETWORK ACTIVITY AND CELL VIABILITY FROM MICROELECTRODE ARRAYSABSTRACT BODY: Microelectrode array (MEA) recordings are increasingly being used as an in vitro method to detect and characte...

  10. Enhanced Viability of Endothelial Colony Forming Cells in Fibrin Microbeads for Sensor Vascularization.

    PubMed

    Gandhi, Jarel K; Zivkovic, Lada; Fisher, John P; Yoder, Mervin C; Brey, Eric M

    2015-09-18

    Enhanced vascularization at sensor interfaces can improve long-term function. Fibrin, a natural polymer, has shown promise as a biomaterial for sensor coating due to its ability to sustain endothelial cell growth and promote local vascularization. However, the culture of cells, particularly endothelial cells (EC), within 3D scaffolds for more than a few days is challenging due to rapid loss of EC viability. In this manuscript, a robust method for developing fibrin microbead scaffolds for long-term culture of encapsulated ECs is described. Fibrin microbeads are formed using sodium alginate as a structural template. The size, swelling and structural properties of the microbeads were varied with needle gauge and composition and concentration of the pre-gel solution. Endothelial colony-forming cells (ECFCs) were suspended in the fibrin beads and cultured within a perfusion bioreactor system. The perfusion bioreactor enhanced ECFCs viability and genome stability in fibrin beads relative to static culture. Perfusion bioreactors enable 3D culture of ECs within fibrin beads for potential application as a sensor coating.

  11. Thermoresponsive polymers as gene delivery vectors: cell viability, DNA transport and transfection studies.

    PubMed

    Twaites, Beverley R; de Las Heras Alarcón, Carolina; Lavigne, Matthieu; Saulnier, Annabelle; Pennadam, Sivanand S; Cunliffe, David; Górecki, Dariusz C; Alexander, Cameron

    2005-11-28

    A range of gene delivery vectors containing the thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAm) was evaluated for effects on cell viability, intracellular trafficking and transgene expression in C2C12 mouse muscle cells. Polymers were complexed with plasmid DNA at pH 7.4 and the ability of the resulting particles to transfect cells was assessed via confocal microscopy and protein expression studies in tissue culture. Cell viability assays indicated that these polymers were toxic at high concentrations when not complexed to DNA or at certain polymer:DNA ratios. Poly(ethyleneimine) co-polymers with side-chain grafted PNIPAm were shown to be less toxic than poly(ethyleneimine) alone or PNIPAm-co-(N,N'-dimethylaminoethylmethacrylate) linear co-polymers and the effects were concentration dependent. Confocal micrographs of labeled polymers and DNA indicated rapid cellular entry for all the complexes but expression of Green Fluorescent Protein was achieved only when the branched PEI-PNIPAm co-polymers were used as vectors. The results indicate that design of appropriate co-polymer components and overall polymer architecture can be used to mediate, and perhaps ultimately control, DNA transport and transgene expression. PMID:16214254

  12. Enhanced Viability of Endothelial Colony Forming Cells in Fibrin Microbeads for Sensor Vascularization

    PubMed Central

    Gandhi, Jarel K.; Zivkovic, Lada; Fisher, John P.; Yoder, Mervin C.; Brey, Eric M.

    2015-01-01

    Enhanced vascularization at sensor interfaces can improve long-term function. Fibrin, a natural polymer, has shown promise as a biomaterial for sensor coating due to its ability to sustain endothelial cell growth and promote local vascularization. However, the culture of cells, particularly endothelial cells (EC), within 3D scaffolds for more than a few days is challenging due to rapid loss of EC viability. In this manuscript, a robust method for developing fibrin microbead scaffolds for long-term culture of encapsulated ECs is described. Fibrin microbeads are formed using sodium alginate as a structural template. The size, swelling and structural properties of the microbeads were varied with needle gauge and composition and concentration of the pre-gel solution. Endothelial colony-forming cells (ECFCs) were suspended in the fibrin beads and cultured within a perfusion bioreactor system. The perfusion bioreactor enhanced ECFCs viability and genome stability in fibrin beads relative to static culture. Perfusion bioreactors enable 3D culture of ECs within fibrin beads for potential application as a sensor coating. PMID:26393602

  13. Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability.

    PubMed

    Stepanenko, A A; Dmitrenko, V V

    2015-12-15

    The MTT assay (to a less degree MTS, XTT or WST) is a widely exploited approach for measuring cell viability/drug cytotoxicity. MTT reduction occurs throughout a cell and can be significantly affected by a number of factors, including metabolic and energy perturbations, changes in the activity of oxidoreductases, endo-/exocytosis and intracellular trafficking. Over/underestimation of cell viability by the MTT assay may be due to both adaptive metabolic and mitochondrial reprogramming of cells subjected to drug treatment-mediated stress and inhibitor off-target effects. Previously, imatinib, rottlerin, ursolic acid, verapamil, resveratrol, genistein nanoparticles and some polypeptides were shown to interfere with MTT reduction rate resulting in inconsistent results between the MTT assay and alternative assays. Here, to test the under/overestimation of viability by the MTT assay, we compared results derived from the MTT assay with the trypan blue exclusion assay after treatment of glioblastoma U251, T98G and C6 cells with three widely used inhibitors with the known direct and side effects on energy and metabolic homeostasis - temozolomide (TMZ), a DNA-methylating agent, temsirolimus (TEM), an inhibitor of mTOR kinase, and U0126, an inhibitor of MEK1/2 kinases. Inhibitors were applied shortly as in IC50 evaluating studies or long as in studies focusing on drug resistance acquisition. We showed that over/underestimation of cell viability by the MTT assay and its significance depends on a cell line, a time point of viability measurement and other experimental parameters. Furthermore, we provided a comprehensive survey of factors that should be accounted in the MTT assay. To avoid result misinterpretation, supplementation of the tetrazolium salt-based assays with other non-metabolic assays is recommended. PMID:26260013

  14. The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability.

    PubMed

    Billiet, Thomas; Gevaert, Elien; De Schryver, Thomas; Cornelissen, Maria; Dubruel, Peter

    2014-01-01

    In the present study, we report on the combined efforts of material chemistry, engineering and biology as a systemic approach for the fabrication of high viability 3D printed macroporous gelatin methacrylamide constructs. First, we propose the use and optimization of VA-086 as a photo-initiator with enhanced biocompatibility compared to the conventional Irgacure 2959. Second, a parametric study on the printing of gelatins was performed in order to characterize and compare construct architectures. Hereby, the influence of the hydrogel building block concentration, the printing temperature, the printing pressure, the printing speed, and the cell density were analyzed in depth. As a result, scaffolds could be designed having a 100% interconnected pore network in the gelatin concentration range of 10-20 w/v%. In the last part, the fabrication of cell-laden scaffolds was studied, whereby the application for tissue engineering was tested by encapsulation of the hepatocarcinoma cell line (HepG2). Printing pressure and needle shape was revealed to impact the overall cell viability. Mechanically stable cell-laden gelatin methacrylamide scaffolds with high cell viability (>97%) could be printed.

  15. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.

    PubMed

    Chen, Guokai; Hou, Zhonggang; Gulbranson, Daniel R; Thomson, James A

    2010-08-01

    Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs.

  16. Effect of NPC15199 on [Ca²⁺]i and viability in SCM1 human gastric cancer cells.

    PubMed

    Cheng, He-Hsiung; Chou, Chiang-Ting; Liang, Wei-Zhe; Cheng, Jin-Shiung; Chang, Hong-Tai; Kuo, Chun-Chi; Chen, I-S; Lu, Ti; Yu, C-C; Chen, Fu-An; Kuo, Daih-Huang; Shieh, Pochuen; Jan, Chung-Ren

    2016-10-31

    NPC15199 is a synthesized compound that inhibits inflammation in some models. However, whether NPC15199 affects Ca²⁺ homeostasis in human gastric cancer is unclear. This study examined the effect of NPC15199 on cytosolic free Ca²⁺ concentrations ([Ca²⁺]i) and viability in SCM1 human gastric cancer cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]i. NPC15199 evoked [Ca²⁺]i rises concentration-dependently. The response was reduced by removing extracellular Ca²⁺. NPC15199-evoked Ca²⁺ entry was not inhibited by store-operated channel inhibitors (nifedipine, econazole and SKF96365) and protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate, PMA), or PKC inhibitor (GF109203X). In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished NPC15199-evoked [Ca²⁺]i rises. Conversely, treatment with NPC15199 also nearly abolished thapsigargin or BHQ-evoked [Ca²⁺]i rises. Inhibition of phospholipase C (PLC) with U73122 did not affect NPC15199-evoked [Ca²⁺]i rises. NPC15199 at concentrations of 100-900 μM induced concentration-dependent, Ca²⁺-independent decrease in viability. Together, in SCM1 cells, NPC15199 induced [Ca²⁺]i rises that involved Ca²⁺ entry through PKC-insensitive non-store-operated Ca²⁺ channels and PLC-independent Ca²⁺ release from the endoplasmic reticulum. NPC15199 also induced Ca²⁺-independent cell death.

  17. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    SciTech Connect

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M.; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.

  18. MicroRNA-29a inhibits mesenchymal stem cell viability and proliferation by targeting Roundabout 1.

    PubMed

    Zhang, Yudong; Zhou, Shenghua

    2015-10-01

    Secreted Slit glycoproteins and their Roundabout (Robo) receptors have been identified as important axon guidance molecules. The pivotal role of Slit‑Robo signaling is in regulating cell proliferation. MicroRNAs (miRNAs), a class of small non‑coding RNAs, function as critical regulators of gene expression by binding to the 3'‑untranslated region of mRNAs and causing mRNA degradation or translational repression. The present study demonstrated that downregulation of Robo1 using small interfering RNA inhibited mesenchymal stem cell (MSC) proliferation. Additionally, four miRNAs (miR), including miR‑218, miR‑29a, miR‑146 and miR‑148, inhibited the protein expression of Robo1 in the MSCs, with miR‑29 having the most marked effect. A luciferase reporter assay identified Robo1 as a novel target of miR‑29a. Overexpression of miR‑29a suppressed the protein expression levels of Robo1 and Slit2 and inhibited the viability and proliferation of the MSCs. By contrast, overexpression of Robo1 partly rescued these inhibitory effects of miR‑29a on the MSCs confirming that miR‑29a inhibited MSC viability and proliferation, at least partially, by directly targeting Robo1. These results indicated that the miR‑29a/Robo1 axis is crucial for the regulation of MSC viability and proliferation, suggesting that miR‑29a may serve as a potential clinical target for MSC expansion and stem cell transplantation.

  19. High Modulus Biodegradable Polyurethanes for Vascular Stents: Evaluation of Accelerated in vitro Degradation and Cell Viability of Degradation Products

    PubMed Central

    Sgarioto, Melissa; Adhikari, Raju; Gunatillake, Pathiraja A.; Moore, Tim; Patterson, John; Nagel, Marie-Danielle; Malherbe, François

    2015-01-01

    We have recently reported the mechanical properties and hydrolytic degradation behavior of a series of NovoSorb™ biodegradable polyurethanes (PUs) prepared by varying the hard segment (HS) weight percentage from 60 to 100. In this study, the in vitro degradation behavior of these PUs with and without extracellular matrix (ECM) coating was investigated under accelerated hydrolytic degradation (phosphate buffer saline; PBS/70°C) conditions. The mass loss at different time intervals and the effect of aqueous degradation products on the viability and growth of human umbilical vein endothelial cells (HUVEC) were examined. The results showed that PUs with HS 80% and below completely disintegrated leaving no visual polymer residue at 18 weeks and the degradation medium turned acidic due to the accumulation of products from the soft segment (SS) degradation. As expected the PU with the lowest HS was the fastest to degrade. The accumulated degradation products, when tested undiluted, showed viability of about 40% for HUVEC cells. However, the viability was over 80% when the solution was diluted to 50% and below. The growth of HUVEC cells is similar to but not identical to that observed with tissue culture polystyrene standard (TCPS). The results from this in vitro study suggested that the PUs in the series degraded primarily due to the SS degradation and the cell viability of the accumulated acidic degradation products showed poor viability to HUVEC cells when tested undiluted, however particles released to the degradation medium showed cell viability over 80%. PMID:26000274

  20. Histone Deacetylase (HDAC) Inhibitor Kinetic Rate Constants Correlate with Cellular Histone Acetylation but Not Transcription and Cell Viability

    PubMed Central

    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

    2013-01-01

    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. PMID:23897821

  1. 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

  2. Different methods to quantify Listeria monocytogenes biofilms cells showed different profile in their viability

    PubMed Central

    Winkelströter, Lizziane Kretli; Martinis, Elaine C.P. De

    2015-01-01

    Listeria monocytogenes is a foodborne pathogen able to adhere and to form biofilms in several materials commonly present in food processing plants. The aim of this study was to evaluate the resistance of Listeria monocytogenes attached to abiotic surface, after treatment with sanitizers, by culture method, microscopy and Quantitative Real Time Polymerase Chain Reaction (qPCR). Biofilms of L. monocytogenes were obtained in stainless steel coupons immersed in Brain Heart Infusion Broth, under agitation at 37 °C for 24 h. The methods selected for this study were based on plate count, microscopic count with the aid of viability dyes (CTC-DAPI), and qPCR. Results of culture method showed that peroxyacetic acid was efficient to kill sessile L. monocytogenes populations, while sodium hypochlorite was only partially effective to kill attached L. monocytogenes (p < 0.05). When, viability dyes (CTC/DAPI) combined with fluorescence microscopy and qPCR were used and lower counts were found after treatments (p < 0.05). Selective quantification of viable cells of L. monocytogenes by qPCR using EMA revelead that the pre-treatment with EMA was not appropriate since it also inhibited amplification of DNA from live cells by ca. 2 log. Thus, the use of CTC counts was the best method to count viable cells in biofilms. PMID:26221112

  3. Sodium functionalized graphene oxide coated titanium plates for improved corrosion resistance and cell viability

    NASA Astrophysics Data System (ADS)

    Marimuthu, Mohana; Veerapandian, Murugan; Ramasundaram, Subramaniyan; Hong, Seok Won; Sudhagar, P.; Nagarajan, Srinivasan; Raman, V.; Ito, Eisuke; Kim, Sanghyo; Yun, Kyusik; Kang, Yong Soo

    2014-02-01

    Surface functionalization is an important process that has been adopted to well explore the applications of nanomaterials. In this context, we demonstrate the sodium functionalized graphene oxide (NaGO) as an excellent candidate for increasing the life time of titanium (Ti) based ortho-implants. As-prepared aqueous dispersion of NaGO was used to assemble NaGO sheets on commercially pure Ti (CpTi) plates by heat controlled spin coating. The resulting wrinkled NaGO sheets play a dual role in implant material, i.e., passive layer against corrosion and biocompatible scaffold for cell viability. The preparation, physicochemical properties, and biocompatibility of NaGO coatings formed on CpTi were reported. The electrochemical polarization studies demonstrate the relative susceptibility of control GO and NaGO coatings to corrosion, which outline that the NaGO coating act as a geometric blocking layer and hence prevent the implant surface from contacting corrosive media. The immunofluorescence and cell proliferation studies performed using human dermal fibroblasts cells showed that NaGO coatings significantly (P < 0.05) enhanced the cellular viability for longer in vitro culture period (15 days) than control GO and pristine CpTi.

  4. Ischemia-reperfusion model in rat spinal cord: cell viability and apoptosis signaling study

    PubMed Central

    de Lavor, Mário Sérgio Lima; Binda, Nancy Scardua; Fukushima, Fabíola Bono; Caldeira, Fátima Maria Caetano; da Silva, Juliana Figueira; Silva, Carla Maria Osório; de Oliveira, Karen Maciel; Martins, Bernardo de Caro; Torres, Bruno Benetti Junta; Rosado, Isabel Rodrigues; Gomez, Renato Santiago; Gomez, Marcus Vinícius; de Melo, Eliane Gonçalves

    2015-01-01

    This work aimed at determining the ideal ischemia time in an in vitro ischemia-reperfusion model of spinal cord injury. Rat spinal cord slices were prepared and then exposed or not to oxygen deprivation and low glucose (ODLG) for 30, 45, 60, 75 and 90 minutes. Cell viability was assessed by triphenyltetrazolium (TTC), lactate dehydrogenase (LDH) release, and fluorochrome dyes specific for cell dead (ethidium homodimer) using the apotome system. Glutamate release was enzymatically measured by a fluorescent method. Gene expression of apoptotic factors was assessed by real time RT-PCR. Whereas spinal cord slices exposed to ODLG exhibited mild increase in fluorescence for 30 minutes after the insult, the 45, 60, 75 and 90 minutes caused a 2-fold increase. ODLG exposure for 45, 60, 75 or 90 minutes, glutamate and LDH release were significantly elevated. nNOS mRNA expression was overexpressed for 45 minutes and moderately increased for 60 minutes in ODLG groups. Bax/bcl-xl ratio, caspase 9 and caspase 3 mRNA expressions were significantly increased for 45 minutes of ODLG, but not for 30, 60, 75 and 90 minutes. Results showed that cell viability reduction in the spinal cord was dependent on ischemic time, resulting in glutamate and LDH release. ODLG for 45 minutes was adequate for gene expression evaluation of proteins and proteases involved in apoptosis pathways. PMID:26617703

  5. Irradiation Can Selectively Kill Tumor Cells while Preserving Erythrocyte Viability in a Co-Culture System

    PubMed Central

    Liu, Yun-Qing; Tang, Li-Hui; Wang, Yin; Wang, Lie-Ju; Zhang, Feng-Jiang; Yan, Min

    2015-01-01

    An understanding of how to safely apply intraoperative blood salvage (IBS) in cancer surgery has not yet been obtained. Here, we investigated the optimal dose of 137Cs gamma-ray irradiation for killing human hepatocarcinoma (HepG2), gastrocarcinoma (SGC7901), and colonic carcinoma (SW620) tumor cells while preserving co-cultured erythrocytes obtained from 14 healthy adult volunteers. HepG2, SGC7901, or SW620 cells were mixed into the aliquots of erythrocytes. After the mixed cells were treated with 137Cs gamma-ray irradiation (30, 50, and 100 Gy), tumor cells and erythrocytes were separated by density gradient centrifugation in Percoll with a density of 1.063 g/ml. The viability, clonogenicity, DNA synthesis, tumorigenicity, and apoptosis of the tumor cells were determined by MTT assay, plate colony formation, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, subcutaneous xenograft implantation into immunocompromised mice, and annexin V/7-AAD staining, respectively. The ATP concentration, 2,3-DPG level, free Hb concentration, osmotic fragility, membrane phosphatidylserine externalization, blood gas variables, reactive oxygen species levels, and superoxide dismutase levels in erythrocytes were analyzed. We found that 137Cs gamma-ray irradiation at 50 Gy effectively inhibited the viability, proliferation, and tumorigenicity of HepG2, SGC7901, and SW620 cells without markedly damaging the oxygen-carrying ability or membrane integrity or increasing the oxidative stress of erythrocytes in vitro. These results demonstrated that 50 Gy irradiation in a standard 137Cs blood irradiator might be a safe and effective method of inactivating HepG2, SGC7901, and SW620 cells mixed with erythrocytes, which might help to safely allow IBS in cancer surgery. PMID:26018651

  6. Irradiation Can Selectively Kill Tumor Cells while Preserving Erythrocyte Viability in a Co-Culture System.

    PubMed

    Gong, Ming; Yang, Jin-Ting; Liu, Yun-Qing; Tang, Li-Hui; Wang, Yin; Wang, Lie-Ju; Zhang, Feng-Jiang; Yan, Min

    2015-01-01

    An understanding of how to safely apply intraoperative blood salvage (IBS) in cancer surgery has not yet been obtained. Here, we investigated the optimal dose of 137Cs gamma-ray irradiation for killing human hepatocarcinoma (HepG2), gastrocarcinoma (SGC7901), and colonic carcinoma (SW620) tumor cells while preserving co-cultured erythrocytes obtained from 14 healthy adult volunteers. HepG2, SGC7901, or SW620 cells were mixed into the aliquots of erythrocytes. After the mixed cells were treated with 137Cs gamma-ray irradiation (30, 50, and 100 Gy), tumor cells and erythrocytes were separated by density gradient centrifugation in Percoll with a density of 1.063 g/ml. The viability, clonogenicity, DNA synthesis, tumorigenicity, and apoptosis of the tumor cells were determined by MTT assay, plate colony formation, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, subcutaneous xenograft implantation into immunocompromised mice, and annexin V/7-AAD staining, respectively. The ATP concentration, 2,3-DPG level, free Hb concentration, osmotic fragility, membrane phosphatidylserine externalization, blood gas variables, reactive oxygen species levels, and superoxide dismutase levels in erythrocytes were analyzed. We found that 137Cs gamma-ray irradiation at 50 Gy effectively inhibited the viability, proliferation, and tumorigenicity of HepG2, SGC7901, and SW620 cells without markedly damaging the oxygen-carrying ability or membrane integrity or increasing the oxidative stress of erythrocytes in vitro. These results demonstrated that 50 Gy irradiation in a standard 137Cs blood irradiator might be a safe and effective method of inactivating HepG2, SGC7901, and SW620 cells mixed with erythrocytes, which might help to safely allow IBS in cancer surgery. PMID:26018651

  7. The effect of automobile exhaust particulates on cell viability, plating efficiency and cell division of mammalian tissue culture cells.

    PubMed

    Seemayer, N H; Hadnagy, W; Tomingas, R

    1987-03-01

    Extract of particulate matter (EPM) of gasoline engine exhaust induced only a slight loss of cell viability of mouse macrophages (line IC-21) in vitro, while a strong dose-dependent reduction of plating efficiency of human cell line A-549 and of Syrian hamster line 14-1b occurred. Cytological investigations of exposed macrophages of line IC-21 revealed an increase in the mitotic index from 1.5% of control values up to 14.6% at the highest tested concentration of EPM. Mitotic arrest is based almost exclusively on C-type mitoses occurring dose-dependently in the presence of EPM. Results indicate disturbances of the spindle apparatus in the presence of EPM. PMID:2437649

  8. The effect of UV-filters on the viability of neuroblastoma (SH-SY5Y) cell line.

    PubMed

    Broniowska, Żaneta; Pomierny, Bartosz; Smaga, Irena; Filip, Małgorzata; Budziszewska, Bogusława

    2016-05-01

    Topical application of cosmetic products, containing ultraviolet filters (UV filters) are recommended as a protection against sunburns and in order to reduce the risk of skin cancer. However, some UV filters can be absorbed through skin and by consuming contaminated food. Among the chemical UV filters, benzophenone-3 (BP-3), 3-(4-methylbenzylidene)camphor (4-MBC) and 2-ethylhexyl-4-methoxycinnamate (OMC) are absorbed through the skin to the greatest extent. So far, these lipophilic compounds were demonstrated to influence the gonadal and thyroid hormone function, but their effect on central nervous system cells has not been investigated, yet. In the present study, we investigated the effect of some UV filters on cell viability and caspase-3 activity in SH-SY5Y cells. It has been found that benzophenone-2 (BP-2), BP-3, 4-methylbenzophenone (4-MBP) and OMC present in the culture medium for 72h in high concentration (10(-5) and 10(-4)M) and 4-MBC only 10(-4)M produced a significant cytotoxic effect, as determined both by the MTT reduction test and LDH release assay. In contrast to necrotic changes, all tested UV filters increased caspase-3 activity in much lower concentrations (from 10(-8) to 10(-7)M). Proapoptotic properties of the test compounds were positively verified by Hoechst staining. The obtained results indicated that UV filters adversely affected the viability of nerve cells, most likely by enhancing the process of apoptosis. The most potent effect was exerted by BP-3 and 4-MBC and at concentrations that may be reached in vivo. Since human exposure to UV filters is significant these compound should be taken into consideration as one of the possible factors involved in pathogenesis of neurodegenerative diseases. PMID:26965011

  9. The effect of UV-filters on the viability of neuroblastoma (SH-SY5Y) cell line.

    PubMed

    Broniowska, Żaneta; Pomierny, Bartosz; Smaga, Irena; Filip, Małgorzata; Budziszewska, Bogusława

    2016-05-01

    Topical application of cosmetic products, containing ultraviolet filters (UV filters) are recommended as a protection against sunburns and in order to reduce the risk of skin cancer. However, some UV filters can be absorbed through skin and by consuming contaminated food. Among the chemical UV filters, benzophenone-3 (BP-3), 3-(4-methylbenzylidene)camphor (4-MBC) and 2-ethylhexyl-4-methoxycinnamate (OMC) are absorbed through the skin to the greatest extent. So far, these lipophilic compounds were demonstrated to influence the gonadal and thyroid hormone function, but their effect on central nervous system cells has not been investigated, yet. In the present study, we investigated the effect of some UV filters on cell viability and caspase-3 activity in SH-SY5Y cells. It has been found that benzophenone-2 (BP-2), BP-3, 4-methylbenzophenone (4-MBP) and OMC present in the culture medium for 72h in high concentration (10(-5) and 10(-4)M) and 4-MBC only 10(-4)M produced a significant cytotoxic effect, as determined both by the MTT reduction test and LDH release assay. In contrast to necrotic changes, all tested UV filters increased caspase-3 activity in much lower concentrations (from 10(-8) to 10(-7)M). Proapoptotic properties of the test compounds were positively verified by Hoechst staining. The obtained results indicated that UV filters adversely affected the viability of nerve cells, most likely by enhancing the process of apoptosis. The most potent effect was exerted by BP-3 and 4-MBC and at concentrations that may be reached in vivo. Since human exposure to UV filters is significant these compound should be taken into consideration as one of the possible factors involved in pathogenesis of neurodegenerative diseases.

  10. Effect of intervertebral disc degeneration on disc cell viability: a numerical investigation.

    PubMed

    Galbusera, Fabio; Mietsch, Antje; Schmidt, Hendrik; Wilke, Hans-Joachim; Neidlinger-Wilke, Cornelia

    2013-01-01

    Degeneration of the intervertebral disc may be initiated and supported by impairment of the nutrition processes of the disc cells. The effects of degenerative changes on cell nutrition are, however, only partially understood. In this work, a finite volume model was used to investigate the effect of endplate calcification, water loss, reduction of disc height and cyclic mechanical loading on the sustainability of the disc cell population. Oxygen, lactate and glucose diffusion, production and consumption were modelled with non-linear coupled partial differential equations. Oxygen and glucose consumption and lactate production were expressed as a function of local oxygen concentration, pH and cell density. The cell viability criteria were based on local glucose concentration and pH. Considering a disc with normal water content, cell death was initiated in the centre of the nucleus for oxygen, glucose, and lactate diffusivities in the cartilaginous endplate below 20% of the physiological values. The initial cell population could not be sustained even in the non-calcified endplates when a reduction of diffusion inside the disc due to water loss was modelled. Alterations in the disc shape such as height loss, which shortens the transport route between the nutrient sources and the cells, and cyclic mechanical loads, could enhance cell nutrition processes. PMID:21970697

  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. PMID:27315332

  12. Magnetically induced electrostimulation of human osteoblasts results in enhanced cell viability and osteogenic differentiation.

    PubMed

    Hiemer, Bettina; Ziebart, Josefin; Jonitz-Heincke, Anika; Grunert, Philip Christian; Su, Yukun; Hansmann, Doris; Bader, Rainer

    2016-07-01

    The application of electromagnetic fields to support the bone-healing processes is a therapeutic approach for patients with musculoskeletal disorders. The ASNIS-III s-series screw is a bone stimulation system providing electromagnetic stimulation; however, its influence on human osteoblasts (hOBs) has not been extensively investigated. Therefore, in the present study, the impact of this system on the viability and differentiation of hOBs was examined. We used the ASNIS-III s screw system in terms of a specific experimental test set-up. The ASNIS-III s screw system was used for the application of electromagnetic fields (EMF, 3 mT, 20 Hz) and electromagnetic fields combined with an additional alternating electric field (EMF + EF) (3 mT, 20 Hz, 700 mV). The stimulation of primary hOBs was conducted 3 times per day for 45 min over a period of 72 h. Unstimulated cells served as the controls. Subsequently, the viability, the gene expression of differentiation markers and pro-collagen type 1 synthesis of the stimulated osteoblasts and corresponding controls were investigated. The application of both EMF and EMF + EF using the ASNIS-III s screw system revealed a positive influence on bone cell viability and moderately increased the synthesis of pro-collagen type 1 compared to the unstimulated controls. Stimulation with EMF resulted in a slightly enhanced gene expression of type 1 collagen and osteocalcin; however, stimulation with EMF + EF resulted in a significant increase in alkaline phosphatase (1.4-fold) and osteocalcin (1.6-fold) levels, and a notable increase in the levels of runt-related transcription factor 2 (RUNX-2; 1.54-fold). Our findings demonstrate that stimulation with electromagnetic fields and an additional alternating electric field has a positive influence on hOBs as regards cell viability and the expression of osteoblastic differentiation markers.

  13. 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

  14. Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts

    PubMed Central

    Chen, Joseph C.; Johnson, Brittni A.; Erikson, David W.; Piltonen, Terhi T.; Barragan, Fatima; Chu, Simon; Kohgadai, Nargis; Irwin, Juan C.; Greene, Warner C.; Giudice, Linda C.; Roan, Nadia R.

    2014-01-01

    STUDY QUESTION How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? SUMMARY ANSWER Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. WHAT IS KNOWN ALREADY Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductive tract (FRT), which may participate in promoting reproductive success. STUDY DESIGN, SIZE, DURATION This is a cross sectional study involving control samples versus treatment. SP (pooled from twenty donors) was first tested for dose- and time-dependent cytotoxic effects on eEC and eSF (n = 4). As exposure of eEC or eSF to 1% SP for 6 h proved to be non-toxic, a second set of eEC/eSF samples (n = 4) was treated under these conditions for transcriptome, protein and functional analysis. With a third set of samples (n = 3), we further compared the transcriptional response of the cells to SP versus fresh semen. PARTICIPANTS/MATERIALS, SETTING, METHODS eEC and eSF were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. RNA was isolated and processed for microarray studies to analyze global transcriptomic changes. Secreted factors in conditioned media from SP-treated cells were analyzed by Luminex and for the ability to stimulate migration of CD14+ monocytes and CD4+ T cells. MAIN RESULTS AND THE ROLE OF CHANCE Pathway identifications were determined using the Z-scoring system in Ingenuity Pathways Analysis (Z scores ≥|1.5|). SP induced transcriptomic changes (P < 0.05) associated with promoting leukocyte and endothelial cell recruitment, and proliferation of eEC and eSF. Cell viability pathways were induced, while those associated with cell death were suppressed (P < 0.05). SP and fresh semen induced

  15. Ex vivo non-invasive assessment of cell viability and proliferation in bio-engineered whole organ constructs.

    PubMed

    Ren, Xi; Tapias, Luis F; Jank, Bernhard J; Mathisen, Douglas J; Lanuti, Michael; Ott, Harald C

    2015-06-01

    Decellularized organ scaffolds allow whole organ regeneration and study of cell behavior in three-dimensional culture conditions. Cell viability within the bio-engineered organ constructs is an essential parameter reflecting the performance of participating cells during long-term ex vivo culture, and is a prerequisite for further functional performance. Resazurin-based redox metabolic assays have been used to monitor cell viability in both two- and three-dimensional cell cultures. Here we developed a method for monitoring cell viability and proliferation in bio-engineered organ constructs using a resazurin perfusion assay. This method allows non-invasive, repetitive and rapid estimation of viable cell numbers during long-term ex vivo culture. As a proof-of-principle, we assessed the performance of two different endothelial sources and the impact of different perfusion programs on endothelial viability after re-endothelialization of decellularized lung scaffolds. The resazurin-based perfusion assay revealed changes in endothelial viability and proliferation during long-term ex vivo culture, which was consistent with histological assessment at different time points. Finally, we showed that this method could be used for assessment of proliferation and cytotoxicity after pharmacological treatment on a three-dimensional non-small cell lung cancer culture model.

  16. Modeling the developmental neurotoxicity of nicotine in vitro: cell acquisition, growth and viability in PC12 cells.

    PubMed

    Abreu-Villaça, Yael; Seidler, Frederic J; Qiao, Dan; Slotkin, Theodore A

    2005-02-01

    Although nicotine is a developmental neurotoxicant, it also can exert neuroprotective effects. In the current study, we used PC12 cells to determine the developmental phases in which these disparate actions are expressed and to compare the concentrations required for each. In undifferentiated cells, 1 or 10 microM nicotine had little or no effect on cell number (assessed by measuring DNA) but exerted positive trophic actions, characterized by transient enhancement of cell growth (increased total protein/DNA ratio) and persistent enhancement of cell viability (decreased proportions of cells stained with trypan blue). When differentiation was initiated with nerve growth factor, nicotine elicited a different spectrum of actions, with decreases in cell number, impaired neuritic outgrowth (reduced ratio of membrane/total protein) and weakened viability. In either undifferentiated or differentiating cells, nicotine increased lipid peroxidation (determined as thiobarbituric acid reactive species), providing evidence for oxidative damage. Our results indicate that nicotine exerts positive trophic effects primarily on undifferentiated cells, whereas with differentiation the effects undergo a transition to neurotoxicity. These findings support the view that the neurodevelopmental actions of nicotine depend not only upon the magnitude and duration of the exposure, but most importantly on the developmental stage (e.g., differentiation state) in which exposure occurs. PMID:15707677

  17. Impact of Axis of GHRH and GHRH Receptor on Cell Viability and Apoptosis of the Placental Choriocarcinoma Cell Line.

    PubMed

    Liu, A-X; Zhang, D; Zhu, Y-M; Gao, H-J; Jiang, J-Y; Hu, X-L; Lv, P-P; Leung, P C K; Huang, H-F

    2016-01-01

    Although GHRH and GHRH-R are recognized as key factors in placental development, little is known about the mechanism(s) of the regulation in trophoblastic cells during placental development. The objective of this study is to determine the potential relationship between the expression levels of GHRH-R and the placental and JEG-3 cell function. Furthermore, we aim to investigate the downstream pathways of GHRH/GHRH-R axis in the control of the JEG-3 cell viability and apoptosis. In this study, we detected the expression pattern of GHRH-R in human chorionic villous tissues and JEG-3 cell. Then, we evaluated the effects of GHRH/GHRH-R and the downstream pathways by using GHRH antagonist (JMR-132) on JEG-3 cell. Our present study found the expressions of GHRH-R in placental villous tissues and JEG-3 cell, and the expression levels of GHRH-R was significantly lower in villous tissues of early pregnancy loss when compared to normal controls. JMR-132 inhibited cellular viability and induced apoptosis in JEG-3 cell in a time and dosedependent manners through activation of caspase-3, p38, and p53, as well as inhibition of phosphorylation of Akt. Interestingly, ER stress markers such as GRP78, ubiquitinated proteins and phospho-eIF2α were significantly increased in JEG-3 cell after being treated with JMR-132. Conversely, pretreated with salubrinal (a selective inhibition of protein phosphatase 1-mediated eIF2α dephosphorylation), JEG-3 cells were rescued from JMR-132-mediated cell growth inhibition, and abolished JMR-132-induced cleaved caspase-3, CHOP, phospho-p53, and ubiquitinated proteins accumulation. Knockdown of endogenous GHRH-R significantly abolished the JMR-132-induced cleaved caspase-3 and activation of p38. In conclusion, our results, for the first time, demonstrated the expression levels of GHRH-R were closely related to the placental function. Inhibition of GHRH-R by using GHRH antagonist in JEG-3 cell may reduce cell viability and induce apoptosis through

  18. A novel dual luciferase assay for the simultaneous monitoring of HIV infection and cell viability.

    PubMed

    Mitsuki, Yu-Ya; Yamamoto, Takuya; Mizukoshi, Fuminori; Momota, Masatoshi; Terahara, Kazutaka; Yoshimura, Kazuhisa; Harada, Shigeyoshi; Tsunetsugu-Yokota, Yasuko

    2016-05-01

    Human immunodeficiency virus type 1 (HIV-1) reporter cell lines are critical tools for drug development. However, one disadvantage of HIV-1 reporter cell lines is that reductions in reporter gene activity need to be normalized to cytotoxicity, i.e., live cell numbers. Here, we developed a dual luciferase assay based on a R. reniformis luciferase (hRLuc)-expressing R5-type HIV-1 (NLAD8-hRLuc) and a CEM cell line expressing CCR5 and firefly luciferase (R5CEM-FiLuc). The NLAD8-hRLuc reporter virus was replication competent in peripheral blood mononuclear cells. The level of hRLuc was correlated with p24 antigen levels (p<0.001, R=0.862). The target cell line, R5CEM-FiLuc, stably expressed the firefly luciferase (FiLuc) reporter gene and allowed the simultaneous monitoring of compound cytotoxicity. The dual reporter assay combining a NLAD8-hRLuc virus with R5CEM-FiLuc cells permitted the accurate determination of drug susceptibility for entry, reverse transcriptase, integrase, and protease inhibitors at different multiplicities of infection. This dual reporter assay provides a rapid and direct method for the simultaneous monitoring of HIV infection and cell viability. PMID:26898957

  19. QSAR model for predicting cell viability of human embryonic kidney cells exposed to SiO₂ nanoparticles.

    PubMed

    Manganelli, Serena; Leone, Caterina; Toropov, Andrey A; Toropova, Alla P; Benfenati, Emilio

    2016-02-01

    A predictive model for the viability (%) of cultured human embryonic kidney cells (HEK293) exposed to 20 and 50 nm silica nanoparticles was built using 'optimal descriptors' as mathematical functions of size, concentration and exposure time. The calculation was carried out with CORAL software (http://www.insilico.eu/coral/) on five random splits of combined systems (particle size-particle concentration-cell exposure time) into training, calibration, and validation sets. The R(2) values of the best models were above 0.68. The average statistical quality of the model for the viability (%) of HEK293 exposed to different concentrations of silica nanoparticles measured by MTT assay is satisfactory. PMID:26439516

  20. Pressurized liquid extraction of Aglaonema sp. iminosugars: Chemical composition, bioactivity, cell viability and thermal stability.

    PubMed

    Rodríguez-Sánchez, S; Martín-Ortiz, A; Carrero-Carralero, C; Ramos, S; Sanz, M L; Soria, A C

    2016-08-01

    Pressurized liquid extraction of Aglaonema sp. iminosugars has been optimized. A single cycle under optimal conditions (80mg, 100°C, 2min) was enough to extract ⩾96% of most iminosugars. Further incubation with Saccharomyces cerevisiae for 5h removed coextracted interfering low molecular weight carbohydrates from extracts of different Aglaonema cultivars. A complete characterization of these extracts was carried out by gas chromatography-mass spectrometry: three iminosugars were tentatively identified for the first time; α-homonojirimycin and 2,5-dideoxy-2,5-imino-d-mannitol were the major iminosugars determined. α-Glucosidase inhibition activity, cell viability and thermal stability of Aglaonema extracts were also evaluated. Extracts with IC50 for α-glucosidase activity in the 0.010-0.079mgmL(-1) range showed no decrease of Caco-2 cell viability at concentrations lower than 125μgmL(-1) and were stable at 50°C for 30days. These results highlight the potential of Aglaonema extracts as a source of bioactives to be used as functional ingredients.

  1. In vitro Effects of Lemongrass Extract on Candida albicans Biofilms, Human Cells Viability, and Denture Surface.

    PubMed

    Madeira, Petrus L B; Carvalho, Letícia T; Paschoal, Marco A B; de Sousa, Eduardo M; Moffa, Eduardo B; da Silva, Marcos A Dos Santos; Tavarez, Rudys de Jesus Rodolfo; Gonçalves, Letícia M

    2016-01-01

    The purpose of this study was to investigate whether immersion of a denture surface in lemongrass extract (LGE) has effects on C. albicans biofilms, human cell viability and denture surface. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were performed for LGE against C. albicans. For biofilm analysis, discs were fabricated using a denture acrylic resin with surface roughness standardization. C. albicans biofilms were developed on saliva-coated discs, and the effects of LGE at MIC, 5XMIC, and 10XMIC were investigated during biofilm formation and after biofilm maturation. Biofilms were investigated for cell counting, metabolic activity, and microscopic analysis. The cytotoxicity of different concentrations of LGE to peripheral blood mononuclear cells (PBMC) was analyzed using MTT. The effects of LGE on acrylic resin were verified by measuring changes in roughness, color and flexural strength after 28 days of immersion. Data were analyzed by ANOVA, followed by a Tukey test at a 5% significance level. The minimal concentration of LGE required to inhibit C. albicans growth was 0.625 mg/mL, while MFC was 2.5 mg/mL. The presence of LGE during biofilm development resulted in a reduction of cell counting (p < 0.05), which made the MIC sufficient to reduce approximately 90% of cells (p < 0.0001). The exposure of LGE after biofilm maturation also had a significant antifungal effect at all concentrations (p < 0.05). When compared to the control group, the exposure of PBMC to LGE at MIC resulted in similar viability (p > 0.05). There were no verified differences in color perception, roughness, or flexural strength after immersion in LGE at MIC compared to the control (p > 0.05). It could be concluded that immersion of the denture surface in LGE was effective in reducing C. albicans biofilms with no deleterious effects on acrylic properties at MIC. MIC was also an effective and safe concentration for use. PMID:27446818

  2. In vitro Effects of Lemongrass Extract on Candida albicans Biofilms, Human Cells Viability, and Denture Surface

    PubMed Central

    Madeira, Petrus L. B.; Carvalho, Letícia T.; Paschoal, Marco A. B.; de Sousa, Eduardo M.; Moffa, Eduardo B.; da Silva, Marcos A. dos Santos; Tavarez, Rudys de Jesus Rodolfo; Gonçalves, Letícia M.

    2016-01-01

    The purpose of this study was to investigate whether immersion of a denture surface in lemongrass extract (LGE) has effects on C. albicans biofilms, human cell viability and denture surface. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were performed for LGE against C. albicans. For biofilm analysis, discs were fabricated using a denture acrylic resin with surface roughness standardization. C. albicans biofilms were developed on saliva-coated discs, and the effects of LGE at MIC, 5XMIC, and 10XMIC were investigated during biofilm formation and after biofilm maturation. Biofilms were investigated for cell counting, metabolic activity, and microscopic analysis. The cytotoxicity of different concentrations of LGE to peripheral blood mononuclear cells (PBMC) was analyzed using MTT. The effects of LGE on acrylic resin were verified by measuring changes in roughness, color and flexural strength after 28 days of immersion. Data were analyzed by ANOVA, followed by a Tukey test at a 5% significance level. The minimal concentration of LGE required to inhibit C. albicans growth was 0.625 mg/mL, while MFC was 2.5 mg/mL. The presence of LGE during biofilm development resulted in a reduction of cell counting (p < 0.05), which made the MIC sufficient to reduce approximately 90% of cells (p < 0.0001). The exposure of LGE after biofilm maturation also had a significant antifungal effect at all concentrations (p < 0.05). When compared to the control group, the exposure of PBMC to LGE at MIC resulted in similar viability (p > 0.05). There were no verified differences in color perception, roughness, or flexural strength after immersion in LGE at MIC compared to the control (p > 0.05). It could be concluded that immersion of the denture surface in LGE was effective in reducing C. albicans biofilms with no deleterious effects on acrylic properties at MIC. MIC was also an effective and safe concentration for use. PMID:27446818

  3. Inhibition of mitochondrial 2-oxoglutarate dehydrogenase impairs viability of cancer cells in a cell-specific metabolism-dependent manner.

    PubMed

    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

    2016-05-01

    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

  4. 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

  5. Activation of salt-inducible kinase 2 promotes the viability of peritoneal mesothelial cells exposed to stress of peritoneal dialysis

    PubMed Central

    Wang, H-H; Lin, C-Y; Su, S-H; Chuang, C-T; Chang, Y-L; Lee, T-Y; Lee, S-C; Chang, C-J

    2016-01-01

    Maintaining mesothelial cell viability is critical to long-term successful peritoneal dialysis (PD) treatment. To clarify the viability mechanism of peritoneal mesothelial cells under PD solutions exposure, we examined the mechanisms of cellular response to this stress conditions. Here we report that the proteasome activity is inhibited when treated with PD solutions. Proteasome inhibition-mediated activation of salt-inducible kinase 2 (SIK2), an endoplasmic reticulum-resident protein, is important for mesothelial cell viability. SIK2 is mobilized to promote autophagy and protect the cells from apoptosis under PD solution or MG132 treatment. Immunofluorescence staining showed that SIK2 is colocalized with LC3B in the autophagosomes of mesothelial cells treated with PD solution or derived from patients undergoing PD treatment. SIK2 activation is likely via a two-step mechanism, upstream kinases relieving the autoinhibitory conformation of SIK2 molecule followed by autophosphorylation of Thr175 and activation of kinase activity. These results suggest that activation of SIK2 is required for the cell viability when proteasome activity is inhibited by PD solutions. Maintaining or boosting the activity of SIK2 may promote peritoneal mesothelial cell viability and evolve as a potential therapeutic target for maintaining or restoring peritoneal membrane integrity in PD therapy. PMID:27441650

  6. Viability study of HL60 cells in contact with commonly used microchip materials.

    PubMed

    Wolbers, Floor; ter Braak, Paul; Le Gac, Severine; Luttge, Regina; Andersson, Helene; Vermes, Istvan; van den Berg, Albert

    2006-12-01

    This paper presents a study in which different commonly used microchip materials (silicon oxide, borosilicate glass, and PDMS) were analyzed for their effect on human promyelocytic leukemic (HL60) cells. Copper-coated silicon was analyzed for its toxicity and therefore served as a positive control. With quantitative PCR, the expression of the proliferation marker Cyclin D1 and the apoptosis marker tissue transglutaminase were measured. Flow cytometry was used to analyze the distribution through the different phases of the cell cycle (propidium iodide, PI) and the apoptotic cascade (Annexin V in combination with PI). All microchip materials, with the exception of Cu, appeared to be suitable for HL60 cells, showing a ratio apoptosis/proliferation (R(ap)) comparable to materials used in conventional cell culture (polystyrene). These results were confirmed with cell cycle analysis and apoptosis studies. Precoating the microchip material surfaces with serum favor the proliferation, as demonstrated by a lower R(ap) as compared to uncoated surfaces. The Cu-coated surface appeared to be toxic for HL60 cells, showing over 90% decreased viability within 24 h. From these results, it can be concluded that the chosen protocol is suitable for selection of the cell culture material, and that the most commonly used microchip materials are compatible with HL60 culturing. PMID:17124709

  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. PMID:25193657

  8. Mycolic Acid Cyclopropanation is Essential for Viability, Drug Resistance, and Cell Wall Integrity of Mycobacterium tuberculosis

    SciTech Connect

    Barkan, Daniel; Liu, Zhen; Sacchettini, James C.; Glickman, Michael S.

    2009-12-01

    Mycobacterium tuberculosis infection remains a major global health problem complicated by escalating rates of antibiotic resistance. Despite the established role of mycolic acid cyclopropane modification in pathogenesis, the feasibility of targeting this enzyme family for antibiotic development is unknown. We show through genetics and chemical biology that mycolic acid methyltransferases are essential for M. tuberculosis viability, cell wall structure, and intrinsic resistance to antibiotics. The tool compound dioctylamine, which we show acts as a substrate mimic, directly inhibits the function of multiple mycolic acid methyltransferases, resulting in loss of cyclopropanation, cell death, loss of acid fastness, and synergistic killing with isoniazid and ciprofloxacin. These results demonstrate that mycolic acid methyltransferases are a promising antibiotic target and that a family of virulence factors can be chemically inhibited with effects not anticipated from studies of each individual enzyme.

  9. Aquaporin-1 down regulation associated with inhibiting cell viability and inducing apoptosis of human lens epithelial cells

    PubMed Central

    Zheng, Hong-Hua; Xu, Guo-Xing; Guo, Jian; Fu, Li-Cheng; Yao, Yao

    2016-01-01

    AIM To investigate the role of Aquaporin-1 (AQP-1) in lens epithelial cells (LECs) and its potential target genes. AQP-1 is specifically expressed in LECs of eyes and is significant for lens homeostasis and transparency maintenance. Herein, AQP-1 expression in LECs was investigated to evaluate its influence on cell survival in association with its potential role in cataract formation. METHODS LECs were transfected with lentivirus carrying AQP-1 small interfering RNA (siRNA). Real-time polymerase chain reaction (PCR) and Western blotting were conducted to detect AQP-1 expression in LECs from different groups. Meanwhile, cell counting kit-8 (CCK-8) assay and flow cytometry were performed to measure LEC proliferation and apoptosis, respectively. RESULTS AQP-1 expression was significantly reduced in LECs, both at mRNA and protein levels (P<0.05), after siRNA treatment. Decreased cell viability was detected by CCK-8 assay in LECs with siRNA interference, compared to control cells (P<0.05). The apoptosis rate significantly increased in cells after siRNA interference (P<0.05). CONCLUSION The decreased cell viability following AQP-1 down regulation is largely due to its induction of apoptosis of LECs. AQP-1 reduction might lead to changes of physiological functions in LECs, which might be associated with the occurrence and development of cataracts. PMID:26949604

  10. A Novel Small Molecular STAT3 Inhibitor, LY5, Inhibits Cell Viability, Cell Migration, and Angiogenesis in Medulloblastoma Cells*

    PubMed Central

    Xiao, Hui; Bid, Hemant Kumar; Jou, David; Wu, Xiaojuan; Yu, Wenying; Li, Chenglong; Houghton, Peter J.; Lin, Jiayuh

    2015-01-01

    Signal transducers and activators of transcription 3 (STAT3) signaling is persistently activated and could contribute to tumorigenesis of medulloblastoma. Numerous studies have demonstrated that inhibition of the persistent STAT3 signaling pathway results in decreased proliferation and increased apoptosis in human cancer cells, indicating that STAT3 is a viable molecular target for cancer therapy. In this study, we investigated a novel non-peptide, cell-permeable small molecule, named LY5, to target STAT3 in medulloblastoma cells. LY5 inhibited persistent STAT3 phosphorylation and induced apoptosis in human medulloblastoma cell lines expressing constitutive STAT3 phosphorylation. The inhibition of STAT3 signaling by LY5 was confirmed by down-regulating the expression of the downstream targets of STAT3, including cyclin D1, bcl-XL, survivin, and micro-RNA-21. LY5 also inhibited the induction of STAT3 phosphorylation by interleukin-6 (IL-6), insulin-like growth factor (IGF)-1, IGF-2, and leukemia inhibitory factor in medulloblastoma cells, but did not inhibit STAT1 and STAT5 phosphorylation stimulated by interferon-γ (IFN-γ) and EGF, respectively. In addition, LY5 blocked the STAT3 nuclear localization induced by IL-6, but did not block STAT1 and STAT5 nuclear translocation mediated by IFN-γ and EGF, respectively. A combination of LY5 with cisplatin or x-ray radiation also showed more potent effects than single treatment alone in the inhibition of cell viability in human medulloblastoma cells. Furthermore, LY5 demonstrated a potent inhibitory activity on cell migration and angiogenesis. Taken together, these findings indicate LY5 inhibits persistent and inducible STAT3 phosphorylation and suggest that LY5 is a promising therapeutic drug candidate for medulloblastoma by inhibiting persistent STAT3 signaling. PMID:25313399

  11. 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.

  12. 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. PMID:27069145

  13. 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

  14. Perivascular Stem Cells Diminish Muscle Atrophy and Retain Viability in a Rotator Cuff Tear Model

    PubMed Central

    Eliasberg, Claire; Jensen, Andrew; Dar, Ayelet; Kowalski, Tomasz J.; Murray, Iain; Khan, Adam Z.; Natsuhara, Kyle; Garagozlo, Cameron; McAllister, David R.; Petrigliano, Frank A.

    2016-01-01

    Objectives: Rotator cuff tears (RCTs) are a common cause of shoulder pain and often necessitate surgical repair. Muscle changes including atrophy, fibrosis, and fatty degeneration can develop after RCTs, which may compromise surgical repair and clinical outcomes. Lipoaspirate-derived human perivascular stem cells (PSCs) have demonstrated myogenic and angiogenic potential in other small animal models of muscle injury. We hypothesized that the administration of PSCs following massive RCTs may help to diminish these muscle changes in a small animal model. Methods: A total of 90 immunodeficient mice were used (15 groups, N=6). Each was assigned to one of three surgical groups: i) sham, ii) supraspinatus and infraspinatus tendon transection (TT), or iii) TT and suprascapular nerve denervation (TT+DN). PSCs were harvested from human lipoaspirate and sorted using fluorescence-activated cell sorting into small blood vessel residing pericytes (CD146+ CD34- CD45- CD31-) and large blood perivascular adventitial cells (CD146- CD34+ CD45- CD31-). Mice received either a) no injection, b) saline injection, c) pericyte injection, or d) adventitial cell injection at the time of the index procedure or at two weeks following index surgery. The supraspinatus muscles were harvested six weeks after the index procedure. Muscle atrophy was assessed by measuring percent wet muscle weight change for each sample. Muscle fiber cross-sectional area (CSA), fibrosis, and fatty degeneration were analyzed using Image J™. Additionally, pericytes and adventitial cells were transduced with a luciferase-containing construct. Animals were given injections of luciferin and imaged using IVIS to track in vivo bioluminescence following injections to assess cell viability. Results: Treatment with PSC injection after TT resulted in less wet weight loss and greater muscle fiber CSA than control groups (P<0.05). The TT+DN groups treated with PSC injections two weeks post-op also had less muscle weight loss

  15. The Effect of Saturated Fatty Acids on Methanogenesis and Cell Viability of Methanobrevibacter ruminantium

    PubMed Central

    Zhou, Xuan; Meile, Leo; Kreuzer, Michael; Zeitz, Johanna O.

    2013-01-01

    Saturated fatty acids (SFAs) are known to suppress ruminal methanogenesis, but the underlying mechanisms are not well known. In the present study, inhibition of methane formation, cell membrane permeability (potassium efflux), and survival rate (LIVE/DEAD staining) of pure ruminal Methanobrevibacter ruminantium (DSM 1093) cell suspensions were tested for a number of SFAs. Methane production rate was not influenced by low concentrations of lauric (C12; 1 μg/mL), myristic (C14; 1 and 5 μg/mL), or palmitic (C16; 3 and 5 μg/mL) acids, while higher concentrations were inhibitory. C12 and C14 were most inhibitory. Stearic acid (C18), tested at 10–80 μg/mL and ineffective at 37°C, decreased methane production rate by half or more at 50°C and ≥50 μg/mL. Potassium efflux was triggered by SFAs (C12 = C14 > C16 > C18 = control), corroborating data on methane inhibition. Moreover, the exposure to C12 and C14 decreased cell viability to close to zero, while 40% of control cells remained alive after 24 h. Generally, tested SFAs inhibited methanogenesis, increased cell membrane permeability, and decreased survival of M. ruminantium in a dose- and time-dependent way. These results give new insights into how the methane suppressing effect of SFAs could be mediated in methanogens. PMID:23710130

  16. GDP-mannose pyrophosphorylase is essential for cell wall integrity, morphogenesis and viability of Aspergillus fumigatus.

    PubMed

    Jiang, Hechun; Ouyang, Haomiao; Zhou, Hui; Jin, Cheng

    2008-09-01

    GDP-mannose pyrophosphorylase (GMPP) catalyses the synthesis of GDP-mannose, which is the precursor for the mannose residues in glycoconjugates, using mannose 1-phosphate and GTP as substrates. Repression of GMPP in yeast leads to phenotypes including cell lysis, defective cell wall, and failure of polarized growth and cell separation. Although several GMPPs have been isolated and characterized in filamentous fungi, the physiological consequences of their actions are not clear. In this study, Afsrb1, which is a homologue of yeast SRB1/PSA1/VIG9, was identified in the Aspergillus fumigatus genome. The Afsrb1 gene was expressed in Escherichia coli, and recombinant AfSrb1 was functionally confirmed as a GMPP. By the replacement of the native Afsrb1 promoter with an inducible Aspergillus nidulans alcA promoter, the conditional inactivation mutant strain YJ-gmpp was constructed. The presence of 3 % glucose completely blocked transcription of P(alcA)-Afsrb1, and was lethal to strain YJ-gmpp. Repression of Afsrb1 expression in strain YJ-gmpp led to phenotypes including hyphal lysis, defective cell wall, impaired polarity maintenance, and branching site selection. Also, rapid germination and reduced conidiation were documented. However, in contrast to yeast, strain YJ-gmpp retained the ability to direct polarity establishment and septation. Our results showed that the Afsrb1 gene is essential for cell wall integrity, morphogenesis and viability of Aspergillus fumigatus.

  17. Effects of selective somatostatin analogs and cortistatin on cell viability in cultured human non-functioning pituitary adenomas.

    PubMed

    Padova, H; Rubinfeld, H; Hadani, M; Cohen, Z R; Nass, D; Taylor, J E; Culler, M D; Shimon, I

    2008-05-14

    Clinically "non-functioning" human pituitary adenomas (NFPA) constitute about 35% of pituitary adenomas. Somatostatin receptors (SSTR) expression in these adenomas has previously been described both in vitro and in vivo, without evidence for a correlation with tumor volume or the therapeutic efficacy of somatostatin analogs. This study was performed on 13 surgically removed pituitary macroadenomas, diagnosed before surgery as "non-functioning". In addition, 3 growth hormone (GH)-secreting adenomas served as controls. A specimen from each tumor was dispersed and digested to isolate and culture the tumor cells, and the in vitro effects of SSTR2 and SSTR5 selective analogs and Cortistatin (CST) (100nM) on cell viability were studied. The quantity of viable cells was estimated using the XTT method. RNA purification of tumor samples and subsequent RT-PCR studies for SSTR2 and SSTR5 expression were performed. Somatostatin analog with high affinity for SSTR2 reduced cell viability by 20-80% in 8 of 13 NFPAs studied, all expressing the SSTR2. The inhibitory effect on cell viability of SSTR5-selective analog was 15-80% in 10 of 13 NFPAs studied, all but three expressing the SSTR5. CST, however, effectively reduced cell viability in only 6 NFPAs. Cell viability was inhibited by all peptides studied in 2 out of 3 GH-secreting adenomas, expressing both receptors. The third adenoma responded to SSTR2 analog and expressed only SSTR2. These results suggest the involvement of SSTR2 and SSTR5 in the anti-proliferative effects of somatostatin; however, CST is less potent in reducing cell viability in these tumors. PMID:18276067

  18. 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

  19. Essential oils--their antimicrobial activity against Escherichia coli and effect on intestinal cell viability.

    PubMed

    Fabian, Dusan; Dusan, Fabian; Sabol, Marián; Marián, Sabol; Domaracká, Katarína; Katarína, Domaracká; Bujnáková, Dobroslava; Dobroslava, Bujnáková

    2006-12-01

    Essential oils are known to possess antimicrobial activity against a wide spectrum of bacteria. The main objective of this study was to evaluate possible harmful effects of four commonly used essential oils and their major components on intestinal cells. Antimicrobial activity of selected plant extracts against enteroinvasive Escherichia coli was dose dependent. However, doses of essential oils with the ability to completely inhibit bacterial growth (0.05%) showed also relatively high cytotoxicity to intestinal-like cells cultured in vitro. Lower doses of essential oils (0.01%) had only partial antimicrobial activity and their damaging effect on Caco-2 cells was only modest. Cell death assessment based on morphological and viability staining followed by fluorescence microscopy showed that essential oils of cinnamon and clove and their major component eugenol had almost no cytotoxic effect at lower doses. Although essential oil of oregano and its component carvacrol slightly increased the incidence of apoptotic cell death, they showed extensive antimicrobial activity even at lower concentrations. Relatively high cytotoxicity was demonstrated by thyme oil, which increased both apoptotic and necrotic cell death incidence. In contrast, its component thymol showed no cytotoxic effect as well as greatly-reduced ability to inhibit visible growth of the chosen pathogen in the doses used. On the other hand, the addition of all essential oils and their components at lower doses, with the exception of thyme oil, to bacterial suspension significantly reduced the cytotoxic effect of E. coli on Caco-2 cells after 1h culture. In conclusion, it is possible to find appropriate doses of essential oils showing both antimicrobial activity and very low detrimental effect on intestinal cells. PMID:16919909

  20. Hydrochloric acid alters the effect of L-glutamic acid on cell viability in human neuroblastoma cell cultures.

    PubMed

    Croce, Nicoletta; Bernardini, Sergio; Di Cecca, Stefano; Caltagirone, Carlo; Angelucci, Francesco

    2013-07-15

    l-Glutamic acid (l-glutamate) is used to induce excitotoxicity and test neuroprotective compounds in cell cultures. However, because l-glutamate powder is nearly insoluble in water, many manufacturers recommend reconstituting l-glutamate in hydrochloric acid (HCl) prior to successive dilutions. Nevertheless, HCl, even at low concentrations, may alter the pH of the cell culture medium and interfere with cell activity. Thus, the aim of this study was to evaluate whether the reconstitution of l-glutamate powder in HCl alters its capacity to induce neurotoxicity in different human neuroblastoma cell lines. SH-SY5Y, IMR-32 and SK-N-BE(2) cells were exposed to various concentrations of l-glutamate, which was either reconstituted in HCl (1M) or post re-equilibrated to the pH of the culture medium (7.5). After 24 and 48h of incubation, changes in the cell viability of treated versus untreated cells were evaluated. The effect of an identical amount of HCl present in the l-glutamate dilutions on neuroblastoma cell survival was also investigated. Our data showed that the neurotoxicity of glutamate reconstituted in HCl was comparable to that of HCl alone. Moreover, the pH variations induced by glutamate or HCl in the culture medium were similar. When the pH of the glutamate stock solution was re-equilibrated, l-glutamate induced variation in cell viability to a lower extent and after a longer incubation time. This study demonstrated that HCl used to reconstitute l-glutamate powder might alter the effect of glutamate itself in neuroblastoma cell cultures. Thus, this information might be useful to scientists who use l-glutamate to induce excitotoxicity or to test neuroprotective agents.

  1. Overexpression of stress-related genes enhances cell viability and velum formation in Sherry wine yeasts.

    PubMed

    Fierro-Risco, Jesús; Rincón, Ana María; Benítez, Tahía; Codón, Antonio C

    2013-08-01

    Flor formation and flor endurance have been related to ability by Saccharomyces cerevisiae flor yeasts to resist hostile conditions such as oxidative stress and the presence of acetaldehyde and ethanol. Ethanol and acetaldehyde toxicity give rise to formation of reactive oxygen species (ROS) and loss of cell viability. Superoxide dismutases Sod1p and Sod2p and other proteins such as Hsp12p are involved in oxidative stress tolerance. In this study, genes SOD1, SOD2, and HSP12 were overexpressed in flor yeast strains FJF206, FJF414 and B16. In the SOD1 and SOD2 transformant strains superoxide dismutases encoded by genes SOD1 and SOD2 increased their specific activity considerably as a direct result of overexpression of genes SOD1 and SOD2, indirectly, catalase, glutathione reductase, and glutathione peroxidase activities increased too. The HSP12 transformant strains showed higher levels of glutathione peroxidase and reductase activities. These transformant strains showed an increase in intracellular glutathione content, a reduction in peroxidized lipid concentration, and higher resistance to oxidative stress conditions. As a result, flor formation by these strains took place more rapidly than by their parental strains, velum being thicker and with higher percentages of viable cells. In addition, a slight decrease in ethanol and glycerol concentrations, and an increase in acetaldehyde were detected in wines matured under velum formed by transformant strains, as compared to their parental strains. In the industry, velum formed by transformant strains with increased viability may result in acceleration of both metabolism and wine aging, thus reducing time needed for wine maturation. PMID:23553032

  2. Biphasic regulation of InsP3 receptor gating by dual Ca2+ release channel BH3-like domains mediates Bcl-xL control of cell viability

    PubMed Central

    Yang, Jun; Vais, Horia; Gu, Wenen; Foskett, J. Kevin

    2016-01-01

    Antiapoptotic Bcl-2 family members interact with inositol trisphosphate receptor (InsP3R) Ca2+ release channels in the endoplasmic reticulum to modulate Ca2+ signals that affect cell viability. However, the molecular details and consequences of their interactions are unclear. Here, we found that Bcl-xL activates single InsP3R channels with a biphasic concentration dependence. The Bcl-xL Bcl-2 homology 3 (BH3) domain-binding pocket mediates both high-affinity channel activation and low-affinity inhibition. Bcl-xL activates channel gating by binding to two BH3 domain-like helices in the channel carboxyl terminus, whereas inhibition requires binding to one of them and to a previously identified Bcl-2 interaction site in the channel-coupling domain. Disruption of these interactions diminishes cell viability and sensitizes cells to apoptotic stimuli. Our results identify BH3-like domains in an ion channel and they provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP3R that play critical roles in Ca2+ signaling and cell viability. PMID:26976600

  3. Organic-inorganic hybrid material for the cells immobilization: long-term viability mechanism and application in BOD sensors.

    PubMed

    Liu, Ling; Shang, Li; Guo, Shaojun; Li, Dan; Liu, Changyu; Qi, Li; Dong, Shaojun

    2009-10-15

    In this paper, organic-inorganic hybrid material, which is composed of silica and the grafting copolymer of poly (vinyl alcohol) and 4-vinylpyridine (PVA-g-P(4-VP)), was employed to immobilize Trichosporon cutaneum strain 2.570 cells. Cells entrapped into the hybrid material were found to keep a long-term viability. The mechanism of such a long-term viability was investigated by using confocal laser scanning microscopy (CLSM). Our studies revealed that arthroconidia produced in the extracellular material might play an important role in keeping the long-term viability of the immobilized microorganism. After the arthroconidia were activated, an electrochemical biochemical oxygen demand (BOD) sensor based on cell/hybrid material-modified supporting membrane was constructed for verifying the proposed mechanism. The results and insight gained from the present experiments can be widely used to various biosensor designs.

  4. Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines.

    PubMed

    Große-Kreul, Jan; Busch, Maike; Winter, Claudia; Pikos, Stefanie; Stephan, Harald; Dünker, Nicole

    2016-01-01

    Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma. PMID:27626280

  5. Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines.

    PubMed

    Große-Kreul, Jan; Busch, Maike; Winter, Claudia; Pikos, Stefanie; Stephan, Harald; Dünker, Nicole

    2016-01-01

    Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma.

  6. Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines

    PubMed Central

    Winter, Claudia; Pikos, Stefanie; Stephan, Harald; Dünker, Nicole

    2016-01-01

    Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma. PMID:27626280

  7. In vitro viability of human periodontal ligament cells in green tea extract

    PubMed Central

    Ghasempour, Maryam; Moghadamnia, Ali Akbar; Abedian, Zeynab; Amir, Mahdi Pour; Feizi, Farideh; Gharekhani, Samane

    2015-01-01

    Context: Delayed replantation of avulsed teeth may be successful if the majority of periodontal ligament cells (PDL) survive. A proper transport medium is required when immediate replantation is not possible. Green tea extract (GTE) may be effective in preserving the cells because of its special properties. Aims: This study was done to evaluate the potential of GTE in periodontal ligament cells preservation. Materials and Methods: Fifty-four extracted human teeth with closed apices were randomly divided into three groups each with 18 teeth as follow: GTE, water (negative control), and Hank's balanced salt solution (HBSS) (positive control). The specimens were immersed in the media for 1, 3, and 15 hours at 4°C (n = 6) and treated with collagenase 1A for 45 minutes. Cell viability was determined using the trypan blue exclusion technique. Statistical Analysis: Data were analyzed by one-way analysis of variance (ANOVA), post hoc Tukey and paired t-test at significance level of P < 0.05. Results: Means (standard deviation, SD) of viable cells in HBSS, water, and GTE were estimated 348.33 ± 88.49, 101 ± 14.18, and 310.56 ± 56.97 at 1 hours; 273.4 ± 44.80, 64.16 ± 16.44, and 310.2 ± 11.21 at 3 hours; and 373.72 ± 67.81, 14.41 ± 2.88 and 315.24 ± 34.48 at 15 hours; respectively. No significant differences were found between HBSS and GTE at all the time intervals. Both these solutions could preserve the cells more than water significantly. Conclusion: GTE and HBSS were equally effective in preserving the cells and were significantly superior to water. PMID:25657527

  8. Effects of Different Zinc Species on Cellar Zinc Distribution, Cell Cycle, Apoptosis and Viability in MDAMB231 Cells.

    PubMed

    Wang, Yan-hong; Zhao, Wen-jie; Zheng, Wei-juan; Mao, Li; Lian, Hong-zhen; Hu, Xin; Hua, Zi-chun

    2016-03-01

    Intracellular metal elements exist in mammalian cells with the concentration range from picomoles per litre to micromoles per litre and play a considerable role in various biological procedures. Element provided by different species can influence the availability and distribution of the element in a cell and could lead to different biological effects on the cell's growth and function. Zinc as an abundant and widely distributed essential trace element, is involved in numerous and relevant physiological functions. Zinc homeostasis in cells, which is regulated by metallothioneins, zinc transporter/SLC30A, Zrt-/Irt-like proteins/SLC39A and metal-response element-binding transcription factor-1 (MTF-1), is crucial for normal cellular functioning. In this study, we investigated the influences of different zinc species, zinc sulphate, zinc gluconate and bacitracin zinc, which represented inorganic, organic and biological zinc species, respectively, on cell cycle, viability and apoptosis in MDAMB231 cells. It was found that the responses of cell cycle, apoptosis and death to different zinc species in MDAMB231 cells are different. Western blot analysis of the expression of several key proteins in regulating zinc-related transcription, cell cycle, apoptosis, including MTF-1, cyclin B1, cyclin D1, caspase-8 and caspase-9 in treated cells further confirmed the observed results on cell level.

  9. T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death

    PubMed Central

    Chiu, Chang-Fang; Weng, Jing-Ru; Jadhav, Appaso; Wu, Chia-Yung; Sargeant, Aaron M.; Bai, Li-Yuan

    2016-01-01

    T315, an integrin-linked kinase (ILK) inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt) and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy. PMID:27537872

  10. T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death.

    PubMed

    Chiu, Chang-Fang; Weng, Jing-Ru; Jadhav, Appaso; Wu, Chia-Yung; Sargeant, Aaron M; Bai, Li-Yuan

    2016-01-01

    T315, an integrin-linked kinase (ILK) inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt) and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy. PMID:27537872

  11. The effect of modified polysialic acid based hydrogels on the adhesion and viability of primary neurons and glial cells.

    PubMed

    Haile, Yohannes; Berski, Silke; Dräger, Gerald; Nobre, Andrè; Stummeyer, Katharina; Gerardy-Schahn, Rita; Grothe, Claudia

    2008-04-01

    In this study we present the enzymatic and biological analysis of polysialic acid (polySia) based hydrogel in terms of its degradation and cytocompatibility. PolySia based hydrogel is completely degradable by endosialidase enzyme which may avoid second surgery after tissue recovery. Viability assay showed that soluble components of polySia hydrogel did not cause any toxic effect on cultured Schwann cells. Moreover, green fluorescence protein transfected neonatal and adult Schwann cells, neural stem cells and dorsal root ganglionic cells (unlabelled) were seeded on polySia hydrogel modified with poly-L-lysine (Pll), poly-L-ornithine-laminin (porn-laminin) or collagen. Water soluble tetrazolium salt assay revealed that modification of the hydrogel significantly improved cell adhesion and viability. These results infer that polySia based scaffolds in combination with cell adhesion molecules and cells genetically modified to express growth factors would potentially be promising alternative in reconstructive therapeutic strategies. PMID:18255143

  12. The effect of modified polysialic acid based hydrogels on the adhesion and viability of primary neurons and glial cells.

    PubMed

    Haile, Yohannes; Berski, Silke; Dräger, Gerald; Nobre, Andrè; Stummeyer, Katharina; Gerardy-Schahn, Rita; Grothe, Claudia

    2008-04-01

    In this study we present the enzymatic and biological analysis of polysialic acid (polySia) based hydrogel in terms of its degradation and cytocompatibility. PolySia based hydrogel is completely degradable by endosialidase enzyme which may avoid second surgery after tissue recovery. Viability assay showed that soluble components of polySia hydrogel did not cause any toxic effect on cultured Schwann cells. Moreover, green fluorescence protein transfected neonatal and adult Schwann cells, neural stem cells and dorsal root ganglionic cells (unlabelled) were seeded on polySia hydrogel modified with poly-L-lysine (Pll), poly-L-ornithine-laminin (porn-laminin) or collagen. Water soluble tetrazolium salt assay revealed that modification of the hydrogel significantly improved cell adhesion and viability. These results infer that polySia based scaffolds in combination with cell adhesion molecules and cells genetically modified to express growth factors would potentially be promising alternative in reconstructive therapeutic strategies.

  13. 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.

  14. Effects of extracts of Salvadora persica on proliferation and viability of human dental pulp stem cells

    PubMed Central

    Tabatabaei, Fahimeh sadat; Moezizadeh, Maryam; Javand, Fateme

    2015-01-01

    Objectives: Efficacy of an ideal antimicrobial agent depends on its ability to eliminate microorganisms while causing minimal toxicity to host cells. The purpose of this study was to assess the effect of ethanolic and water extracts of Salvadora persica (SP) on proliferation and viability of human dental pulp stem cells (hDPSCs). Materials and Methods: In this in-vitro study, the effects of seven concentrations of ethanolic and water extracts of SP (ranging from 5.75 mg/ml to 0.08 mg/ml) on hDPSCs were evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The results were analyzed using one-way ANOVA and Tukey's post-hoc test. P < 0.05 was considered statistically significant. Results: Water extract of SP only had cytotoxic effect at 5.75 mg/ml concentration; and caused significant cell proliferation at 1.43-0.08 mg/ml concentrations at 24 h (P < 0.05). At 48 h, only 0.17 and 0.08 mg/ml concentrations caused significant cell proliferation (P < 0.05). Ethanolic extract of SP at 5.75-1.43 mg/ml concentrations showed severe cytotoxic effects at 24 and 48 h. Other concentrations had no significant effects on cells (P > 0.05). Conclusion: The highest concentrations of both water and ethanolic extracts of SP had cytotoxic effects on hDPSCs. Water extract of SP has favorable effects on cell proliferation at specific concentrations in a time-dependent manner. PMID:26180418

  15. Formation of size-controllable spheroids using gingiva-derived stem cells and concave microwells: Morphology and viability tests

    PubMed Central

    LEE, SUNG-IL; YEO, SEONG-IL; KIM, BO-BAE; KO, YOUNGKYUNG; PARK, JUN-BEOM

    2016-01-01

    Human mesenchymal stem cells have previously been isolated and characterized from the gingiva, and gingiva-derived stem cells have been applied for tissue engineering purposes. The present study was performed to generate size-controllable stem cell spheroids using concave microwells. Gingiva-derived stem cells were isolated, and the stem cells of 1×105 (group A) or 2×105 (group B) cells were seeded in polydimethylsiloxane-based, concave micromolds with 600 µm diameters. The morphology of the microspheres was viewed under an inverted microscope, and the changes in the diameter and cell viability were analyzed. The gingiva-derived stem cells formed spheroids in the concave microwells. The diameters of the spheroids were larger in group A compared to group B. No significant changes in shape or diameter were noted with increases in incubation time. Cell viability was higher in group B at each time point when compared with group A. Within the limits of the study, the size-controllable stem cell spheroids could be generated from gingival cells using microwells. The shape of the spheroids and their viability were clearly maintained during the experimental periods. PMID:26870343

  16. Oxidative Stress and DNA Lesions: The Role of 8-Oxoguanine Lesions in Trypanosoma cruzi Cell Viability

    PubMed Central

    Aguiar, Pedro H. N.; Furtado, Carolina; Repolês, Bruno M.; Ribeiro, Grazielle A.; Mendes, Isabela C.; Peloso, Eduardo F.; Gadelha, Fernanda R.; Macedo, Andrea M.; Franco, Glória R.; Pena, Sérgio D. J.; Teixeira, Santuza M. R.; Vieira, Leda Q.; Guarneri, Alessandra A.; Andrade, Luciana O.; Machado, Carlos R.

    2013-01-01

    The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability. PMID:23785540

  17. Effect of STI-571 (imatinib mesylate) in combination with retinoic acid and {gamma}-irradiation on viability of neuroblastoma cells

    SciTech Connect

    Roessler, Jochen . E-mail: jochen.roessler@uniklinik-freiburg.de; Zambrzycka, Izabella; Lagodny, Jeanette; Kontny, Udo; Niemeyer, Charlotte Marie

    2006-04-21

    Neuroblastoma (NB) expresses the tyrosine kinase receptors c-Kit, PDGFR-{alpha} and -{beta}-targets for STI-571.We investigated a possible combination therapy of STI-571 with retinoic acid (RA) and {gamma}-irradiation on NB cell viability in vitro. Expression of tyrosine kinase receptors and their ligands was examined in 6 NB cell lines by RT-PCR and FACS. The effect on cell viability was determined by MTT assay. Cell viability of all 6 NB cell lines was significantly inhibited after treatment with 20 {mu}M STI-571 for 72 h, two cell lines responding already to 10 {mu}M. Cell lines responded irrespective of their mRNA status or cell surface expression of c-Kit, PDGFR-{alpha} and -{beta}. Co-incubation with 9-cis RA sensitized cells to the inhibitory effects of STI-571. However, pre-treatment with 9-cis RA resulted in resistance of NB cell lines to STI-571 and {gamma}-irradiation. Treatment of NB with STI-571 in combination with 9-cis RA might be a therapeutic strategy for patients in consolidation therapy who have completed {gamma}-irradiation therapy.

  18. 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

  19. The effects of arginine glutamate, a promising excipient for protein formulation, on cell viability: Comparisons with NaCl.

    PubMed

    Kheddo, Priscilla; Golovanov, Alexander P; Mellody, Kieran T; Uddin, Shahid; van der Walle, Christopher F; Dearman, Rebecca J

    2016-06-01

    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

  20. Combination of carbonic anhydrase inhibitor, acetazolamide, and sulforaphane, reduces the viability and growth of bronchial carcinoid cell lines

    PubMed Central

    2013-01-01

    Background Bronchial carcinoids are pulmonary neuroendocrine cell-derived tumors comprising typical (TC) and atypical (AC) malignant phenotypes. The 5-year survival rate in metastatic carcinoid, despite multiple current therapies, is 14-25%. Hence, we are testing novel therapies that can affect the proliferation and survival of bronchial carcinoids. Methods In vitro studies were used for the dose–response (AlamarBlue) effects of acetazolamide (AZ) and sulforaphane (SFN) on clonogenicity, serotonin-induced growth effect and serotonin content (LC-MS) on H-727 (TC) and H-720 (AC) bronchial carcinoid cell lines and their derived NOD/SCID mice subcutaneous xenografts. Tumor ultra structure was studied by electron microscopy. Invasive fraction of the tumors was determined by matrigel invasion assay. Immunohistochemistry was conducted to study the effect of treatment(s) on proliferation (Ki67, phospho histone-H3) and neuroendocrine phenotype (chromogranin-A, tryptophan hydroxylase). Results Both compounds significantly reduced cell viability and colony formation in a dose-dependent manner (0–80 μM, 48 hours and 7 days) in H-727 and H-720 cell lines. Treatment of H-727 and H-720 subcutaneous xenografts in NOD/SCID mice with the combination of AZ + SFN for two weeks demonstrated highly significant growth inhibition and reduction of 5-HT content and reduced the invasive capacity of H-727 tumor cells. In terms of the tumor ultra structure, a marked reduction in secretory vesicles correlated with the decrease in 5-HT content. Conclusions The combination of AZ and SFN was more effective than either single agent. Since the effective doses are well within clinical range and bioavailability, our results suggest a potential new therapeutic strategy for the treatment of bronchial carcinoids. PMID:23927827

  1. Cell viability of bovine spermatozoa subjected to DNA electroporation and DNAse I treatment.

    PubMed

    Cavalcanti, Paulo Varoni; Milazzotto, Marcella Pecora; Simões, Renata; Nichi, Marcilio; de Oliveira Barros, Flavia Regina; Visintin, Jose Antonio; Assumpção, Mayra Elena Ortiz D'Avila

    2016-04-15

    Many mechanisms involved in sperm-mediated gene transfer (SMGT) are still unknown. It is still a matter of debate whether exogenous DNA fragments incorporated by the embryo are originated from those bound to the sperm membrane or by those that penetrated the intracellular compartment. In an attempt to elucidate the transmission mechanism of exogenous DNA molecules by sperm, some authors suggested a treatment with DNAse I to remove DNA molecules outside the sperm. But little is known regarding the effects of DNAse I treatment on sperm viability and its impact on sperm organelles. An important aspect of the SMGT technique is the amount of exogenous DNA incubated with sperm, which may influence the internalization rate. Due to the inconsistencies found in literature, this work aimed to contribute to bovine sperm physiology knowledge evaluating the effects of different DNA concentrations, electroporation, and DNAse I treatments on sperm viability characteristics, DNA uptake, and IVF. For that, the effects of different concentrations of exogenous DNA (250, 500 and 1000 ng/10(6) cells) and incubation or electroporation were tested on sperm functional characteristics and in vitro embryo production. No effect of DNA concentration was observed on uptake, plasma membrane integrity, and mitochondrial membrane potential. The addition of exogenous DNA induced a decrease on acrosomal lesion in the 500-ng group when compared to the control. Cells incubated with DNA, electroporated, and treated with DNAse I presented a deleterious influence on mitochondrial membrane potential. In vitro fertilization was made with 1000 ng of DNA, sperm cells incubated or electroporated followed by DNAse I treatment. No significant difference was found in cleavage rate. Blastocyst rates were 24.36% for the control; 19.65% for incubated; 3.5% for electroporated control; and 17.40% for electroporated. There is a significant difference in blastocyst rate between the control and electroporated control

  2. Specific phase arrest of cell cycle restores cell viability against tRNA cleavage by killer toxin.

    PubMed

    Shigematsu, Megumi; Ogawa, Tetsuhiro; Kitamoto, Hiroko K; Hidaka, Makoto; Masaki, Haruhiko

    2012-04-20

    Zymocin and PaT are killer toxins that induce cell cycle arrest of sensitive yeast cells in G1 and S phase, respectively. Recent studies have revealed that these two toxins cleave specific tRNAs, indicating that the cell growth impairment is due to the tRNA cleavage. Additionally, we have previously shown that the active domain of colicin D (D-CRD), which also cleaves specific Escherichia coli tRNAs, statically impairs growth when expressed in yeast cells. To verify that phase-specific cell cycle arrest is also induced by the expression of D-CRD, D-CRD and the subunits of zymocin and PaT that have tRNA cleaving activity were expressed in yeast cells and cell cycle status was analyzed. Our results indicate that phase-specific arrest does not commonly occur by tRNA cleavage, and it saves the cell viability. Furthermore, the extent of protein synthesis impairment may determine the phase specificity of cell cycle arrest. PMID:22450321

  3. Effect of low-level laser irradiation on proliferation and viability of human dental pulp stem cells.

    PubMed

    Zaccara, Ivana Maria; Ginani, Fernanda; Mota-Filho, Haroldo Gurgel; Henriques, Águida Cristina Gomes; Barboza, Carlos Augusto Galvão

    2015-12-01

    A positive effect of low-level laser irradiation (LLLI) on the proliferation of some cell types has been observed, but little is known about its effect on dental pulp stem cells (DPSCs). The aim of this study was to identify the lowest energy density able to promote the proliferation of DPSCs and to maintain cell viability. Human DPSCs were isolated from two healthy third molars. In the third passage, the cells were irradiated or not (control) with an InGaAlP diode laser at 0 and 48 h using two different energy densities (0.5 and 1.0 J/cm²). Cell proliferation and viability and mitochondrial activity were evaluated at intervals of 24, 48, 72, and 96 h after the first laser application. Apoptosis- and cell cycle-related events were analyzed by flow cytometry. The group irradiated with an energy density of 1.0 J/cm² exhibited an increase of cell proliferation, with a statistically significant difference (p < 0.05) compared to the control group at 72 and 96 h. No significant changes in cell viability were observed throughout the experiment. The distribution of cells in the cell cycle phases was consistent with proliferating cells in all three groups. We concluded that LLLI, particularly a dose of 1.0 J/cm², contributed to the growth of DPSCs and maintenance of its viability. This fact indicates this therapy to be an important future tool for tissue engineering and regenerative medicine involving stem cells.

  4. Harpagophytum procumbens prevents oxidative stress and loss of cell viability in vitro.

    PubMed

    Schaffer, Larissa Finger; Peroza, Luis Ricardo; Boligon, Aline Augusti; Athayde, Margareth Linde; Alves, Sydney Hartz; Fachinetto, Roselei; Wagner, Caroline

    2013-11-01

    Harpagophytum procumbens, popularly known as devil's claw, is a plant commonly used in the treatment of diseases of inflammatory origin. The anti-inflammatory effects of H. procumbens have been studied; however, the mechanism of action is not elucidated. It is known that excess of reactive oxygen and nitrogen species may contribute to increasing tissue damage due to inflammation. In the present study, we examined the effects of H. procumbens infusion, crude extract and fractions on lipid peroxidation (brain homogenates) induced by different pro-oxidants (Fe(2+) or sodium nitroprusside) and the effects of ethyl acetate fraction (rich in phenolic compounds) on antioxidant defenses (catalase activity and thiol levels) and cell damage (brain cortical slices) induced by different pro-oxidants. All tested extracts of H. procumbens inhibited lipid peroxidation in a concentration-dependent manner. Furthermore, the ethyl acetate fraction had the highest antioxidant effects either by decreasing lipid peroxidation and cellular damage or restoring thiols levels and catalase activity. Taken together, our results showed that H. procumbens acts either by preventing oxidative stress or loss of cell viability. Thus, the previously reported anti-inflammatory effect of H. procumbens could also be attributed to its antioxidant activity. PMID:23982320

  5. Cell viability of Candida albicans against the antifungal activity of thymol.

    PubMed

    de Vasconcelos, Laís César; Sampaio, Fabio Correia; Albuquerque, Allan de Jesus dos Reis; Vasconcelos, Laurylene César de Souza

    2014-01-01

    Candida albicans is a commensal fungus, but circumstantially it may cause superficial infections of the mucous membranes, such as denture stomatitis, when a biofilm is formed on the surface of dental prostheses. This study evaluated the cell viability of C. albicans biofilms against the antifungal activity of thymol when compared with miconazole, by the fluorescence imaging using SYTO 9 and propidium iodide dyes, and counting of colony forming units. C. albicans standard strains (ATCC 11006) were used. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of drugs were determined by broth microdilution tests and the inoculum was standardized to match 0.5 on the McFarland scale (106 cfu/mL). Biofilms were grown on the surface of acrylic resin disks in parallel flow chambers from Sabouraud broth supplemented with 10% dextrose. For counting of colony forming units, the fungal solution was sequentially diluted and plated in Sabouraud dextrose agar. Data were analyzed using two-way ANOVA and Tukey's test (a=5%). Biofilms treated with thymol and miconazole presented low numbers of viable cells at the evaluated exposure times. There was statistically significant difference (p<0.05) when compared with control, and the mean value of the exposure times between miconazole and thymol did not differ significantly (p>0.05). In conclusion, both drugs have similar efficiency as antifungal agents against biofilms of C. albicans formed on acrylic surfaces. PMID:25250489

  6. The aryl hydrocarbon receptor nuclear translocator is an essential regulator of murine hematopoietic stem cell viability

    PubMed Central

    Krock, Bryan L.; Eisinger-Mathason, Tzipora S.; Giannoukos, Dionysios N.; Shay, Jessica E.; Gohil, Mercy; Lee, David S.; Nakazawa, Michael S.; Sesen, Julie; Skuli, Nicolas

    2015-01-01

    Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to low oxygen and play essential roles in embryonic development, tissue homeostasis, and disease. Recent studies have demonstrated that hematopoietic stem cells (HSCs) within the bone marrow localize to a hypoxic niche and that HIF-1α promotes HSC adaptation to stress. Because the related factor HIF-2α is also expressed in HSCs, the combined role of HIF-1α and HIF-2α in HSC maintenance is unclear. To this end, we have conditionally deleted the HIF-α dimerization partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) in the hematopoietic system to ablate activity of both HIF-1α and HIF-2α and assessed the functional consequence of ARNT deficiency on fetal liver and adult hematopoiesis. We determined that ARNT is essential for adult and fetal HSC viability and homeostasis. Importantly, conditional knockout of both Hif-1α and Hif-2α phenocopied key aspects of these HSC phenotypes, demonstrating that the impact of Arnt deletion is primarily HIF dependent. ARNT-deficient long-term HSCs underwent apoptosis, potentially because of reduced B-cell lymphoma 2 (BCL-2) and vascular endothelial growth factor A (VEGF-A) expression. Our results suggest that HIF activity may regulate HSC homeostasis through these prosurvival factors. PMID:25855602

  7. Phosphatidylserine exposure and red cell viability in red cell aging and in hemolytic anemia

    PubMed Central

    Boas, Franz Edward; Forman, Linda; Beutler, Ernest

    1998-01-01

    Phosphatidylserine (PS) normally localizes to the inner leaflet of cell membranes but becomes exposed in abnormal or apoptotic cells, signaling macrophages to ingest them. Along similar lines, it seemed possible that the removal of red cells from circulation because of normal aging or in hemolytic anemias might be triggered by PS exposure. To investigate the role of PS exposure in normal red cell aging, we used N-hydroxysuccinimide-biotin to tag rabbit red cells in vivo, then used phycoerythrin-streptavidin to label the biotinylated cells, and annexin V-fluorescein isothiocyanate (FITC) to detect the exposed PS. Flow cytometric analysis of these cells drawn at 10-day intervals up to 70 days after biotinylation indicated that older, biotinylated cells expose more PS. Furthermore, our data match a simple model of red cell senescence that assumes both an age-dependent destruction of senescent red cells preceded by several hours of PS exposure and a random destruction of red cells without PS exposure. By using this model, we demonstrated that the exposure of PS parallels the rate at which biotinylated red cells are removed from circulation. On the other hand, using an annexin V-FITC label and flow cytometry demonstrates that exposed PS does not cause the reduced red cell life span of patients with hemolytic anemia, with the possible exception of those with unstable hemoglobins or sickle cell anemia. Thus, in some cases PS exposure on the cell surface may signal the removal of red cells from circulation, but in other cases some other signal must trigger the sequestration of cells. PMID:9501218

  8. Effect of thymol on Ca2+ homeostasis and viability in human glioblastoma cells.

    PubMed

    Hsu, Shu-Shong; Lin, Ko-Long; Chou, Chiang-Ting; Chiang, An-Jen; Liang, Wei-Zhe; Chang, Hong-Tai; Tsai, Jeng-Yu; Liao, Wei-Chuan; Huang, Fong-Dee; Huang, Jong Khing; Chen, I-Shu; Liu, Shuih-Inn; Kuo, Chun-Chi; Jan, Chung-Ren

    2011-11-16

    The effect of the natural essential oil thymol on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in human glioblastoma cells was examined. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Thymol at concentrations of 400-1000 μM induced a [Ca(2+)](i) rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca(2+). Thymol-induced Ca(2+) signal was not altered by nifedipine, econazole, SK&F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca(2+) was removed, incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished thymol-induced [Ca(2+)](i) rise. Incubation with thymol also abolished thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished thymol-induced [Ca(2+)](i) rise. At concentrations of 200-800 μM, thymol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that thymol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. Collectively, in human glioblastoma cells, thymol induced a [Ca(2+)](i) rise by inducing phospholipase C- and protein kinase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via non store-operated Ca(2+) channels. Thymol induced cell death that may involve apoptosis. PMID:21914442

  9. Survivin Modulates Squamous Cell Carcinoma-Derived Stem-Like Cell Proliferation, Viability and Tumor Formation in Vivo.

    PubMed

    Lotti, Roberta; Palazzo, Elisabetta; Petrachi, Tiziana; Dallaglio, Katiuscia; Saltari, Annalisa; Truzzi, Francesca; Quadri, Marika; Puviani, Mario; Maiorana, Antonino; Marconi, Alessandra; Pincelli, Carlo

    2016-01-01

    Squamous Cell Carcinoma-derived Stem-like Cells (SCC-SC) originate from alterations in keratinocyte stem cells (KSC) gene expression and sustain tumor development, invasion and recurrence. Since survivin, a KSC marker, is highly expressed in SCC-SC, we evaluate its role in SCC-SC cell growth and SCC models. Survivin silencing by siRNA decreases clonal growth of SCC keratinocytes and viability of total, rapidly adhering (RAD) and non-RAD (NRAD) cells from primary SCC. Similarly, survivin silencing reduces the expression of stem cell markers (OCT4, NOTCH1, CD133, β₁-integrin), while it increases the level of differentiation markers (K10, involucrin). Moreover, survivin silencing improves the malignant phenotype of SCC 3D-reconstruct, as demonstrated by reduced epidermal thickness, lower Ki-67 positive cell number, and decreased expression of MMP9 and psoriasin. Furthermore, survivin depletion by siRNA in Ras(G12V)-IκBα-derived tumors leads to smaller tumor formation characterized by lower mitotic index and reduced expression of the tumor-associated marker HIF1α, VEGF and CD51. Therefore, our results indicate survivin as a key gene in regulating SCC cancer stem cell formation and cSCC development.

  10. Survivin Modulates Squamous Cell Carcinoma-Derived Stem-Like Cell Proliferation, Viability and Tumor Formation in Vivo

    PubMed Central

    Lotti, Roberta; Palazzo, Elisabetta; Petrachi, Tiziana; Dallaglio, Katiuscia; Saltari, Annalisa; Truzzi, Francesca; Quadri, Marika; Puviani, Mario; Maiorana, Antonino; Marconi, Alessandra; Pincelli, Carlo

    2016-01-01

    Squamous Cell Carcinoma-derived Stem-like Cells (SCC-SC) originate from alterations in keratinocyte stem cells (KSC) gene expression and sustain tumor development, invasion and recurrence. Since survivin, a KSC marker, is highly expressed in SCC-SC, we evaluate its role in SCC-SC cell growth and SCC models. Survivin silencing by siRNA decreases clonal growth of SCC keratinocytes and viability of total, rapidly adhering (RAD) and non-RAD (NRAD) cells from primary SCC. Similarly, survivin silencing reduces the expression of stem cell markers (OCT4, NOTCH1, CD133, β1-integrin), while it increases the level of differentiation markers (K10, involucrin). Moreover, survivin silencing improves the malignant phenotype of SCC 3D-reconstruct, as demonstrated by reduced epidermal thickness, lower Ki-67 positive cell number, and decreased expression of MMP9 and psoriasin. Furthermore, survivin depletion by siRNA in RasG12V-IκBα-derived tumors leads to smaller tumor formation characterized by lower mitotic index and reduced expression of the tumor-associated marker HIF1α, VEGF and CD51. Therefore, our results indicate survivin as a key gene in regulating SCC cancer stem cell formation and cSCC development. PMID:26771605

  11. MicroRNA-101 regulates the viability and invasion of cervical cancer cells

    PubMed Central

    Lin, Chen; Huang, Fei; Shen, Guqun; Yiming, Adilijiang

    2015-01-01

    Background: Cervical cancer has the second highest morbidity and mortality rates of any malignancy in women worldwide, and it is one of the leading causes of death in Uygur women in Xinjiang China. MicroRNAs are involved in cancer development and progression. Previously, we found that miR-101 is significantly down-regulated in cervical cancer tissues from Uyghur women. The underlying pathophysiology and relevance to tumorigenesis of miR-101 is still largely unknown. The purpose of this study was to elucidate the molecular mechanisms of miR-101 regulation of cervical cancer cell viability and invasion. Materials and methods: The expression of miR-101 in cervical cancer cell line (SiHa) was detected by real-time PCR. A miR-101 mimic was overexpressed in SiHa cells, and MTT assays were performed to determine the impact on cell proliferation. Cell would heal assays and flow cytometry were used to detect migratory ability and cellular apoptosis, respectively. Immunohistochemistry was performed to assess protein expression of the miR-101 target gene COX-2. Results: MiR-101 was endogenously expressed in SiHa cells, and alterations in its expression had profound effects on cellular migration and invasion efficiency. Overexpression of miR-101 decreased proliferation in the MTT assay (the mimics at 490 nm absorbance is lower 60% than normal, and decreased cellular motility in the cell would healing assay (transfected: 37 ± 2 m, pre-transfected 184 ± 2 m). Apoptosis rate was significantly higher with overexpression of miR-101 relative to control (transfected: 76.6%, pre-transfected: 3.5%) (P < 0.05). The expression of Cox-2 was decreased in transfected cells. Conclusions: MiR-101 likely acts as a tumor suppressor in cervical cancer. Overexpression of miR-101 decreased expression of its target gene Cox-2 and inhibited proliferation and invasion, and promoted apoptosis to suppress tumorigenicity. MiR-101 is a promising new target for the development of therapeutic strategies

  12. The effect of pulsed electromagnetic fields and dehydroepiandrosterone on viability and osteo-induction of human mesenchymal stem cells.

    PubMed

    Kaivosoja, Emilia; Sariola, Veikko; Chen, Yan; Konttinen, Yrjö T

    2015-01-01

    The hypothesis of this work was that human bone marrow-derived mesenchymal stem cells (MSCs) are regulated by pulsed electromagnetic fields (PEMFs) and by intracrine conversion of an adrenal prohormone to dihydrotestosterone. The effect of PEMF and dehydroepiandrosterone (DHEA) on viability and osteogenic differentiation of human MSCs and on the viability of osteoblastic SaOS-2 cells was evaluated. It was found that PEMF promoted the viability rate of both cell types, whereas DHEA decreased the viability rate in a concentration-dependent manner. PEMF did not have major effects on osteo-induction at this low seeding density level (3000 cells/cm(2) ). Instead, DHEA, after MSC-mediated and 5α-reductase-dependent conversion to dihydrotestosterone, clearly promoted the osteo-induction of MSCs induced with β-glyserophosphate, ascorbate and dexamethasone. Alkaline phosphatase (ALP), SMAD1, RUNX2, osteopontin (OP) and osteocalcin (OC) RNA levels were increased and alizarin red S- and hydroxyapatite-specific OsteoImage(TM) stainings disclosed a promoted mineralization process. In addition, DHEA increased OP and OC mRNA levels of non-induced MSCs. A sequential use of mitogenic PEMF early during the fracture healing, followed by later administration of DHEA with osteogenic differentiating effect, might be worth subjecting to a randomized clinical trial.

  13. Effects of trehalose supplementation on cell viability and oxidative stress variables in frozen-thawed bovine calf testicular tissue.

    PubMed

    Zhang, Xiao-Gang; Wang, Yan-Hua; Han, Cong; Hu, Shan; Wang, Li-Qiang; Hu, Jian-Hong

    2015-06-01

    Trehalose is widely used for cryopreservation of various cells and tissues. Until now, the effect of trehalose supplementation on cell viability and antioxidant enzyme activity in frozen-thawed bovine calf testicular tissue remains unexplored. The objective of the present study was to compare the effect of varying doses of trehalose in cryomedia on cell viability and key antioxidant enzymes activities in frozen-thawed bovine calf testicular tissue. Bovine calf testicular tissue samples were collected and cryopreserved in the cryomedias containing varying doses (0, 5, 10, 15, 20 and 25%; v/v) of trehalose, respectively. Cell viability, total antioxidant capacity (T-AOC) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, glutathione (GSH) content and malondialdehyde (MDA) content were measured and analyzed. The results showed that cell viability, T-AOC activity, SOD activity, CAT activity and GSH content of frozen-thawed bovine calf testicular tissue was decreased compared with that of fresh group (P<0.05). MDA content in frozen-thawed bovine calf testicular tissue was significantly increased compared with that of fresh group (P<0.05). The cryomedia added 15% trehalose exhibited the greatest percentage of cell viability and antioxidant enzyme activity (SOD and CAT) among frozen-thawed groups (P<0.05). Meanwhile, GSH content was the lowest among frozen-thawed groups (P<0.05). However, there were no significance differences in MDA content among the groups added 10, 15 and 20% trehalose (P>0.05). In conclusion, the cryomedia added 15% trehalose reduced the oxidative stress and improved the cryoprotective effect of bovine calf testicular tissue. Further studies are required to obtain more concrete results on the determination of antioxidant capacity of trehalose in frozen-thawed bovine calf testicular tissue. PMID:25818604

  14. Acid adaptation affects the viability of Listeria monocytogenes BCRC 14846 and Salmonella Typhimurium BCRC 10747 exposed to disinfectants at 25°C and 40°C.

    PubMed

    Lin, Meng-Hsuan; Lee, Shiow-Ling; Chou, Cheng-Chun

    2011-10-01

    In the present study, Listeria monocytogenes BCRC 14846 and Salmonella Typhimurium BCRC 10747 were subjected to acid adaptation at pH 5.5 at 37°C for 1 and 4 h, respectively. The viability of the acid-adapted cells of test organisms exposed to Clidox-S, a chlorine-containing disinfectant, and Quatricide, a quaternary ammonium compound, was examined and compared with that of the control cells at 25°C and 40°C. Results revealed that acid adaptation significantly enhanced the viability of L. monocytogenes and Salmonella Typhimurium exposed to the disinfectants under investigation. Both pathogens examined were more susceptible to Clidox-S and Quatricide at 40°C than at 25°C. Further, L. monocytogenes was more susceptible to Quatricide than Salmonella Typhimurium, whereas Salmonella Typhimurium was more susceptible to Clidox-S than L. monocytogenes.

  15. The cell wall amidase AmiB is essential for Pseudomonas aeruginosa cell division, drug resistance, and viability

    PubMed Central

    Yakhnina, Anastasiya A.; McManus, Heather R.; Bernhardt, Thomas G.

    2015-01-01

    SUMMARY The physiological function of cell wall amidases has been investigated in several proteobacterial species. In all cases, they have been implicated in the cleavage of cell wall material synthesized by the cytokinetic ring. Although typically non-essential, this activity is critical for daughter cell separation and outer membrane invagination during division. In Escherichia coli, proteins with LytM domains also participate in cell separation by stimulating amidase activity. Here, we investigated the function of amidases and LytM proteins in the opportunistic pathogen Pseudomonas aeruginosa. In agreement with studies in other organisms, PaAmiB and three LytM proteins were found to play crucial roles in P. aeruginosa cell separation, envelope integrity, and antibiotic resistance. Importantly, the phenotype of amidase-defective P. aeruginosa cells also differed in informative ways from the E. coli paradigm; PaAmiB was found to be essential for viability and the successful completion of cell constriction. Our results thus reveal a key role for amidase activity in cytokinetic ring contraction. Furthermore, we show that the essential function of PaAmiB can be bypassed in mutants activated for a Cpx-like envelope stress response, suggesting that this signaling system may elicit the repair of division machinery defects in addition to general envelope damage. PMID:26032134

  16. Differential electrophoretic separation of cells and its effect on cell viability

    NASA Technical Reports Server (NTRS)

    Leise, E. M.; Lesane, F.

    1974-01-01

    An electrophoretic separation method was applied to the separation of cells. To determine the efficiency of the separation, it was necessary to apply existing methodology and develop new methods to assess the characteristics and functions of the separated subpopulations. Through appropriate application of the widely used isoelectric focusing procedure, a reproducible separation method was developed. Cells accumulated at defined pH and 70-80% remained viable. The cells were suitable for further biologic, biochemical and immunologic studies.

  17. 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. PMID:27428423

  18. Long Term and Standard Incubations of WST-1 Reagent Reflect the Same Inhibitory Trend of Cell Viability in Rat Airway Smooth Muscle Cells

    PubMed Central

    Yin, Lei-Miao; Wei, Yin; Wang, Yu; Xu, Yu-Dong; Yang, Yong-Qing

    2013-01-01

    The WST-1 assay is an efficient test for cell viability measurement and the standard incubation time is 2h. In order to test if one-time addition of WST-1 reagent can reflect the relative cell viability trend of the testing agents at different time points, the effects of 2h standard incubation time and long term incubation time (2h+24h, 2h+48h) of WST-1 were compared in the rat airway smooth muscle cells (ASM cells) after adding of the testing protein MRP-14. Our study demonstrated that the effect of different dosages of the protein after 2h WST-1 incubation on ASM cells showed a tendency of inhibition and achieved the maximal inhibition effect at 72h. The relative cell viability trend of the 2h+24h group was the same to that of the 2h WST-1 incubation, which means that 24h prolonged incubation time of WST-1 reagent could still reflect the relative cell viability trend. In conclusion, the study suggested that the WST-1 is a proper candidate reagent for continuous monitation of cell viability. PMID:23289007

  19. Simulated Microgravity and 3D Culture Enhance Induction, Viability, Proliferation and Differentiation of Cardiac Progenitors from Human Pluripotent Stem Cells

    PubMed Central

    Jha, Rajneesh; Wu, Qingling; Singh, Monalisa; Preininger, Marcela K.; Han, Pengcheng; Ding, Gouliang; Cho, Hee Cheol; Jo, Hanjoong; Maher, Kevin O.; Wagner, Mary B.; Xu, Chunhui

    2016-01-01

    Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes. PMID:27492371

  20. Simulated Microgravity and 3D Culture Enhance Induction, Viability, Proliferation and Differentiation of Cardiac Progenitors from Human Pluripotent Stem Cells.

    PubMed

    Jha, Rajneesh; Wu, Qingling; Singh, Monalisa; Preininger, Marcela K; Han, Pengcheng; Ding, Gouliang; Cho, Hee Cheol; Jo, Hanjoong; Maher, Kevin O; Wagner, Mary B; Xu, Chunhui

    2016-01-01

    Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes. PMID:27492371

  1. DCT protects human melanocytic cells from UVR and ROS damage and increases cell viability.

    PubMed

    Ainger, Stephen A; Yong, Xuan L; Wong, Shu S; Skalamera, Dubravka; Gabrielli, Brian; Leonard, J Helen; Sturm, Richard A

    2014-12-01

    Dopachrome tautomerase (DCT) is involved in the formation of the photoprotective skin pigment eumelanin and has also been shown to have a role in response to apoptotic stimuli and oxidative stress. The effect of DCT on UVR DNA damage responses and survival pathways in human melanocytic cells was examined by knockdown experiments using melanoma cells, neonatal foreskin melanoblasts (MB) in monoculture and in co-culture with human keratinocytes. MB cell strains genotyped as either MC1R WT or MC1R RHC homozygotes, which are known to be deficient in DCT, were transduced with lentivirus vectors for either DCT knockdown or overexpression. We found melanoma cell survival was reduced by DCT depletion and by UVR over time. UVR-induced p53 and pp53-Ser15 levels were reduced with DCT depletion. Knockdown of DCT in MC1R WT and MC1R RHC MB cells reduced their survival after UVR exposure, whereas increased DCT protein levels enhanced survival. DCT depletion reduced p53 and pp53-Ser15 levels in WM266-4 melanoma and MC1R WT MB cells, while MC1R RHC MB cells displayed variable levels. Both MC1R WT and RHC genotypes of MB cells were responsive to UVR at 3 h with increases in both p53 and pp53-Ser15 proteins. MC1R WT MB cell strains in coculture with keratinocytes have an increased cell survival after UVR exposure when compared to those in monoculture, a protective effect which appears to be conferred by the keratinocytes.

  2. 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

  3. Kalanchoe tubiflora extract inhibits cell proliferation by affecting the mitotic apparatus

    PubMed Central

    2012-01-01

    Background Kalanchoe tubiflora (KT) is a succulent plant native to Madagascar, and is commonly used as a medicinal agent in Southern Brazil. The underlying mechanisms of tumor suppression are largely unexplored. Methods Cell viability and wound-healing were analyzed by MTT assay and scratch assay respectively. Cell cycle profiles were analyzed by FACS. Mitotic defects were analyzed by indirect immunofluoresence images. Results An n-Butanol-soluble fraction of KT (KT-NB) was able to inhibit cell proliferation. After a 48 h treatment with 6.75 μg/ml of KT, the cell viability was less than 50% of controls, and was further reduced to less than 10% at higher concentrations. KT-NB also induced an accumulation of cells in the G2/M phase of the cell cycle as well as an increased level of cells in the subG1 phase. Instead of disrupting the microtubule network of interphase cells, KT-NB reduced cell viability by inducing multipolar spindles and defects in chromosome alignment. KT-NB inhibits cell proliferation and reduces cell viability by two mechanisms that are exclusively involved with cell division: first by inducing multipolarity; second by disrupting chromosome alignment during metaphase. Conclusion KT-NB reduced cell viability by exclusively affecting formation of the proper structure of the mitotic apparatus. This is the main idea of the new generation of anti-mitotic agents. All together, KT-NB has sufficient potential to warrant further investigation as a potential new anticancer agent candidate. PMID:22963191

  4. Effects of glass ionomers and dental resin composites on viability of beta-cells and insulin release in isolated islets of Langerhans.

    PubMed

    Persson-Sjögren, Solveig; Sjögren, Göran

    2003-09-01

    Information on the biocompatibility of glass ionomers and resin composites is sparse. To extend the scale of biological testing we evaluated the influence of those materials on insulin secretion at whole organ level in vitro. The effects on insulin secretion of three glass ionomers and two resin composites, aged for 1 week, were studied in isolated mouse islets of Langerhans at basal (5.5mM) and at stimulatory (11.1mM) D-glucose concentrations. In addition, viability of single mouse beta-cells was evaluated. The effect of glass ionomer specimens aged for 1 and 4 months on insulin secretion at 11.1mM D-glucose was also studied. None of the materials affected the viability of the beta-cells. At 5.5mM D-glucose none of the materials affected the insulin secretion. At 11.1mM D-glucose, the glass ionomers only decreased the secretion and glass ionomers aged for 1 month still decreased insulin release whereas after 4 months ageing only one of the glass ionomers affected the release. The result shows a dynamic effect on insulin release of the elements and/or compounds released from the specimens.

  5. Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

    SciTech Connect

    Gikas, P.; Livingston, A.G. . Dept. of Chemical Engineering)

    1993-12-01

    This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bio-reactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h[sup [minus]1] in the CST bioreactor and between 0.111 and 0.500h[sup [minus]1] in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g[sup [minus]1] dry weight (dw) as dilution rate increases from 0.027 to 0.115 h[sup [minus]1]. At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g[sup [minus]1] dw, which is assumed to be the quantity of ATP in 100% viable biomass, In the TPAL bioreactor, the ATP level increased with dilution rat in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g[sup [minus]1] dw at dilution rates between 0.111 and 0.200 h[sup [minus]1] to approximately 0.119 mg ATP g[sup [minus]1] dw at dilution rates between 0.300 and 0.500 h[sup [minus]1].

  6. Transport and viability of Escherichia coli cells in clean and iron oxide coated sand following coating with silver nanoparticles.

    PubMed

    Ngwenya, Bryne T; Curry, Philip; Kapetas, Leon

    2015-08-01

    A mechanistic understanding of processes controlling the transport and viability of bacteria in porous media is critical for designing in situ bioremediation and microbiological water decontamination programs. We investigated the combined influence of coating sand with iron oxide and silver nanoparticles on the transport and viability of Escherichia coli cells under saturated conditions. Results showed that iron oxide coatings increase cell deposition which was generally reversed by silver nanoparticle coatings in the early stages of injection. These observations are consistent with short-term, particle surface charge controls on bacteria transport, where a negatively charged surface induced by silver nanoparticles reverses the positive charge due to iron oxide coatings, but columns eventually recovered irreversible cell deposition. Silver nanoparticle coatings significantly increased cell inactivation during transit through the columns. However, when viability data is normalised to volume throughput, only a small improvement in cell inactivation is observed for silver nanoparticle coated sands relative to iron oxide coating alone. This counterintuitive result underscores the importance of net surface charge in controlling cell transport and inactivation and implies that the extra cost for implementing silver nanoparticle coatings on porous beds coated with iron oxides may not be justified in designing point of use water filters in low income countries. PMID:26042624

  7. Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast, Pichia anomala, for aflatoxin reduction.

    PubMed

    Hua, Sui Sheng T; Hernlem, Bradley J; Yokoyama, Wallace; Sarreal, Siov Bouy L

    2015-05-01

    Pichia anomala (Wickerhamomyces anomalus) WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of aflatoxin in the food chain. Maintaining the viability of biocontrol agents in formulated products is a great challenge for commercial applications. Four media, NYG, NYGS, NYGT and NYGST are described which support good growth of yeast cells and were tested as storage formulations. Post growth supplement of 5 % trehalose to NYGST resulted in 83 % viable yeast cells after 12 months in cold storage. Intracellular sorbitol and trehalose concentrations were determined by HPLC analysis at the beginning of the storage and at the end of 12 month. Correlation of cell viability to both trehalose and sorbitol suggested a synergistic effect. Bonferroni (Dunn) t Test, Tukey's Studentized Range (HSD) Test and Duncan's Multiple Range Test, all showed that yeast cell viability in samples with both intracellular trehalose and sorbitol were significantly higher than those with either or none, at a 95 % confidence level. DiBAC4(5) and CFDA-AM were used as the membrane integrity fluorescent stains to create a two-color vital staining scheme with red and green fluorescence, respectively. Yeast cells stored in formulations NYG and NYGS with no detectable trehalose, displayed mostly red fluorescence. Yeast cells in NYGST+5T showed mostly green fluorescence. PMID:25700743

  8. Transport and viability of Escherichia coli cells in clean and iron oxide coated sand following coating with silver nanoparticles.

    PubMed

    Ngwenya, Bryne T; Curry, Philip; Kapetas, Leon

    2015-08-01

    A mechanistic understanding of processes controlling the transport and viability of bacteria in porous media is critical for designing in situ bioremediation and microbiological water decontamination programs. We investigated the combined influence of coating sand with iron oxide and silver nanoparticles on the transport and viability of Escherichia coli cells under saturated conditions. Results showed that iron oxide coatings increase cell deposition which was generally reversed by silver nanoparticle coatings in the early stages of injection. These observations are consistent with short-term, particle surface charge controls on bacteria transport, where a negatively charged surface induced by silver nanoparticles reverses the positive charge due to iron oxide coatings, but columns eventually recovered irreversible cell deposition. Silver nanoparticle coatings significantly increased cell inactivation during transit through the columns. However, when viability data is normalised to volume throughput, only a small improvement in cell inactivation is observed for silver nanoparticle coated sands relative to iron oxide coating alone. This counterintuitive result underscores the importance of net surface charge in controlling cell transport and inactivation and implies that the extra cost for implementing silver nanoparticle coatings on porous beds coated with iron oxides may not be justified in designing point of use water filters in low income countries.

  9. Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast, Pichia anomala, for aflatoxin reduction.

    PubMed

    Hua, Sui Sheng T; Hernlem, Bradley J; Yokoyama, Wallace; Sarreal, Siov Bouy L

    2015-05-01

    Pichia anomala (Wickerhamomyces anomalus) WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of aflatoxin in the food chain. Maintaining the viability of biocontrol agents in formulated products is a great challenge for commercial applications. Four media, NYG, NYGS, NYGT and NYGST are described which support good growth of yeast cells and were tested as storage formulations. Post growth supplement of 5 % trehalose to NYGST resulted in 83 % viable yeast cells after 12 months in cold storage. Intracellular sorbitol and trehalose concentrations were determined by HPLC analysis at the beginning of the storage and at the end of 12 month. Correlation of cell viability to both trehalose and sorbitol suggested a synergistic effect. Bonferroni (Dunn) t Test, Tukey's Studentized Range (HSD) Test and Duncan's Multiple Range Test, all showed that yeast cell viability in samples with both intracellular trehalose and sorbitol were significantly higher than those with either or none, at a 95 % confidence level. DiBAC4(5) and CFDA-AM were used as the membrane integrity fluorescent stains to create a two-color vital staining scheme with red and green fluorescence, respectively. Yeast cells stored in formulations NYG and NYGS with no detectable trehalose, displayed mostly red fluorescence. Yeast cells in NYGST+5T showed mostly green fluorescence.

  10. Effects of air pollution-related heavy metals on the viability and inflammatory responses of human airway epithelial cells.

    PubMed

    Honda, Akiko; Tsuji, Kenshi; Matsuda, Yugo; Hayashi, Tomohiro; Fukushima, Wataru; Sawahara, Takahiro; Kudo, Hitomi; Murayama, Rumiko; Takano, Hirohisa

    2015-01-01

    Various metals produced from human activity are ubiquitously detected in ambient air. The metals may lead to induction and/or exacerbation of respiratory diseases, but the significant metals and factors contributing to such diseases have not been identified. To compare the effects of each metal and different oxidation states of metals on human airway, we examined the viability and production of interleukin (IL)-6 and IL-8 using BEAS-2B cell line, derived from human airway epithelial cells. Airway epithelial cells were exposed to Mn(2+), V(4+), V(5+), Cr(3+), Cr(6+), Zn(2+), Ni(2+), and Pb(2+) at a concentration of 0.5, 5, 50, or 500 μmol/L for 24 hours. Mn and V decreased the cell viability in a concentration-dependent manner, and V(5+) tended to have a greater effect than V(4+). The Cr decreased the cell viability, and (Cr(+6)) at concentrations of 50 and 500 μmol/L was more toxic than (Cr(+3)). Zn at a concentration of 500 μmol/L greatly decreased the cell viability, whereas Ni at the same concentration increased it. Pb produced fewer changes. Mn and Ni at a concentration of 500 μmol/L induced the significant production of IL-6 and IL-8. However, most of the metals including (V(+4), V(+5)), (Cr(+3), Cr(+6)), Zn, and Pb inhibited the production of both IL-6 and IL-8. The present results indicate that various heavy metals have different effects on toxicity and the proinflammatory responses of airway epithelial cells, and those influences also depend on the oxidation states of the metals.

  11. Ethanol Extracts of Selected Cyathea Species Decreased Cell Viability and Inhibited Growth in MCF 7 Cell Line Cultures.

    PubMed

    Janakiraman, Narayanan; Johnson, Marimuthu

    2016-06-01

    Cancer is the cause of more than 6 million deaths worldwide every year. For centuries, medicinal plants have been used in the treatment of cancer. Chemotherapy, radiotherapy, surgery and acupuncture point stimulation are also used to treat cancer. The present study was intended to reveal the cytotoxic and anticancer potential of selected Cyathea species and to highlight their importance in the pharmaceutical industry for the development of cost-effective drugs. Cytotoxic studies using brine shrimp lethality bioassays and MCF 7 cell line cultures were carried out. Compared to petroleum ether, chloroform and acetone extracts, the ethanol extracts of selected Cyathea species were found to be more effective against brine shrimps. The ethanol extracts were further subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assays. A decrease in cell viability and an increase in growth inhibition were observed for the MCF 7 cell line. The maximum percentage of cell inhibition was observed in Cyathea crinit, followed by Cyathea nilgirensis and Cyathea gigantea. The results of the present study suggest that Cyathea species are an effective source of cytotoxic compounds. PMID:27342889

  12. Lithium improves cell viability in psychosine-treated MO3.13 human oligodendrocyte cell line via autophagy activation.

    PubMed

    Del Grosso, Ambra; Antonini, Sara; Angella, Lucia; Tonazzini, Ilaria; Signore, Giovanni; Cecchini, Marco

    2016-11-01

    Globoid cell leukodystrophy (GLD) is a rare, rapidly progressing childhood leukodystrophy triggered by deficit of the lysosomal enzyme galactosylceramidase (GALC) and characterized by the accumulation of galactosylsphingosine (psychosine; PSY) in the nervous system. PSY is a cytotoxic sphingolipid, which leads to widespread degeneration of oligodendrocytes and Schwann cells, causing demyelination. Here we report on autophagy in the human oligodendrocyte cell line MO3.13 treated with PSY and exploitation of Li as an autophagy modulator to rescue cell viability. We demonstrate that PSY causes upregulation of the autophagic flux at the level of autophagosome and autolysosome formation and LC3-II expression. We show that pretreatment with Li, a drug clinically used to treat bipolar disorders, can further stimulate autophagy, improving cell tolerance to PSY. This Li protective effect is found not to be linked to reduction of PSY-induced oxidative stress and might not stem from a reduction of PSY accumulation. These data provide novel information on the intracellular pathways activated during PSY-induced toxicity and suggest the autophagy pathway as a promising novel therapeutic target for ameliorating the GLD phenotype. © 2016 Wiley Periodicals, Inc. PMID:27638607

  13. Spiclomazine Induces Apoptosis Associated with the Suppression of Cell Viability, Migration and Invasion in Pancreatic Carcinoma Cells

    PubMed Central

    Liu, Zuojia; Zheng, Xiliang; Wang, Jin; Wang, Erkang

    2013-01-01

    The effective treatment for pancreatic carcinoma remains critically needed. Herein, this current study showed that spiclomazine treatment caused a reduction in viability in pancreatic carcinoma cell lines CFPAC-1 and MIA PaCa-2 in vitro. It was notable in this regard that, compared with pancreatic carcinoma cells, normal human embryonic kidney (HEK-293) and liver (HL-7702) cells were more resistant to the antigrowth effect of spiclomazine. Biochemically, spiclomazine treatment regulated the expression of protein levels in the apoptosis related pathways. Consistent with this effect, spiclomazine reduced the mitochondria membrane potential, elevated reactive oxygen species, and activated caspase-3/9. In addition, a key finding from this study was that spiclomazine suppressed migration and invasion of cancer cells through down-regulation of MMP-2/9. Collectively, the proposed studies did shed light on the antiproliferation effect of spiclomazine on pancreatic carcinoma cell lines, and further clarified the mechanisms that spiclomazine induced apoptosis associated with the suppression of migration and invasion. PMID:23840452

  14. Equol, an isoflavone metabolite, regulates cancer cell viability and protein synthesis initiation via c-Myc and eIF4G.

    PubMed

    de la Parra, Columba; Borrero-Garcia, Luis D; Cruz-Collazo, Ailed; Schneider, Robert J; Dharmawardhane, Suranganie

    2015-03-01

    Epidemiological studies implicate dietary soy isoflavones as breast cancer preventives, especially due to their anti-estrogenic properties. However, soy isoflavones may also have a role in promoting breast cancer, which has yet to be clarified. We previously reported that equol, a metabolite of the soy isoflavone daidzein, may advance breast cancer potential via up-regulation of the eukaryotic initiation factor 4GI (eIF4GI). In estrogen receptor negative (ER-) metastatic breast cancer cells, equol induced elevated levels of eIF4G, which were associated with increased cell viability and the selective translation of mRNAs that use non-canonical means of initiation, including internal ribosome entry site (IRES), ribosome shunting, and eIF4G enhancers. These mRNAs typically code for oncogenic, survival, and cell stress molecules. Among those mRNAs translationally increased by equol was the oncogene and eIF4G enhancer, c-Myc. Here we report that siRNA-mediated knockdown of c-Myc abrogates the increase in cancer cell viability and mammosphere formation by equol, and results in a significant down-regulation of eIF4GI (the major eIF4G isoform), as well as reduces levels of some, but not all, proteins encoded by mRNAs that are translationally stimulated by equol treatment. Knockdown of eIF4GI also markedly reduces an equol-mediated increase in IRES-dependent mRNA translation and the expression of specific oncogenic proteins. However, eIF4GI knockdown did not reciprocally affect c-Myc levels or cell viability. This study therefore implicates c-Myc as a potential regulator of the cancer-promoting effects of equol via up-regulation of eIF4GI and selective initiation of translation on mRNAs that utilize non-canonical initiation, including certain oncogenes. PMID:25593313

  15. Data on cell viability of human lung fibroblasts treated with polyphenols-rich extract from Plinia trunciflora (O. Berg) Kausel).

    PubMed

    Calloni, Caroline; Silva Santos, Luciana Fernandes; Martínez, Luana Soares; Salvador, Mirian

    2016-03-01

    Jaboticaba (Plinia trunciflora (O. Berg) Kausel) is a Brazilian native berry, which presents high levels of polyphenols. Here we provide data related to the effects of the polyphenols-rich extract from jaboticaba on the cell viability, mitochondrial complex I (nicotinamide adenine dinucleotide/CoQ oxidoreductase) activity and ATP biosynthesis of human lung fibroblast cells (MRC-5) treated with amiodarone. The data presented in this article demonstrate that the polyphenols-rich extract from jaboticaba was able to reduce cell death as well as the decrease in complex I activity and ATP biosynthesis caused by amiodarone in MRC-5 cells.

  16. Data on cell viability of human lung fibroblasts treated with polyphenols-rich extract from Plinia trunciflora (O. Berg) Kausel)

    PubMed Central

    Calloni, Caroline; Silva Santos, Luciana Fernandes; Martínez, Luana Soares; Salvador, Mirian

    2016-01-01

    Jaboticaba (Plinia trunciflora (O. Berg) Kausel) is a Brazilian native berry, which presents high levels of polyphenols. Here we provide data related to the effects of the polyphenols-rich extract from jaboticaba on the cell viability, mitochondrial complex I (nicotinamide adenine dinucleotide/CoQ oxidoreductase) activity and ATP biosynthesis of human lung fibroblast cells (MRC-5) treated with amiodarone. The data presented in this article demonstrate that the polyphenols-rich extract from jaboticaba was able to reduce cell death as well as the decrease in complex I activity and ATP biosynthesis caused by amiodarone in MRC-5 cells. PMID:26870757

  17. Effect of extracellular calcium on regucalcin expression and cell viability in neoplastic and non-neoplastic human prostate cells.

    PubMed

    Vaz, Cátia V; Rodrigues, Daniel B; Socorro, Sílvia; Maia, Cláudio J

    2015-10-01

    Extracellular calcium (Ca2+o) and its receptor, the Ca2+-sensing receptor (CaSR), play an important role in prostate physiology, and it has been shown that the deregulation of Ca2+ homeostasis and the overexpression of CaSR are involved in prostate cancer (PCa). Regucalcin (RGN), a Ca2+-binding protein that plays a relevant role in intracellular Ca2+ homeostasis, was identified as an under-expressed protein in human PCa. Moreover, RGN was associated with suppression of cell proliferation, suggesting that the loss of RGN may favor development and progression of PCa. This work aims to unveil the role of Ca2+o on RGN expression and viability of non-neoplastic (PNT1A) and neoplastic (LNCaP) prostate cell lines. It was demonstrated that Ca2+o up-regulates RGN expression in both cell lines, but important differences were found between cells for dose- and time-responses to Ca2+o treatment. It was also shown that high [Ca2+]o triggers different effects on cell proliferation of neoplastic and non-neoplastic PCa cells, which seems to be related with RGN expression levels. This suggests the involvement of RGN in the regulation of cell proliferation in response to Ca2+o treatment. Also, the effect of Ca2+o on CaSR expression seems to be dependent of RGN expression, which is strengthened by the fact that RGN-knockdown in PNT1A cells increases the CaSR expression, whereas transgenic rats overexpressing RGN exhibit low levels of CaSR. Overall, our results highlighted the importance of RGN as a regulatory protein in Ca2+-dependent signaling pathways and its deregulation of RGN expression by Ca2+o may contribute for onset and progression of PCa.

  18. Using digital inline holographic microscopy and quantitative phase contrast imaging to assess viability of cultured mammalian cells

    NASA Astrophysics Data System (ADS)

    Missan, Sergey; Hrytsenko, Olga

    2015-03-01

    Digital inline holographic microscopy was used to record holograms of mammalian cells (HEK293, B16, and E0771) in culture. The holograms have been reconstructed using Octopus software (4Deep inwater imaging) and phase shift maps were unwrapped using the FFT-based phase unwrapping algorithm. The unwrapped phase shifts were used to determine the maximum phase shifts in individual cells. Addition of 0.5 mM H2O2 to cell media produced rapid rounding of cultured cells, followed by cell membrane rupture. The cell morphology changes and cell membrane ruptures were detected in real time and were apparent in the unwrapped phase shift images. The results indicate that quantitative phase contrast imaging produced by the digital inline holographic microscope can be used for the label-free real time automated determination of cell viability and confluence in mammalian cell cultures.

  19. 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

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

    PubMed

    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

  1. Automated enumeration and viability measurement of canine stromal vascular fraction cells using fluorescence-based image cytometry method.

    PubMed

    Chan, Leo Li-Ying; Cohen, Donald A; Kuksin, Dmitry; Paradis, Benjamin D; Qiu, Jean

    2014-07-01

    In recent years, the lipoaspirate collected from adipose tissue has been seen as a valuable source of adipose-derived mesenchymal stem cells for autologous cellular therapy. For multiple applications, adipose-derived mesenchymal stem cells are isolated from the stromal vascular fraction (SVF) of adipose tissue. Because the fresh stromal vascular fraction typically contains a heterogeneous mixture of cells, determining cell concentration and viability is a crucial step in preparing fraction samples for downstream processing. Due to a large amount of cellular debris contained in the SVF sample, as well as counting irregularities standard manual counting can lead to inconsistent results. Advancements in imaging and optics technologies have significantly improved the image-based cytometric analysis method. In this work, we validated the use of fluorescence-based image cytometry for SVF concentration and viability measurement, by comparing to standard flow cytometry and manual hemocytometer. The concentration and viability of freshly collected canine SVF samples are analyzed, and the results highly correlated between all three methods, which validated the image cytometry method for canine SVF analysis, and potentially for SVF from other species. PMID:24740550

  2. Activation of the Nrf2-regulated antioxidant cell response inhibits HEMA-induced oxidative stress and supports cell viability.

    PubMed

    Gallorini, Marialucia; Petzel, Christine; Bolay, Carola; Hiller, Karl-Anton; Cataldi, Amelia; Buchalla, Wolfgang; Krifka, Stephanie; Schweikl, Helmut

    2015-07-01

    Oxidative stress due to increased formation of reactive oxygen species (ROS) in target cells of dental resin monomers like 2-hydroxyethyl methacrylate (HEMA) is a major mechanism underlying the disturbance of vital cell functions including mineralization and differentiation, responses of the innate immune system, and the induction of cell death via apoptosis. Although a shift in the equilibrium between cell viability and apoptosis is related to the non-enzymatic antioxidant glutathione (GSH) in HEMA-exposed cells, the major mechanisms of adaptive antioxidant cell responses to maintain cellular redox homeostasis are still unknown. The present study provides insight into the induction of a communicating network of pathways under the control of the redox-sensitive transcription factor Nrf2, a major transcriptional activator of genes coding for enzymatic antioxidants. Here, oxidative stress was indicated by DCF fluorescence in cells after a short exposure (1 h) to HEMA, while DHR123 fluorescence significantly increased about 1.8-fold after a long exposure period (24 h) showing the formation of hydrogen peroxide (H2O2). The corresponding expression of Nrf2 was activated immediately after HEMA exposure (1 h) and remained constant up to 24 h. Nrf2-regulated expression of enzymes of the glutathione metabolism (glutathione peroxidase 1/2, glutathione reductase) decreased in HEMA-exposed cells as a result of GSH depletion, and superoxide dismutase expression was downregulated after H2O2 overproduction. However, the expression of Nrf2-controlled enzymatic antioxidants (catalase, peroxiredoxin, thioredoxin 1, thioredoxin reductase, heme oxygenase-1) and the NADPH-regenerating system (glucose 6-phosphate dehydrogenase, transaldolase) was increased. Phenolic tert-butylhydroquinone (tBHQ), a classic inducer of the Nrf2 pathway, reduced oxidative stress and protected cells from HEMA-induced cell death through a shift in the number of cells in necrosis to apoptosis. The

  3. Optically induced dielectropheresis sorting with automated medium exchange in an integrated optofluidic device resulting in higher cell viability.

    PubMed

    Lee, Gwo-Bin; Wu, Huan-Chun; Yang, Po-Fu; Mai, John D

    2014-08-01

    We demonstrated the integration of a microfluidic device with an optically induced dielectrophoresis (ODEP) device such that the critical medium replacement process was performed automatically and the cells could be subsequently manipulated by using digitally projected optical images. ODEP has been demonstrated to generate sufficient forces for manipulating particles/cells by projecting a light pattern onto photoconductive materials which creates virtual electrodes. The production of the ODEP force usually requires a medium that has a suitable electrical conductivity and an appropriate dielectric constant. Therefore, a 0.2 M sucrose solution is commonly used. However, this requires a complicated medium replacement process before one is able to manipulate cells. Furthermore, the 0.2 M sucrose solution is not suitable for the long-term viability of cells. In comparison to conventional manual processes, our automated medium replacement process only took 25 minutes. Experimental data showed that there was up to a 96.2% recovery rate for the manipulated cells. More importantly, the survival rate of the cells was greatly enhanced due to this faster automated process. This newly developed microfluidic chip provided a promising platform for the rapid replacement of the cell medium and this was also the first time that an ODEP device was integrated with other active flow control components in a microfluidic device. By improving cell viability after cell manipulation, this design may contribute to the practical integration of ODEP modules into other lab-on-a-chip devices and biomedical applications in the future. PMID:24911448

  4. Evaluation of the viability of cultured corneal endothelial cells by quantitative electron probe X-ray microanalysis.

    PubMed

    Alaminos, M; Sanchez-Quevedo, M C; Muñoz-Avila, J I; García, J M; Crespo, P V; González-Andrades, M; Campos, A

    2007-06-01

    Construction of artificial organs and tissues by tissue engineering is strongly dependent on the availability of viable cells. For that reason, the viability and the physiological status of cells kept in culture must be evaluated before the cells can be used for clinical purposes. In this work, we determined the viability of isolated rabbit corneal endothelial cells by trypan blue staining and quantitative electron probe X-ray microanalysis. Our results showed that the ionic content of potassium in cultured corneal endothelial cells tended to rise initially, but significantly decreased in cells in the fifth (and final) subculture, especially in comparison to cells in the fourth subculture (P < 0.001). However, the concentration of sulfur was higher in the fifth subculture than in the fourth subculture (P < 0.001), with a nonsignificant increase in sodium in the fifth subculture (P = 0.031). These data imply a remarkable decrease in the K/Na ratio from the fourth to the fifth subculture. Our microanalytical results, along with the morphological differences between cells in the last two subcultures, are compatible with an early phase of the preapoptotic process in the fifth subculture, and suggest that cells of the first four subcultures would be better candidates for tissue engineering.

  5. Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT

    NASA Astrophysics Data System (ADS)

    Jung, Yookyung; Klein, Oliver J.; Wang, Hequn; Evans, Conor L.

    2016-06-01

    Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation.

  6. Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT

    PubMed Central

    Jung, Yookyung; Klein, Oliver J.; Wang, Hequn; Evans, Conor L.

    2016-01-01

    Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation. PMID:27248849

  7. The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

    SciTech Connect

    Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas; Simos, George

    2010-07-16

    Research highlights: {yields} Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; {yields} Kaempferol causes cytoplasmic mislocalization of HIF-1{alpha} by impairing the MAPK pathway. {yields} Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1{alpha} subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1{alpha} as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC{sub 50} = 5.16 {mu}M). The mechanism of this inhibition did not involve suppression of HIF-1{alpha} protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC{sub 50} = 4.75 {mu}M). Exposure of Huh7 cells to 10 {mu}{Mu} kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 {mu}M) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.

  8. Factors affecting the efficacy of pressure inactivation of Escherichia coli O157:H7 on alfalfa seeds and seed viability.

    PubMed

    Neetoo, Hudaa; Ye, Mu; Chen, Haiqiang

    2009-05-31

    The application of high hydrostatic pressure technology as a seed decontamination technology was evaluated. Alfalfa seeds inoculated with approximately 10(5) CFU/g of Escherichia coli O157:H7 were subjected to oscillatory pressure treatments at 600 MPa and 20 degrees C for up to five cycles with a holding time of 2 min/cycle. However, oscillatory pressurization was not able to eliminate E. coli O157:H7. The application of pressure treatment at 600 MPa for 2 min at 20 degrees C in the presence of chemicals such as calcium hypochlorite, calcium hydroxide, lactic acid or sodium acid sulfate was subsequently investigated and it was demonstrated that this "multiple hurdle" approach was unable to decontaminate alfalfa seeds. Soaking seeds prior to pressure treatment was found to play a critical role on enhancing the pressure inactivation of E. coli O157:H7; seeds soaked in water for 60 min followed by treatment at 600 MPa for 2 min at 20 degrees C were decontaminated and had a germination rate of 91% which was 4% lower than that of the untreated seeds (not statistically significant, P>0.05). It was further demonstrated that a process involving soaking seeds in water for > or = 10 min followed by treatment at 600 MPa for 15 min at 20 degrees C was equally effective with respect to E. coli O157:H7 elimination and viability retention of the seeds.

  9. Evaluation and comparison of efficacy of three different storage media, coconut water, propolis, and oral rehydration solution, in maintaining the viability of periodontal ligament cells

    PubMed Central

    Sanghavi, Tulsi; Shah, Nimisha; Parekh, Vaishali; Singbal, Kiran

    2013-01-01

    Background: Two of the most critical factors affecting the prognosis of an avulsed tooth after replantation are extra oral dry time and the storage medium in which the tooth is placed before treatment is rendered. However, the ability of a storage/transport medium to support cell viability can be more important than the extra oral time to prevent ankylosis and replacement resorption. Aim: Purpose of this study was evaluation and comparison of efficacy of a new storage medium, oral rehydration solution (ORS) with coconut water, and propolis in maintaining the viability of periodontal ligament (PDL) cells by using a collagenase-dispase assay. Materials and Methods: 40 teeth were selected with intact crown which were advised for Orthodontic extraction having healthy PDL. Teeth were then randomly divided into three experimental storage solution groups. Other 10 were divided into positive and negative control groups (5 each). Statistical Analysis and Result: The results were statistically analyzed with analysis of variance and multiple range by using post hoc tests. The results of the prevailing study indicated that coconut water group demonstrated a significantly higher number of viable PDL cells than propolis 50%, and ORS. There was no significant difference between coconut water and propolis 50% groups. PMID:23349581

  10. New strategy for enhancing in situ cell viability of cell-printing process via piezoelectric transducer-assisted three-dimensional printing.

    PubMed

    Koo, YoungWon; Kim, GeunHyung

    2016-06-01

    Tissue engineering has become one of the great applications of three-dimensional cell printing because of the possibility of fabricating complex cell-laden scaffolds. Three typical methods (inkjet, micro-extrusion, and laser-assisted bio-printing) have been used to fabricate structures. Of these, micro-extrusion is a comparatively easy method, but has some drawbacks such as low in situ cell viability after fabricating cell-laden structures because of the high wall shear stress in micro-sized nozzles. To overcome this shortcoming, we suggest an innovative cell printing method, which is assisted by a piezoelectric transducer (PZT). The PZT assistance in the dispensing process enhances the printing efficiency and cell viability by decreasing the wall shear stress within a nozzle because the PZT effect can lower the shear viscosity of the bioink via micro-scale vibration. In this study, 5 wt% cell-laden alginate was used as a bioink, and various PZT conditions (frequencies up to ∼400 Hz and amplitudes up to ∼40.5 μm) were simultaneously applied to the cell-printing process to examine the effectiveness of the PZT. The PZT-assisted cell-printing method was found to be highly effective in direct cell printing and could achieve cell-laden structures with high in situ cell viability. PMID:27203798

  11. Nilotinib reduced the viability of human ovarian cancer cells via mitochondria-dependent apoptosis, independent of JNK activation.

    PubMed

    Chen, Tze-Chien; Yu, Ming-Chih; Chien, Chih-Chiang; Wu, Ming-Shun; Lee, Yu-Chieh; Chen, Yen-Chou

    2016-03-01

    Nilotinib (AMN) induces apoptosis in various cancer cells; however the effect of AMN on human ovarian cancer cells is still unclear. A reduction in cell viability associated with the occurrence of apoptotic characteristics was observed in human SKOV-3 ovarian cancer cells under AMN but not sorafenib (SORA) or imatinib (STI) stimulation. Activation of apoptotic pathway including increased caspase (Casp)-3 and poly(ADP-ribose) polymerase 1 (PARP1) protein cleavage by AMN was detected with disrupted mitochondrial membrane potential (MMP) accompanied by decreased Bcl-2 protein and increased cytosolic cytochrome (Cyt) c/cleaved Casp-9 protein expressions was found, and AMN-induced cell death was inhibited by peptidyl Casp inhibitors, VAD, DEVD and LEHD. Increased phosphorylated c-Jun N-terminal kinase (JNK) protein expression was detected in AMN- but not SORA- or STI-treated SKOV-3 cells, and the JNK inhibitors, SP600125 and JNKI, showed slight but significant enhancement of AMN-induced cell death in SKOV-3 cells. The intracellular peroxide level was elevated by AMN and H2O2, and N-acetylcysteine (NAC) prevented H2O2- but not AMN-induced peroxide production and apoptosis in SKOV-3 cells. AMN induction of apoptosis with increased intracellular peroxide production and JNK protein phosphorylation was also identified in human A2780 ovarian cancer cells, cisplatin-resistant A2780CP cells, and clear ES-2 cells. The evidence supporting AMN effectively reducing the viability of human ovarian cancer cells via mitochondrion-dependent apoptosis is provided.

  12. Real-time direct cell concentration and viability determination using a fully automated microfluidic platform for standalone process monitoring.

    PubMed

    Nunes, P S; Kjaerulff, S; Dufva, M; Mogensen, K B

    2015-06-21

    The industrial production of cells has a large unmet need for greater process monitoring, in addition to the standard temperature, pH and oxygen concentration determination. Monitoring the cell health by a vast range of fluorescence cell-based assays can greatly improve the feedback control and thereby ensure optimal cell production, by prolonging the fermentation cycle and increasing the bioreactor output. In this work, we report on the development of a fully automated microfluidic system capable of extracting samples directly from a bioreactor, diluting the sample, staining the cells, and determining the total cell and dead cells concentrations, within a time frame of 10.3 min. The platform consists of custom made stepper motor actuated peristaltic pumps and valves, fluidic interconnections, sample to waste liquid management and image cytometry-based detection. The total concentration of cells is determined by brightfield microscopy, while fluorescence detection is used to detect propidium iodide stained non-viable cells. This method can be incorporated into facilities with bioreactors to monitor the cell concentration and viability during the cultivation process. Here, we demonstrate the microfluidic system performance by monitoring in real time the cell concentration and viability of yeast extracted directly from an in-house made bioreactor. This is the first demonstration of using the Dean drag force, generated due to the implementation of a curved microchannel geometry in conjunction with high flow rates, to promote passive mixing of cell samples and thus homogenization of the diluted cell plug. The autonomous operation of the fluidics furthermore allows implementation of intelligent protocols for administering air bubbles from the bioreactor in the microfluidic system, so that these will be guided away from the imaging region, thereby significantly improving both the robustness of the system and the quality of the data. PMID:25923294

  13. Real-time direct cell concentration and viability determination using a fully automated microfluidic platform for standalone process monitoring.

    PubMed

    Nunes, P S; Kjaerulff, S; Dufva, M; Mogensen, K B

    2015-06-21

    The industrial production of cells has a large unmet need for greater process monitoring, in addition to the standard temperature, pH and oxygen concentration determination. Monitoring the cell health by a vast range of fluorescence cell-based assays can greatly improve the feedback control and thereby ensure optimal cell production, by prolonging the fermentation cycle and increasing the bioreactor output. In this work, we report on the development of a fully automated microfluidic system capable of extracting samples directly from a bioreactor, diluting the sample, staining the cells, and determining the total cell and dead cells concentrations, within a time frame of 10.3 min. The platform consists of custom made stepper motor actuated peristaltic pumps and valves, fluidic interconnections, sample to waste liquid management and image cytometry-based detection. The total concentration of cells is determined by brightfield microscopy, while fluorescence detection is used to detect propidium iodide stained non-viable cells. This method can be incorporated into facilities with bioreactors to monitor the cell concentration and viability during the cultivation process. Here, we demonstrate the microfluidic system performance by monitoring in real time the cell concentration and viability of yeast extracted directly from an in-house made bioreactor. This is the first demonstration of using the Dean drag force, generated due to the implementation of a curved microchannel geometry in conjunction with high flow rates, to promote passive mixing of cell samples and thus homogenization of the diluted cell plug. The autonomous operation of the fluidics furthermore allows implementation of intelligent protocols for administering air bubbles from the bioreactor in the microfluidic system, so that these will be guided away from the imaging region, thereby significantly improving both the robustness of the system and the quality of the data.

  14. Seasonal factors affecting egg production and viability of eggs of Acartia tonsa Dana from East Lagoon, Galveston, Texas

    NASA Astrophysics Data System (ADS)

    Ambler, Julie Weills

    1985-06-01

    Egg production and hatching success were determined between March 1981 and March 1982 for the copepod Acartia tonsa Dana from East Lagoon, Galveston, Texas. During three-day experiments in the laboratory, field collected females were fed diets of (1) natural particles collected over the water column, (2) some modification of this and (3) the diatom Thalassiosira weissflogii. The latter served to isolate the effects of temperature and salinity from the effects of the natural particle diets on egg production. Specific rates of egg production, i.e. μg egg biomass/μg body biomass/time, were consistently higher per unit carbon than nitrogen. The influence of seasonal factors on egg production was compared. Salinity was inversely correlated with egg production, but had less effect than temperature. Positive correlations with temperature were always higher for specific rates per unit nitrogen than carbon. At 15°C, females produced ˜0·25 of their body carbon (or nitrogen) as eggs per day, whereas at 28°C, they produced at least their own biomass as eggs per day (up to 1·80). These high rates of A. tonsa were probably due to its reproductive biology and adaptation to the subtropical habitat as well as the high temperatures and food concentrations. The correlation between specific egg production rate and temperature was less with the natural particle diets than with the unialgal diet. This indicated that the quantity or quality of natural particle assemblages in East Lagoon influenced egg production. Egg viability was highest in the spring, but was not related to diet or the percentage of females with spermatophores.

  15. Caffeic Acid Reduces the Viability and Migration Rate of Oral Carcinoma Cells (SCC-25) Exposed to Low Concentrations of Ethanol

    PubMed Central

    Dziedzic, Arkadiusz; Kubina, Robert; Kabała-Dzik, Agata; Wojtyczka, Robert D.; Morawiec, Tadeusz; Bułdak, Rafał J.

    2014-01-01

    Alcohol increases the risk of carcinoma originated from oral epithelium, but the biological effects of ultra-low doses of ethanol on existing carcinoma cells in combination with natural substances are still unclear. A role for ethanol (EtOH), taken in small amounts as an ingredient of some beverages or mouthwashes to change the growth behavior of established squamous cell carcinoma, has still not been examined sufficiently. We designed an in vitro study to determine the effect of caffeic acid (CFA) on viability and migration ability of malignant oral epithelial keratinocytes, exposed to ultra-low concentrations (maximum 100 mmol/L) EtOH. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-dimethyltetrazolium bromide) and LDH (lactate dehydrogenase) assays were used to assess the cytotoxic effect of EtOH/CFA and the viability of squamous carcinoma SCC-25 cells (ATCC CRL-1628, mobile part of the tongue). Tested EtOH concentrations were: 2.5, 5, 10, 25, 50, and 100 mmol/L, along with an equal CFA concentration of 50 μmol/L. Carcinoma cells’ migration was investigated by monolayer “wound” healing assay. We demonstrated that very low concentrations of EtOH ranging between 2.5 and 10 mmol/L may induce the viability of oral squamous cell carcinoma cells, while the results following addition of CFA reveal an antagonistic effect, attenuating pro-proliferative EtOH activity. The migration rate of oral squamous carcinoma cells can be significantly inhibited by the biological activity of caffeic acid. PMID:25329614

  16. Modulation of breast cancer cell viability by a cannabinoid receptor 2 agonist, JWH-015, is calcium dependent

    PubMed Central

    Hanlon, Katherine E; Lozano-Ondoua, Alysia N; Umaretiya, Puja J; Symons-Liguori, Ashley M; Chandramouli, Anupama; Moy, Jamie K; Kwass, William K; Mantyh, Patrick W; Nelson, Mark A; Vanderah, Todd W

    2016-01-01

    Introduction Cannabinoid compounds, both nonspecific as well as agonists selective for either cannabinoid receptor 1 (CB1) or cannabinoid receptor 2 (CB2), have been shown to modulate the tumor microenvironment by inducing apoptosis in tumor cells in several model systems. The mechanism of this modulation remains only partially delineated, and activity induced via the CB1 and CB2 receptors may be distinct despite significant sequence homology and structural similarity of ligands. Methods The CB2-selective agonist JWH-015 was used to investigate mechanisms downstream of CB2 activation in mouse and human breast cancer cell lines in vitro and in a murine mammary tumor model. Results JWH-015 treatment significantly reduced primary tumor burden and metastasis of luciferase-tagged murine mammary carcinoma 4T1 cells in immunocompetent mice in vivo. Furthermore, JWH-015 reduced the viability of murine 4T1 and human MCF7 mammary carcinoma cells in vitro by inducing apoptosis. JWH-015-mediated reduction of breast cancer cell viability was not dependent on Gαi signaling in vitro or modified by classical pharmacological blockade of CB1, GPR55, TRPV1, or TRPA1 receptors. JWH-015 effects were calcium dependent and induced changes in MAPK/ERK signaling. Conclusion The results of this work characterize the actions of a CB2-selective agonist on breast cancer cells in a syngeneic murine model representing how a clinical presentation of cancer progression and metastasis may be significantly modulated by a G-protein-coupled receptor. PMID:27186076

  17. Cell viability viscoelastic measurement in a rheometer used to stress and engineer tissues at low sonic frequencies1

    PubMed Central

    Klemuk, Sarah A.; Jaiswal, Sanyukta; Titze, Ingo R.

    2008-01-01

    Effects of vibration on human vocal fold extracellular matrix composition and the resultant tissue viscoelastic properties are difficult to study in vivo. Therefore, an in vitro bioreactor, simulating the in vivo physiological environment, was explored. A stress-controlled commercial rheometer was used to administer shear vibrations to living tissues at stresses and frequencies corresponding to male phonation, while simultaneously measuring tissue viscoelastic properties. Tissue environment was evaluated and adjustments made in order to sustain cell life for short term experimentation up to 6 h. Cell nutrient medium evaporation, osmolality, pH, and cell viability of cells cultured in three-dimensional synthetic scaffolds were quantified under comparably challenging environments to the rheometer bioreactor for 4 or 6 h. The functionality of the rheometer bioreactor was demonstrated by applying three vibration regimes to cell-seeded three-dimensional substrates for 2 h. Resulting strain was quantified throughout the test period. Rheologic data and cell viability are reported for each condition, and future improvements are discussed. PMID:19062871

  18. Comparison of cell viability and morphology of a human osteoblast-like cell line (SaOS-2) seeded on various bone substitute materials: An in vitro study

    PubMed Central

    Ayobian-Markazi, Nader; Fourootan, T.; Kharazifar, M. J.

    2012-01-01

    Background: Many studies have shown favorable results following the use of different bone graft materials. The aim of the present study was to evaluate the biocompatibility of four different bone graft materials regarding cell viability and morphology of Human osteoblast-like cells (SaOS-2) in vitro. Materials and Methods: The effects of Bio-Oss®, Tutodent®, Osteon®, and Cerasorb® were studied on the human osteoblast-like cell line to evaluate various parameters. Human osteoblast-like cells were seeded onto the mentioned bone substitute materials (BSMs). Cell differentiation; cell viability and alkaline phosphatase (ALP) activity of the seeded cells were evaluated by means of scanning electron microscopy, cell viability test and phase contrast microscopy Analysis of variance (ANOVA). Tamhane's post-hoc, Kruskal-Wallis Test, and Dunn's Test were used. The results were considered to be statistically significant at P<0.05. Results: The control group (SaOS-2 cells which were incubated in Dulbecco Modified Eagle Medium without any kind of bone graft materials) had the highest level of cell viability (P<0.001), followed by Tutodent®, Osteon®, Cerasorb®, and Bio-Oss®. There was no significant difference in MTT assay results between Tutodent® and the control group (P=0.032). All tested bone graft materials showed significantly higher ALP activity than the control (P<0.001). The Tutodent® group showed the best cell growth among all experimental groups, followed by the Osteon® group. The former had a higher spindle-like morphology with good attachment to the surface. Cells cultivated on the surfaces of the Cerasorb® and Bio-Oss® granules had more round morphologies. Conclusion: This in vitro study demonstrated that all tested BSMs can provide good cell differentiation but a lower rate of proliferation. PMID:22363369

  19. Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment

    PubMed Central

    Ortiz-Espín, Ana; Locato, Vittoria; Camejo, Daymi; Schiermeyer, Andreas; De Gara, Laura; Sevilla, Francisca; Jiménez, Ana

    2015-01-01

    Background and Aims Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H2O2. Methods Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H2O2 and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H2O2. Analysis of different hallmarks of programmed cell death was also carried out. Key Results Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H2O2, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H2O2 and nitric oxide contents and maintenance of the glutathione redox state were observed. Conclusions A decreased content of endogenous H2O2 may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and

  20. Aptamer–polymer functionalized silicon nanosubstrates for enhanced recovered circulating tumor cell viability and in vitro chemosensitivity testing

    PubMed Central

    Shen, Qinglin; Peng, Caixia; Zhan, Yan; Fan, Liang; Wang, Mengyi; Zhou, Qing; Liu, Jue; Lv, Xiaojuan; Tang, Qiu; Li, Jun; Huang, Xiaodong; Xia, Jiahong

    2016-01-01

    Selection of the optimal chemotherapy regimen for an individual cancer patient is challenging. The existing chemosensitivity tests are costly, time-consuming, and not amenable to wide utilization within a clinic. This limitation might be addressed by the recently proposed use of circulating tumor cells (CTCs), which provide an opportunity to noninvasively monitor response to therapy. Over the past few decades, various techniques were developed to capture and recover CTCs, but these techniques were often limited by a capture and recovery performance tradeoff between high viability and high efficiency. In this work, we used anti-epithelial cell adhesion molecule coated aptamer–poly (N-isopropylacrylamide) functionalized silicon nanowire substrates to capture and release epithelial cell adhesion molecule-positive CTCs at 32°C and 4°C, respectively. Then, we applied the nuclease to digest the aptamer to release the captured CTCs (near or at the end of the polymer brush), which cannot be released by heating/cooling process. High viability and purity CTCs could be achieved by decreasing the heating/cooling cycles and enzymatic treatment rounds. Furthermore, the time-saving process is helpful to maintain the morphology and enhance vitality of the recovered CTCs and is beneficial to the subsequent cell culture in vitro. We validated the feasibility of chemosensitivity testing based on the recovered HCC827 cells using an adenosine triphosphate–tumor chemosensitivity assay, and the results suggested that our method can determine which agent and what concentration have the best chemosensitivity for the culturing recovered CTCs. So, the novel method capable of a highly effective capture and recovery of high viability CTCs will pave the way for chemosensitivity testing. PMID:27274239

  1. Viability of hydroxyethyl methacrylate-methyl methacrylate-microencapsulated PC12 cells after omental pouch implantation within agarose gels.

    PubMed

    Fleming, A J; Sefton, M V

    2003-10-01

    Hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA, 75 mol% HEMA). Microcapsules containing viable PC12 cells (as an allogeneic transplant model) were implanted into omental pouches in Wistar rats. Two different capsule preparations were tested, based on differences in polymer solutions during extrusion: 10% HEMA-MMA in TEG, and 9% HEMA-MMA in TEG with 30% poly(vinyl pyrrolidone) (PVP). The omental pouch proved to be an ideal transplant site in terms of implantation, recovery, and blood vessel proximity (nutrient supply). To minimize the fibrous overgrowth and damaged capsules previously seen on implantation of individual capsules, agarose gels were used to embed the capsules before implantation. Cells proliferated within the microcapsule-agarose device during the first 7 days of implantation, but overall cell viability declined over the 3-week period, when compared with similar capsules maintained in vitro. Nonetheless, approximately 50% of the initial encapsulated cells were still viable after 3 weeks in vivo. This approach to HEMA-MMA microcapsule implantation improved cell viability and capsule integrity after 3 weeks in vivo, compared with capsules implanted without agarose.

  2. Effect of methoxychlor on Ca2+ handling and viability in OC2 human oral cancer cells.

    PubMed

    Tseng, Li-Ling; Shu, Su-Shung; Kuo, Chun-Chi; Chou, Chiang-Ting; Hsieh, Yao-Dung; Chu, Sau-Tung; Chi, Chao-Chuan; Liang, Wei-Zhe; Ho, Chin-Man; Jan, Chung-Ren

    2011-05-01

    The effect of the insecticide methoxychlor on the physiology of oral cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) in human oral cancer cells (OC2) by using the Ca(2+)-sensitive fluorescent dye fura-2. Methoxychlor at 5-20 μM increased [Ca(2+)](i) in a concentration-dependent manner. The signal was reduced by 70% by removing extracellular Ca(2+). Methoxychlor-induced Ca(2+) entry was not affected by nifedipine, econazole, SK&F96365 and protein kinase C modulators but was inhibited by the phospholipase A2 inhibitor aristolochic acid. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished methoxychlor-induced [Ca(2+)](i) rise. Incubation with methoxychlor also inhibited thapsigargin- or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 did not alter methoxychlor-induced [Ca(2+)](i) rise. At 5-20 μM, methoxychlor killed cells in a concentration-dependent manner. The cytotoxic effect of methoxychlor was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM). Annexin V-FITC data suggest that methoxychlor (10 and 20 μM) evoked apoptosis in a concentration-dependent manner. Together, in human OC2, methoxychlor induced a [Ca(2+)](i) rise probably by inducing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive channels. Methoxychlor induced cell death that may involve apoptosis.

  3. Role of different vehicles in carotenoids delivery and their influence on cell viability, cell cycle progression, and induction of apoptosis in HeLa cells.

    PubMed

    Sowmya, Poorigali Raghavendra-Rao; Arathi, Bangalore Prabhashankar; Vijay, Kariyappa; Baskaran, Vallikannan; Lakshminarayana, Rangaswamy

    2015-08-01

    The objective of the present study was to determine the role of different vehicles in carotenoids delivery and their influence on cell viability, cell cycle progression and induction of apoptosis in HeLa cells. Cells (5 × 10(3)) were treated with different concentrations (25-100 µM) of β-carotene (BC) or lutein (L) or astaxanthin (AST) dissolved in 0.5% of tetrahydrofuran (THF), dimethylsulfoxide (DMSO), and fetal bovine serum (FBS), respectively. The effect of delivery vehicle on carotenoids uptake, cytotoxicity, oxidative status, cell cycle distribution, and apoptosis was examined after 48 h of incubation. The results shown that, cell viability reduced significantly in a dose- and time-dependent manner irrespective of carotenoid delivered in vehicles. Cellular uptake of BC delivered in THF was higher by 49.1, 29.7% and L delivered through THF was higher by 41.7 and 37.5% than DMSO and FBS, respectively. While, AST delivered through DMSO was higher by 36.1 and 43.7% than the THF and FBS, respectively. In case of cells treated either with BC or L delivered through THF and AST in DMSO decreased the glutathione and increased the malondialdehyde levels. The net increase in the G 2/M phase percentage of cell cycle progression was observed in carotenoid-treated cells. The % induction of apoptosis by BC or L delivered with THF and AST in DMSO was higher than other treated groups. In conclusion, choice of suitable vehicle for specific carotenoids delivery is essential that in turn may influence on cell proliferation and cell-based assays.

  4. Measuring and modeling of binary mixture effects of pharmaceuticals and nickel on cell viability/cytotoxicity in the human hepatoma derived cell line HepG2

    SciTech Connect

    Rudzok, S.; Schlink, U.; Herbarth, O.; Bauer, M.

    2010-05-01

    The interaction of drugs and non-therapeutic xenobiotics constitutes a central role in human health risk assessment. Still, available data are rare. Two different models have been established to predict mixture toxicity from single dose data, namely, the concentration addition (CA) and independent action (IA) model. However, chemicals can also act synergistic or antagonistic or in dose level deviation, or in a dose ratio dependent deviation. In the present study we used the MIXTOX model (EU project ENV4-CT97-0507), which incorporates these algorithms, to assess effects of the binary mixtures in the human hepatoma cell line HepG2. These cells possess a liver-like enzyme pattern and a variety of xenobiotic-metabolizing enzymes (phases I and II). We tested binary mixtures of the metal nickel, the anti-inflammatory drug diclofenac, and the antibiotic agent irgasan and compared the experimental data to the mathematical models. Cell viability was determined by three different methods the MTT-, AlamarBlue (registered) and NRU assay. The compounds were tested separately and in combinations. We could show that the metal nickel is the dominant component in the mixture, affecting an antagonism at low-dose levels and a synergism at high-dose levels in combination with diclofenac or irgasan, when using the NRU and the AlamarBlue assay. The dose-response surface of irgasan and diclofenac indicated a concentration addition. The experimental data could be described by the algorithms with a regression of up to 90%, revealing the HepG2 cell line and the MIXTOX model as valuable tool for risk assessment of binary mixtures for cytotoxic endpoints. However the model failed to predict a specific mode of action, the CYP1A1 enzyme activity.

  5. Imaging Cell Viability on Non-transparent Scaffolds - Using the Example of a Novel Knitted Titanium Implant.

    PubMed

    Tendulkar, Gauri; Grau, Phillip; Ziegler, Patrick; Buck, Alfred; Buck, Alfred; Badke, Andreas; Kaps, Hans-Peter; Ehnert, Sabrina; Nussler, Andreas K

    2016-01-01

    Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated. This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds. PMID:27684965

  6. Biotransformed soybean extract (BSE) inhibits melanoma cell growth and viability in vitro: involvement of nuclear factor-kappa B signaling.

    PubMed

    Vilela, Fernanda Maria Pinto; Syed, Deeba N; Chamcheu, Jean Christopher; Calvo-Castro, Laura A; Fortes, Vanessa Silveira; Fonseca, Maria José Vieira; Mukhtar, Hasan

    2014-01-01

    Melanoma is recognized as one of the most aggressive cancers with a relatively high propensity for metastasis. The prognosis of melanoma remains poor in spite of treatment advances, emphasizing the importance of additional preventive measures. Isoflavonoids have become not only potential chemopreventive, but also important therapeutic natural agents. We evaluated the antiproliferative and proapoptotic properties of biotransformed soybean extract (BSE) in A375 melanoma cells. Previous analyses demonstrated that the concentration of daidzein, genistein and aminoacids/peptides present in BSE, fermented by Aspergillus awamori is much higher than in the non biotransformed extract (NBSE). Experiments comparing the efficacy of the extracts in preventing cancer cell growth showed that treatment (24 h) of aggressive melanoma cells (A375 and 451Lu) with BSE resulted in a dose-dependent inhibition of growth and viability. In contrast, treatment with similar doses of NBSE failed to inhibit melanoma cell viability. Further studies in A375 cells showed that decrease in cell viability with BSE treatment (1.5-1.9 mg/ml; 24 h) was associated with induction of apoptosis. Immunoblot analysis revealed that BSE treatment resulted in induction of PARP cleavage, activation of caspase-3, -7, and -8 and increased expression of TRAIL and its receptor DR4. BSE did not activate the intrinsic apoptotic pathway in A375 cells, as no change was observed in caspase-9 expression. The expression of Bcl-2 apoptotic proteins such as Bid and Bax remained unaffected with BSE treated cells. Interestingly, we also showed that BSE treatment increased the phosphorylation and activation of IKK, IκBα degradation and p65/NF-κB translocation to the nucleus, and that stimulation of the NF-???B pathway was required for BSE-induced apoptosis of A375 cells. Our findings indicate that the biotransformation of soybean plays a crucial role in the extract anti-cancer effect observed in melanoma cells. However

  7. Viability of Salmonella and Listeria monocytogenes in delicatessen salads and hummus as affected by sodium content and storage temperature.

    PubMed

    Alali, Walid Q; Mann, David A; Beuchat, Larry R

    2012-06-01

    A study was conducted to determine survival and growth behavior of Salmonella and Listeria monocytogenes in commercially prepared mayonnaise-based potato salad, macaroni salad, and coleslaw and in hummus (initial mean pH values were 4.80 to 4.94, 4.18 to 4.31, 3.87, and 4.50 to 4.52, respectively) as affected by sodium concentration (133 to 364, 190 to 336, 146 to 272, and 264 to 728 mg/100 g, respectively) and storage at 4 or 10°C for up to 27 days. Salmonella did not grow in any of the test products. Initial populations (2.02 to 2.38 log CFU/g) decreased in coleslaw to undetectable levels (<1 CFU/25 g) within 13 days and in most formulations of macaroni salad within 20 to 27 days. Salmonella survived in highest numbers in potato salad and hummus. The presence of added sodium in macaroni salad stored at 4°C and hummus stored at 4 or 10°C appeared to protect Salmonella against inactivation. L. monocytogenes, at an initial population of 1.86 to 2.23 log CFU/g, did not grow in test products, but with the exception of coleslaw containing sodium at a concentration used in the standard (control) recipe, this pathogen was detected by direct plating (≥ 1.0 log CFU/g) in all products stored at 4 or 10°C for 27 days. L. monocytogenes populations were significantly (P < 0.05) lower in potato salad and hummus with no added sodium than in test products with added sodium after storage at 4°C. Sodium concentration did not markedly affect aerobic plate counts over the 27-day storage period. Results confirm that the acidic pH of mayonnaise-based salads and hummus is a major factor preventing growth and influencing rates of inactivation of Salmonella and L. monocytogenes. In the absence of added sodium, death of these bacteria may be more rapid. However, in general decreasing or increasing the sodium concentration in selected delicatessen salad and hummus recipes does not markedly affect the behavior of Salmonella and L. monocytogenes when products are stored at 4 or 10

  8. Immunization of mice and baboons with the recombinant Sm28GST affects both worm viability and fecundity after experimental infection with Schistosoma mansoni.

    PubMed

    Boulanger, D; Reid, G D; Sturrock, R F; Wolowczuk, I; Balloul, J M; Grezel, D; Pierce, R J; Otieno, M F; Guerret, S; Grimaud, J A

    1991-09-01

    A member of the glutathione S-transferase family, Sm28GST has previously demonstrated a good ability to protect rodents against experimental infection with Schistosoma mansoni. In order to evaluate its efficacy in a model closer to man, two different protocols of immunization with recombinant Sm28GST were tested on baboons in a large-scale trial. Three injections in the presence of aluminium hydroxide as adjuvant resulted in a significant 38% reduction in the adult worm burden together with a trend for a lower percentage of inflammatory tissue in the liver. Individual levels of protection, ranging from 0 to 80%, underlined the heterogeneity of the immune response to this purified molecule in outbred primates. On the other hand, two injections of Sm28GST in the presence of aluminium hydroxide and Bordetella pertussis reduced female schistosome fecundity by 33%, with a more pronounced effect (66%) on faecal egg output; there was also a trend, in this protocol, for decrease of the mean granuloma surface in the liver. Individual anti-Sm28GST IgG antibodies were apparently unrelated to levels of immunity, but there was partial evidence that cytophilic IgE might play a role in the immune mechanisms affecting worm viability, but not fecundity. In the mouse model, Sm28GST vaccination resulted in a lower hatching ability of tissue eggs recovered from immunized mice whereas passive transfer of specific anti-Sm28GST T-lymphocytes, one day before infection, significantly reduced the number of eggs in the liver of mice. We propose that different protocols of immunization with a recombinant molecule can impede Schistosoma mansoni worm viability and fecundity, but can also affect miracidium physiology, with important consequences for disease transmission and granuloma-derived pathology.

  9. A novel small molecule STAT3 inhibitor, LY5, inhibits cell viability, colony formation, and migration of colon and liver cancer cells

    PubMed Central

    Yu, Wenying; Jou, David; Wang, Yina; Ma, Haiyan; Xiao, Hui; Qin, Hua; Zhang, Cuntai; Lü, Jiagao; Li, Sheng; Li, Chenglong; Lin, Jiayuh; Lin, Li

    2016-01-01

    Signal Transducer and Activator of Transcription 3 (STAT3) is persistently activated in human liver and colon cancer cells and is required for cancer cell viability, survival and migration. Therefore, inhibition of STAT3 signaling may be a viable therapeutic approach for these two cancers. We recently designed a non-peptide small molecule STAT3 inhibitor, LY5, using in silico site-directed Fragment-based drug design (FBDD). The inhibitory effect on STAT3 phosphorylation, cell viability, migration and colony forming ability by LY5 were examined in human liver and colon cancer cells. We demonstrated that LY5 inhibited constitutive Interleukin-6 (IL-6)-induced STAT3 phosphorylation, STAT3 nuclear translocation, decreased STAT3 downstream targeted gene expression and induced apoptosis in liver and colon cancer cells. LY5 had little effect on STAT1 phosphorylation mediated by IFN-γ. Inhibition of persistent STAT3 phosphorylation by LY5 also inhibited colony formation, cell migration, and decreased the viability of liver cancer and colon cancer cells. Furthermore, LY5 inhibited STAT3 phosphorylation and suppressed colon tumor growth in a mouse model in vivo. Our results suggest that LY5 is a potent STAT3 inhibitor and may be a potential drug candidate for liver and colon cancer therapy. PMID:26883202

  10. A novel small molecule STAT3 inhibitor, LY5, inhibits cell viability, colony formation, and migration of colon and liver cancer cells.

    PubMed

    Zhao, Chongqiang; Wang, Wenlong; Yu, Wenying; Jou, David; Wang, Yina; Ma, Haiyan; Xiao, Hui; Qin, Hua; Zhang, Cuntai; Lü, Jiagao; Li, Sheng; Li, Chenglong; Lin, Jiayuh; Lin, Li

    2016-03-15

    Signal Transducer and Activator of Transcription 3 (STAT3) is persistently activated in human liver and colon cancer cells and is required for cancer cell viability, survival and migration. Therefore, inhibition of STAT3 signaling may be a viable therapeutic approach for these two cancers. We recently designed a non-peptide small molecule STAT3 inhibitor, LY5, using in silico site-directed Fragment-based drug design (FBDD). The inhibitory effect on STAT3 phosphorylation, cell viability, migration and colony forming ability by LY5 were examined in human liver and colon cancer cells. We demonstrated that LY5 inhibited constitutive Interleukin-6 (IL-6)-induced STAT3 phosphorylation, STAT3 nuclear translocation, decreased STAT3 downstream targeted gene expression and induced apoptosis in liver and colon cancer cells. LY5 had little effect on STAT1 phosphorylation mediated by IFN-γ. Inhibition of persistent STAT3 phosphorylation by LY5 also inhibited colony formation, cell migration, and decreased the viability of liver cancer and colon cancer cells. Furthermore, LY5 inhibited STAT3 phosphorylation and suppressed colon tumor growth in a mouse model in vivo. Our results suggest that LY5 is a potent STAT3 inhibitor and may be a potential drug candidate for liver and colon cancer therapy. PMID:26883202

  11. Identification of luteolin as enterovirus 71 and coxsackievirus A16 inhibitors through reporter viruses and cell viability-based screening.

    PubMed

    Xu, Lin; Su, Weiheng; Jin, Jun; Chen, Jiawen; Li, Xiaojun; Zhang, Xuyuan; Sun, Meiyan; Sun, Shiyang; Fan, Peihu; An, Dong; Zhang, Huafei; Zhang, Xiguang; Kong, Wei; Ma, Tonghui; Jiang, Chunlai

    2014-07-17

    Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability‑based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 μM. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs.

  12. Cell viability of microencapsulated Bifidobacterium animalis subsp. lactis under freeze-drying, storage and gastrointestinal tract simulation conditions.

    PubMed

    Shamekhi, Fatemeh; Shuhaimi, Mustafa; Ariff, Arbakariya; Manap, Yazid A

    2013-03-01

    The purpose of this study was to improve the survival of Bifidobacterium animalis subsp. lactis 10140 during freeze-drying process by microencapsulation, using a special pediatric prebiotics mixture (galactooligosaccharides and fructooligosaccharides). Probiotic microorganisms were encapsulated with a coat combination of prebiotics-calcium-alginate prior to freeze-drying. Both encapsulated and free cells were then freeze-dried in their optimized combinations of skim milk and prebiotics. Response surface methodology (RSM) was used to produce a coating combination as well as drying medium with the highest cell viability during freeze-drying. The optimum encapsulation composition was found to be 2.1 % Na-alginate, 2.9 % prebiotic, and 21.7 % glycerol. Maximum survival predicted by the model was 81.2 %. No significant (p > 0.05) difference between the predicted and experimental values verified the adequacy of final reduced models. The protection ability of encapsulation was then examined over 120 days of storage at 4 and 25 °C and exposure to a sequential model of infantile GIT conditions including both gastric conditions (pH 3.0 and 4.0, 90 min, 37 °C) and intestinal conditions (pH 7.5, 5 h, 37 °C). Significantly improved cell viability showed that microencapsulation of B. lactis 10140 with the prebiotics was successful in producing a stable symbiotic powdery nutraceutical.

  13. Identification of Luteolin as Enterovirus 71 and Coxsackievirus A16 Inhibitors through Reporter Viruses and Cell Viability-Based Screening

    PubMed Central

    Xu, Lin; Su, Weiheng; Jin, Jun; Chen, Jiawen; Li, Xiaojun; Zhang, Xuyuan; Sun, Meiyan; Sun, Shiyang; Fan, Peihu; An, Dong; Zhang, Huafei; Zhang, Xiguang; Kong, Wei; Ma, Tonghui; Jiang, Chunlai

    2014-01-01

    Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability‑based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 μM. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs. PMID:25036464

  14. Plasma sprayed cerium oxide coating inhibits H2O2-induced oxidative stress and supports cell viability.

    PubMed

    Li, Kai; Xie, Youtao; You, Mingyu; Huang, Liping; Zheng, Xuebin

    2016-06-01

    Oxidative stress is a risk factor in the pathogenesis of osteoporosis, and plays a major role in bone regeneration of osteoporotic patients. Cerium oxide (CeO2) ceramics have the unique ability to protect various types of cells from oxidative damage, making them attractive for biomedical applications. In this study, we developed a plasma sprayed CeO2 coating with a hierarchical topography where ceria nanoparticles were superimposed in the micro-rough coating surface. The protective effects of the CeO2 coating on the response of osteoblasts to H2O2-induced oxidative stress have been demonstrated in terms of cell viability, apoptosis and differentiation. The CeO2 coating reversed the reduced superoxide dismutase activity, decreased reactive oxygen species production and suppressed malondialdehyde formation in H2O2-treated osteoblasts. It indicated that the CeO2 coating can preserve the intracellular antioxidant defense system. The cytocompatibility of the CeO2 coating was further assessed in vitro by cell viability assay and scanning electron microscopy analysis. Taken together, the CeO2 coating could provide an opportunity to be utilized as a potential candidate for bone regeneration under oxidative stress.

  15. Diagnostic performance and application of two commercial cell viability assays in foot-and-mouth disease research.

    PubMed

    Willems, Tom; Lefebvre, David J; Neyts, Johan; De Clercq, Kris

    2011-04-01

    Cell-based assays are still used widely in foot-and-mouth disease (FMD) research, despite the existence of a wide variety of molecular techniques. The aim of this study was to validate an automated, quantitative spectrometric reading to replace the time-consuming and subjective microscopic (MIC) evaluation of the FMD virus-induced cytopathic effect (CPE). Therefore, the diagnostic performance of two commercial cell viability assays (CellTiter 96(®) AQueous One Solution Cell Proliferation Assay (MTS) and CellTiter-Blue(®) Cell Viability Assay (CTB), both from Promega, Leiden, The Netherlands) was evaluated. Following optimization of the assay protocols and using the MIC results as a reference standard, the absorbance-read MTS assay, the fluorescence-read CTB assay and the absorbance-read CTB (CTB(abs)) assay demonstrated similar high sensitivities (97%, 99% and 98%, respectively), specificities (100%, 98% and 99%, respectively), accuracy measures (0.99, 0.98 and 0.98, respectively), precision measures (1.00, 0.98 and 0.99, respectively) and Cohen kappa agreement indices (0.97, 0.97 and 0.96, respectively) for detecting CPE in cell cultures. Due to its performance, cost effectiveness and ease of use, the CTB(abs) assay was selected for further evaluation of its ability to detect virus neutralization and to screen antiviral compounds. The CTB(abs) assay had 99% sensitivity and 100% specificity for the detection of neutralizing antibodies in sera from cattle infected with FMDV and in sera from unvaccinated, uninfected cattle and resulted in a mean Z'-factor of 0.85 for antiviral compound test plates. The CTB(abs) assay is now used routinely in the Belgian FMD reference laboratory for serological testing and high-throughput antiviral compound screening. PMID:21295609

  16. Supplementation of adjuvants for increasing the nutritive value and cell viability of probiotic fermented milk beverage.

    PubMed

    Shobharani, P; Agrawal, Renu

    2009-01-01

    Probiotic are microorganisms that, upon ingestion in adequate amounts, exert a beneficial effect on the host. In the present work, the potent probiotic Leuconostoc mesenteroides was used as a starter culture in the preparation of fermented milk beverage. The product was analyzed for protein, titrable acidity, fat, total sugar, fatty acids and minerals. The viability of culture and nutrition in the product was further enhanced with supplementation of adjuvants like tryptone, casein hydrolysate, cysteine hydrochloride and ascorbic acid. After 5 days, maximum viability was observed on supplementation of tryptone (100 mg/l). The protein content was enhanced by 1.1-fold in the presence of tryptone (100 mg/l) as compared with control after 5 days of storage. Fermented milk supplemented with tryptone (100 mg/l) showed maximum bioavailability of the minerals like iron (92.05%), zinc (95.02%) and magnesium (92.04%) as compared with control. The increase in the composition of beneficial fatty acids on supplementation of adjuvants supports the therapeutic value of the product.

  17. Combined effect of ultrasound/SonoVue microbubble on CD4(+)CD25(+) regulatory T cells viability and optimized parameters for its transfection.

    PubMed

    Shi, Chunying; Zhang, Yu; Yang, Haichao; Dong, Tianxiu; Chen, Yaodong; Xu, Yutong; Yang, Xiuhua

    2015-09-01

    The purpose of this study was to investigate the combined effect of ultrasound and SonoVue microbubble on CD4(+)CD25(+) regulatory T cells (Tregs) viability and to explore the appropriate parameters for Tregs transfection. Tregs were separated from peripheral venous blood of patients with hepatocellular carcinoma and seeded in 96-well plates. The optimal ultrasound exposure time and optimal SonoVue microbubble concentration for Tregs were measured by mechanical index (MI) of 1.2 or 1.4, exposure time of 0, 30, 60, 90, 120, 150, 180s, and 0, 10, 20, 30, 40, 50μL/100μL microbubble per well, respectively. In addition, the combined effect of ultrasound and microbubble on Tregs viability was evaluated according to the following parameters: MI 1.2/1.4+exposure time of 120, 150, 180s+0, 10, 20, 30, 40, 50μL/100μL microbubble per well. Tregs viability investigations were performed in order to explore the optimal transfection condition. The efficiency of plasmid transfer was determined by detection of luciferase activity on the microscopic examinations. The proliferation of Tregs could be promoted by ultrasound exposures, while being decreased with the increasing concentration of microbubbles. Under the current experimental conditions, the optimal ultrasound parameters were MI=1.4 and exposure time=150/180s. The optimal microbubble concentration was 10μL/100μL. Compared with treatment with ultrasound or microbubbles alone, the transfection efficiency of Tregs improved 50% by combining ultrasound and microbubble. The results indicate that both ultrasound and microbubble could affect the Tregs proliferation and the optimal Treg transfection rate was obtained by treating with 10% microbubbles and ultrasound exposure for 150/180s under ultrasound MI of 1.4.

  18. Statins affect ETS1-overexpressing triple-negative breast cancer cells by restoring DUSP4 deficiency

    PubMed Central

    Jung, Hae Hyun; Lee, Soo-Hyeon; Kim, Ji-Yeon; Ahn, Jin Seok; Park, Yeon Hee; Im, Young-Hyuck

    2016-01-01

    We investigated the molecular mechanisms underlying statin-induced growth suppression of triple-negative breast cancer (TNBC) that overexpress the transcription factor ets proto-oncogene 1(ets-1) and downregulate dual specific protein phosphatase 4(dusp4) expression. We examined the gene expression of BC cell lines using the nCounter expression assay, MTT viability assay, cell proliferation assay and Western blot to evaluate the effects of simvastatin. Finally, we performed cell viability testing in TNBC cell line-transfected DUSP4. We demonstrated that ETS1 mRNA and protein were overexpressed in TNBC cells compared with other BC cell lines (P = <0.001) and DUSP4 mRNA was downregulated (P = <0.001). MTT viability assay showed that simvastatin had significant antitumor activity (P = 0.002 in 0.1 μM). In addition, simvastatin could restore dusp4 deficiency and suppress ets-1 expression in TNBC. Lastly, we found that si-DUSP4 RNA transfection overcame the antitumor activity of statins. MAPK pathway inhibitor, U0126 and PI3KCA inhibitor LY294002 also decreased levels of ets-1, phosphor-ERK and phosphor-AKT on Western blot assay. Accordingly, our study indicates that simvastatin potentially affects the activity of transcriptional factors such as ets-1 and dusp4 through the MAPK pathway. In conclusion, statins might be potential candidates for TNBC therapy reducing ets-1 expression via overexpression of dusp4. PMID:27604655

  19. Statins affect ETS1-overexpressing triple-negative breast cancer cells by restoring DUSP4 deficiency.

    PubMed

    Jung, Hae Hyun; Lee, Soo-Hyeon; Kim, Ji-Yeon; Ahn, Jin Seok; Park, Yeon Hee; Im, Young-Hyuck

    2016-01-01

    We investigated the molecular mechanisms underlying statin-induced growth suppression of triple-negative breast cancer (TNBC) that overexpress the transcription factor ets proto-oncogene 1(ets-1) and downregulate dual specific protein phosphatase 4(dusp4) expression. We examined the gene expression of BC cell lines using the nCounter expression assay, MTT viability assay, cell proliferation assay and Western blot to evaluate the effects of simvastatin. Finally, we performed cell viability testing in TNBC cell line-transfected DUSP4. We demonstrated that ETS1 mRNA and protein were overexpressed in TNBC cells compared with other BC cell lines (P = <0.001) and DUSP4 mRNA was downregulated (P = <0.001). MTT viability assay showed that simvastatin had significant antitumor activity (P = 0.002 in 0.1 μM). In addition, simvastatin could restore dusp4 deficiency and suppress ets-1 expression in TNBC. Lastly, we found that si-DUSP4 RNA transfection overcame the antitumor activity of statins. MAPK pathway inhibitor, U0126 and PI3KCA inhibitor LY294002 also decreased levels of ets-1, phosphor-ERK and phosphor-AKT on Western blot assay. Accordingly, our study indicates that simvastatin potentially affects the activity of transcriptional factors such as ets-1 and dusp4 through the MAPK pathway. In conclusion, statins might be potential candidates for TNBC therapy reducing ets-1 expression via overexpression of dusp4.

  20. Statins affect ETS1-overexpressing triple-negative breast cancer cells by restoring DUSP4 deficiency.

    PubMed

    Jung, Hae Hyun; Lee, Soo-Hyeon; Kim, Ji-Yeon; Ahn, Jin Seok; Park, Yeon Hee; Im, Young-Hyuck

    2016-01-01

    We investigated the molecular mechanisms underlying statin-induced growth suppression of triple-negative breast cancer (TNBC) that overexpress the transcription factor ets proto-oncogene 1(ets-1) and downregulate dual specific protein phosphatase 4(dusp4) expression. We examined the gene expression of BC cell lines using the nCounter expression assay, MTT viability assay, cell proliferation assay and Western blot to evaluate the effects of simvastatin. Finally, we performed cell viability testing in TNBC cell line-transfected DUSP4. We demonstrated that ETS1 mRNA and protein were overexpressed in TNBC cells compared with other BC cell lines (P = <0.001) and DUSP4 mRNA was downregulated (P = <0.001). MTT viability assay showed that simvastatin had significant antitumor activity (P = 0.002 in 0.1 μM). In addition, simvastatin could restore dusp4 deficiency and suppress ets-1 expression in TNBC. Lastly, we found that si-DUSP4 RNA transfection overcame the antitumor activity of statins. MAPK pathway inhibitor, U0126 and PI3KCA inhibitor LY294002 also decreased levels of ets-1, phosphor-ERK and phosphor-AKT on Western blot assay. Accordingly, our study indicates that simvastatin potentially affects the activity of transcriptional factors such as ets-1 and dusp4 through the MAPK pathway. In conclusion, statins might be potential candidates for TNBC therapy reducing ets-1 expression via overexpression of dusp4. PMID:27604655

  1. Development of an Aerosol Model of Cryptococcus Reveals Humidity as an Important Factor Affecting the Viability of Cryptococcus during Aerosolization

    PubMed Central

    Springer, Deborah J.; Saini, Divey; Byrnes, Edmond J.; Heitman, Joseph; Frothingham, Richard

    2013-01-01

    Cryptococcus is an emerging global health threat that is annually responsible for over 1,000,000 infections and one third of all AIDS patient deaths. There is an ongoing outbreak of cryptococcosis in the western United States and Canada. Cryptococcosis is a disease resulting from the inhalation of the infectious propagules from the environment. The current and most frequently used animal infection models initiate infection via liquid suspension through intranasal instillation or intravenous injection. These models do not replicate the typically dry nature of aerosol exposure and may hinder our ability to decipher the initial events that lead to clearance or the establishment of infection. We have established a standardized aerosol model of murine infection for the human fungal pathogen Cryptococcus. Aerosolized cells were generated utilizing a Collison nebulizer in a whole-body Madison Chamber at different humidity conditions. The aerosols inside the chamber were sampled using a BioSampler to determine viable aerosol concentration and spray factor (ratio of viable aerosol concentration to total inoculum concentration). We have effectively delivered yeast and yeast-spore mixtures to the lungs of mice and observed the establishment of disease. We observed that growth conditions prior to exposure and humidity within the Madison Chamber during exposure can alter Cryptococcus survival and dose retained in mice. PMID:23894542

  2. Method and apparatus for sustaining viability of biological cells on a substrate

    DOEpatents

    McKnight, Timothy E.; Melechko, Anatoli V.; Simpson, Michael L.

    2013-01-01

    A method for the transient transformation of a living biological cell having an intact cell membrane defining an intracellular domain, and an apparatus for the transient transformation of biological cells. The method and apparatus include introducing a compartmentalized extracellular component fixedly attached to a cellular penetrant structure to the intracellular domain of the cell, wherein the cell is fixed in a predetermined location and wherein the component is expressed within in the cell while being retained within the compartment and wherein the compartment restricts the mobility and interactions of the component within the cell and prevents transference of the component to the cell.

  3. Method and apparatus for sustaining viability of biological cells on a substrate

    DOEpatents

    McKnight, Timothy E.; Melechko, Anatoli V.; Simpson, Michael L.

    2011-12-13

    A method for the transient transformation of a living biological cell having an intact cell membrane defining an intracellular domain, and an apparatus for the transient transformation of biological cells. The method and apparatus include introducing a compartmentalized extracellular component fixedly attached to a cellular penetrant structure to the intracellular domain of the cell, wherein the cell is fixed in a predetermined location and wherein the component is expressed within in the cell while being retained within the compartment and wherein the compartment restricts the mobility and interactions of the component within the cell and prevents transference of the component to the cell.

  4. Divergent branches of mitochondrial signaling regulate specific genes and the viability of specialized cell types of differentiated yeast colonies

    PubMed Central

    Rešetárová, Stanislava; Kučerová, Helena; Hlaváček, Otakar; Váchová, Libuše; Palková, Zdena

    2016-01-01

    Mitochondrial retrograde signaling mediates communication from altered mitochondria to the nucleus and is involved in many normal and pathophysiological changes, including cell metabolic reprogramming linked to cancer development and progression in mammals. The major mitochondrial retrograde pathway described in yeast includes three activators, Rtg1p, Rtg2p and Rtg3p, and repressors, Mks1p and Bmh1p/Bmh2p. Using differentiated yeast colonies, we show that Mks1p-Rtg pathway regulation is complex and includes three branches that divergently regulate the properties and fate of three specifically localized cell subpopulations via signals from differently altered mitochondria. The newly identified RTG pathway-regulated genes ATO1/ATO2 are expressed in colonial upper (U) cells, the cells with active TORC1 that metabolically resemble tumor cells, while CIT2 is a typical target induced in one subpopulation of starving lower (L) cells. The viability of the second L cell subpopulation is strictly dependent on RTG signaling. Additional co-activators of Rtg1p-Rtg3p specific to particular gene targets of each branch are required to regulate cell differentiation. PMID:26992228

  5. Effects of triclosan and triclocarban on the growth inhibition, cell viability, genotoxicity and multixenobiotic resistance responses of Tetrahymena thermophila.

    PubMed

    Gao, Li; Yuan, Tao; Cheng, Peng; Bai, Qifeng; Zhou, Chuanqi; Ao, Junjie; Wang, Wenhua; Zhang, Haimou

    2015-11-01

    The information about adverse effects of emerging contaminants on aquatic protozoa is very scarce. The growth inhibition effect, cell viability, genotoxicity and multixenobiotic resistance (MXR) responses of two commonly used antimicrobial agents, triclosan (TCS) and triclocarban (TCC) to protozoan Tetrahymena thermophila were investigated in this study. The results revealed that TCS and TCC can inhibit the growth of T. thermophila with 24h EC50 values of 1063 and 295μgL(-1), respectively. The impairment of plasma membrane was observed after 2h exposure of TCS or TCC at the level of mg/L. Furthermore, it is noticeable that at environmentally relevant concentration (1.0μgL(-1)), both TCS and TCC can lead to statistically significant DNA damage in T. thermophila, while the inhibition of growth and change of cell viability cannot be observed. Our results firstly provide the evidence for genotoxic effects of TCS and TCC on the freshwater protozoan. Additionally, both TCS and TCC were found to inhibit the efflux transporter activities, with the inhibitory potencies of 39% and 40% (using verapamil as a model inhibitor), respectively. Particularly, TCC could significantly down-regulate the expression of MXR related gene Abcb15, which encodes the membrane efflux protein that acting as P-gp in T. thermophila. The results raise the awareness of potential aquatic ecological and human health risks from the exposure of TCS and TCC, as they might potentiate the toxic effects by chemosensitizing with co-existing toxicants.

  6. Functional physico-chemical, ex vivo permeation and cell viability characterization of omeprazole loaded buccal films for paediatric drug delivery.

    PubMed

    Khan, Sajjad; Trivedi, Vivek; Boateng, Joshua

    2016-03-16

    Buccal films were prepared from aqueous and ethanolic Metolose gels using the solvent casting approach (40°C). The hydration (PBS and simulated saliva), mucoadhesion, physical stability (20°C, 40°C), in vitro drug (omeprazole) dissolution (PBS and simulated saliva), ex vivo permeation (pig buccal mucosa) in the presence of simulated saliva, ex vivo bioadhesion and cell viability using MTT of films were investigated. Hydration and mucoadhesion results showed that swelling capacity and adhesion was higher in the presence of PBS than simulated saliva (SS) due to differences in ionic strength. Omeprazole was more stable at 20°C than 40°C whilst omeprazole release reached a plateau within 1h and faster in PBS than in SS. Fitting release data to kinetic models showed that Korsmeyer-Peppas equation best fit the dissolution data. Drug release in PBS was best described by zero order via non-Fickian diffusion but followed super case II transport in SS attributed to drug diffusion and polymer erosion. The amount of omeprazole permeating over 2h was 275 ug/cm(2) whilst the formulations and starting materials showed cell viability values greater than 95%, confirming their safety for potential use in paediatric buccal delivery. PMID:26802493

  7. Unfolded Protein Response in Cancer: IRE1α Inhibition by Selective Kinase Ligands Does Not Impair Tumor Cell Viability

    PubMed Central

    2014-01-01

    The kinase/endonuclease inositol requiring enzyme 1 (IRE1α), one of the sensors of unfolded protein accumulation in the endoplasmic reticulum that triggers the unfolded protein response (UPR), has been investigated as an anticancer target. We identified potent allosteric inhibitors of IRE1α endonuclease activity that bound to the kinase site on the enzyme. Structure–activity relationship (SAR) studies led to 16 and 18, which were selective in kinase screens and were potent against recombinant IRE1α endonuclease as well as cellular IRE1α. The first X-ray crystal structure of a kinase inhibitor (16) bound to hIRE1α was obtained. Screening of native tumor cell lines (>300) against selective IRE1α inhibitors failed to demonstrate any effect on cellular viability. These results suggest that IRE1α activity is not essential for viability in most tumor cell lines, in vitro, and that interfering with the survival functions of the UPR may not be an effective strategy to block tumorigenesis. PMID:25589933

  8. Listeria monocytogenes PrsA2 Is Required for Virulence Factor Secretion and Bacterial Viability within the Host Cell Cytosol▿

    PubMed Central

    Alonzo, Francis; Freitag, Nancy E.

    2010-01-01

    In the course of establishing its replication niche within the cytosol of infected host cells, the facultative intracellular bacterial pathogen Listeria monocytogenes must efficiently regulate the secretion and activity of multiple virulence factors. L. monocytogenes encodes two predicted posttranslocation secretion chaperones, PrsA1 and PrsA2, and evidence suggests that PrsA2 has been specifically adapted for bacterial pathogenesis. PrsA-like chaperones have been identified in a number of Gram-positive bacteria, where they are reported to function at the bacterial membrane-cell wall interface to assist in the folding of proteins translocated across the membrane; in some cases, these proteins have been found to be essential for bacterial viability. In this study, the contributions of PrsA2 and PrsA1 to L. monocytogenes growth and protein secretion were investigated in vitro and in vivo. Neither PrsA2 nor PrsA1 was found to be essential for L. monocytogenes growth in broth culture; however, optimal bacterial viability was found to be dependent upon PrsA2 for L. monocytogenes located within the cytosol of host cells. Proteomic analyses of prsA2 mutant strains in the presence of a mutationally activated allele of the virulence regulator PrfA revealed a critical requirement for PrsA2 activity under conditions of PrfA activation, an event which normally takes place within the host cell cytosol. Despite a high degree of amino acid similarity, no detectable degree of functional overlap was observed between PrsA2 and PrsA1. Our results indicate a critical requirement for PrsA2 under conditions relevant to host cell infection. PMID:20823208

  9. Viability and MR detectability of iron labeled mesenchymal stem cells used for endoscopic injection into the porcine urethral sphincter.

    PubMed

    Will, Susanne; Martirosian, Petros; Eibofner, Frank; Schick, Fritz; Bantleon, Rüdiger; Vaegler, Martin; Grözinger, Gerd; Claussen, Claus D; Kramer, Ulrich; Schmehl, Jörg

    2015-08-01

    Direct stem cell therapies for functionally impaired tissue require a sufficient number of cells in the target region and a method for verifying the fate of the cells in the subsequent time course. In vivo MRI of iron labeled mesenchymal stem cells has been suggested to comply with these requirements. The study was conducted to evaluate proliferation, migration, differentiation and adhesion effects as well as the obtained iron load of an iron labeling strategy for mesenchymal stem cells. After injection into the porcine urethral sphincter, the labeled cells were monitored for up to six months using MRI. Mesenchymal stem cells were labeled with ferucarbotran (60/100/200 µg/mL) and ferumoxide (200 µg/mL) for the analysis of migration and viability. Phantom MR measurements were made to evaluate effects of iron labeling. For short and long term studies, the iron labeled cells were injected into the porcine urethral sphincter and monitored by MRI. High resolution anatomical images of the porcine urethral sphincter were applied for detection of the iron particles with a turbo-spin-echo sequence and a gradient-echo sequence with multiple TE values. The MR images were then compared with histological staining. The analysis of cell function after iron labeling showed no effects on proliferation or differentiation of the cells. Although the adherence increases with higher iron dose, the ability to migrate decreases as a presumed effect of iron labeling. The iron labeled mesenchymal stem cells were detectable in vivo in MRI and histological staining even six months after injection. Labeling with iron particles and subsequent evaluation with highly resolved three dimensional data acquisition allows sensitive tracking of cells injected into the porcine urethral sphincter for several months without substantial biological effects on mesenchymal stem cells.

  10. Ion channel-mediated uptake of cationic vital dyes into live cells: a potential source of error when assessing cell viability.

    PubMed

    Bukhari, Maurish; Burm, Hayley; Samways, Damien S K

    2016-10-01

    Ionic "vital dyes" are commonly used to assess cell viability based on the idea that their permeation is contingent on a loss of membrane integrity. However, the possibility that dye entry is conducted into live cells by endogenous membrane transporters must be recognized and controlled for. Several cation-selective plasma membrane-localized ion channels, including the adenosine 5'-triphosphate (ATP)-gated P2X receptors, have been reported to conduct entry of the DNA-binding fluorescence dye, YO-PRO-1, into live cells. Extracellular ATP often becomes elevated as a result of release from dying cells, and so it is possible that activation of P2X channels on neighboring live cells could lead to exaggerated estimation of cytotoxicity. Here, we screened a number of fluorescent vital dyes for ion channel-mediated uptake in HEK293 cells expressing recombinant P2X2, P2X7, or TRPV1 channels. Our data shows that activation of all three channels caused substantial uptake and nuclear accumulation of YO-PRO-1, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33258 into transfected cells and did so well within the time period usually used for incubation of cells with vital dyes. In contrast, channel activation in the presence of propidium iodide and SYTOX Green caused no measurable uptake and accumulation during a 20-min exposure, suggesting that these dyes are not likely to exhibit measurable uptake through these particular ion channels during a conventional cell viability assay. Caution is encouraged when choosing and employing cationic dyes for the purpose of cell viability assessment, particularly when there is a likelihood of cells expressing ion channels permeable to large ions. PMID:27423453

  11. Ion channel-mediated uptake of cationic vital dyes into live cells: a potential source of error when assessing cell viability.

    PubMed

    Bukhari, Maurish; Burm, Hayley; Samways, Damien S K

    2016-10-01

    Ionic "vital dyes" are commonly used to assess cell viability based on the idea that their permeation is contingent on a loss of membrane integrity. However, the possibility that dye entry is conducted into live cells by endogenous membrane transporters must be recognized and controlled for. Several cation-selective plasma membrane-localized ion channels, including the adenosine 5'-triphosphate (ATP)-gated P2X receptors, have been reported to conduct entry of the DNA-binding fluorescence dye, YO-PRO-1, into live cells. Extracellular ATP often becomes elevated as a result of release from dying cells, and so it is possible that activation of P2X channels on neighboring live cells could lead to exaggerated estimation of cytotoxicity. Here, we screened a number of fluorescent vital dyes for ion channel-mediated uptake in HEK293 cells expressing recombinant P2X2, P2X7, or TRPV1 channels. Our data shows that activation of all three channels caused substantial uptake and nuclear accumulation of YO-PRO-1, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33258 into transfected cells and did so well within the time period usually used for incubation of cells with vital dyes. In contrast, channel activation in the presence of propidium iodide and SYTOX Green caused no measurable uptake and accumulation during a 20-min exposure, suggesting that these dyes are not likely to exhibit measurable uptake through these particular ion channels during a conventional cell viability assay. Caution is encouraged when choosing and employing cationic dyes for the purpose of cell viability assessment, particularly when there is a likelihood of cells expressing ion channels permeable to large ions.

  12. Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields.

    PubMed

    Wang, Jian; Xiang, Bo; Deng, Jixian; Freed, Darren H; Arora, Rakesh C; Tian, Ganghong

    2016-01-01

    After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF), Adipose-derived Stem Cells (ASCs) and those labeled by superparamagnetic iron oxide (SPIO) nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT) assay, proliferation by cell counting and bromodeoxyuridine (BrdU) incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor-1 (IGF-1), Transforming Growth Factor Beta 1 (TGF-β1), genetic markers comprising Stem Cell Antigen-1 (Sca1), Octamer-4 (Oct-4), ATP-binding Cassette Subfamily B Member 1 (ABCB1), adipogenic marker genes containing Lipoprotein Lipase (LPL), Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ), and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1) and Osterix (OSX). Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling. PMID:26880984

  13. Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields

    PubMed Central

    Wang, Jian; Xiang, Bo; Deng, Jixian; Freed, Darren H.; Arora, Rakesh C.; Tian, Ganghong

    2016-01-01

    After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF), Adipose-derived Stem Cells (ASCs) and those labeled by superparamagnetic iron oxide (SPIO) nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT) assay, proliferation by cell counting and bromodeoxyuridine (BrdU) incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor-1 (IGF-1), Transforming Growth Factor Beta 1 (TGF-β1), genetic markers comprising Stem Cell Antigen-1 (Sca1), Octamer-4 (Oct-4), ATP-binding Cassette Subfamily B Member 1 (ABCB1), adipogenic marker genes containing Lipoprotein Lipase (LPL), Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ), and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1) and Osterix (OSX). Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling. PMID:26880984

  14. Effect of single-walled carbon nanotubes on tumor cells viability and formation of multicellular tumor spheroids

    NASA Astrophysics Data System (ADS)

    Yakymchuk, Olena M.; Perepelytsina, Olena M.; Dobrydnev, Alexey V.; Sydorenko, Mychailo V.

    2015-03-01

    This paper describes the impact of different concentrations of single-walled carbon nanotubes (SWCNTs) on cell viability of breast adenocarcinoma, MCF-7 line, and formation of multicellular tumor spheroids (MTS). Chemical composition and purity of nanotubes is controlled by Fourier transform infrared spectroscopy. The strength and direction of the influence of SWCNTs on the tumor cell population was assessed by cell counting and measurement of the volume of multicellular tumor spheroids. Effect of SWCNTs on the formation of multicellular spheroids was compared with the results obtained by culturing tumor cells with ultra dispersed diamonds (UDDs). Our results demonstrated that SWCNTs at concentrations ranging from 12.5 to 50 μg/ml did not have cytotoxic influence on tumor cells; instead, they had weak cytostatic effect. The increasing of SWCNTs concentration to 100 to 200 μg/ml stimulated proliferation of tumor cells, especially in suspension fractions. The result of this influence was in formation of more MTS in cell culture with SWCNTs compared with UDDs and control samples. In result, the median volume of MTS after cultivation with SWCNTs at 100 to 200 μg/ml concentrations is 3 to 5 times greater than that in samples which were incubated with the UDDs and is 2.5 times greater than that in control cultures. So, if SWCNTs reduced cell adhesion to substrate and stimulated formation of tumor cell aggregates volume near 7 · 10-3 mm3, at the same time, UDDs reduced adhesion and cohesive ability of cells and stimulated generation of cell spheroids volume no more than 4 · 10-3 mm3. Our results could be useful for the control of cell growth in three-dimensional culture.

  15. Inadequate Processing of Decellularized Dermal Matrix Reduces Cell Viability In Vitro and Increases Apoptosis and Acute Inflammation In Vivo

    PubMed Central

    Morris, Aaron H.; Chang, Julie; Kyriakides, Themis R.

    2016-01-01

    Abstract Decellularized tissue scaffolds are commonly used in the clinic because they can be used as substitutes for more traditional biomaterials, while imparting additional physiological effects. Nevertheless, reports of complications associated with their use are widespread and poorly understood. This study probes possible causes of these complications by examining cell viability and apoptosis in response to eluents from decellularized dermis. Using multiple sources of decellularized dermis, this study shows that typical decellularized scaffolds (prepared with commonly used laboratory techniques, as well as purchased from commercial sources) contain soluble components that are cytotoxic and that these components can be removed by extensive washes in cell culture media. In addition, this study demonstrates that these observed in vitro phenotypes correlate with increased apoptosis and acute inflammation when implanted subcutaneously in mice. PMID:27500014

  16. Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation.

    PubMed

    Wang, Zhihao; Wang, Yun; Zhang, Dongxu; Li, Jianghua; Hua, Zhaozhe; Du, Guocheng; Chen, Jian

    2010-02-01

    Alkaline polygalacturonate lyase (PGL) production by Pichia pastoris GS115 was used as a model to study the mechanism and strategy for enhancing heterologous protein production. In order to enhance cell viability and volumetric recombinant protein productivity, sorbitol, which had been confirmed to be a non-repressive carbon source, was added together with methanol during the induction phase. The resultant PGL activity was up to 1593 U mL(-1), which was enhanced 1.85-fold compared to the control (863 U mL(-1)) cultured with sorbitol added at a constant rate of 3.6 g h(-1)L(-1) after an induction period of 100 h. Further results revealed that an appropriate sorbitol co-feeding strategy not only decreased the cell mortality to 8.8% (the control is about 23.1%) in the end of fermentation, but also reduced the proteolytic degradation of PGL.

  17. Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability.

    PubMed

    Park, Jina; Jin, Sung Il; Kim, Hyung Min; Ahn, Junhyoung; Kim, Yeon-Gu; Lee, Eun Gyo; Kim, Min-Gon; Shin, Yong-Beom

    2015-02-15

    We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers.

  18. Hepatocyte-like cells derived from human amniotic epithelial cells can be encapsulated without loss of viability or function in vitro.

    PubMed

    Vaghjiani, Vijesh; Vaithilingam, Vijayaganapathy; Saraswati, Indah; Sali, Adnan; Murthi, Padma; Kalionis, Bill; Tuch, Bernard E; Manuelpillai, Ursula

    2014-04-15

    Placenta derived human amniotic epithelial cells (hAEC) are an attractive source of stem cells for the generation of hepatocyte-like cells (HLC) for therapeutic applications to treat liver diseases. During hAEC differentiation into HLC, they become increasingly immunogenic, which may result in immune cell-mediated rejection upon transplantation into allogeneic recipients. Placing cells within devices such as alginate microcapsules can prevent immune cell-mediated rejection. The aim of this study was to investigate the characteristics of HLC generated from hAEC and to examine the effects of encapsulation on HLC viability, gene expression, and function. hAEC were differentiated for 4 weeks and evaluated for hepatocyte-specific gene expression and function. Differentiated cells were encapsulated in barium alginate microcapsules and cultured for 7 days and the effect of encapsulation on cell viability, function, and hepatocyte related gene expression was determined. Differentiated cells performed key functions of hepatocytes including urea synthesis, drug-metabolizing cytochrome P450 (CYP)3A4 activity, indocyanine green (ICG) uptake, low-density lipoprotein (LDL) uptake, and exhibited glutathione antioxidant capacity. A number of hepatocyte-related genes involved in fat, cholesterol, bile acid synthesis, and xenobiotic metabolism were also expressed showing that the hAEC had differentiated into HLC. Upon encapsulation, the HLC remained viable for at least 7 days in culture, continued to express genes involved in fat, cholesterol, bile acid, and xenobiotic metabolism and had glutathione antioxidant capacity. CYP3A4 activity and urea synthesis by the encapsulated HLC were higher than that of monolayer HLC cultures. Functional HLC can be derived from hAEC, and HLC can be encapsulated within alginate microcapsules without losing viability or function in vitro. PMID:24295364

  19. Hepatocyte-Like Cells Derived from Human Amniotic Epithelial Cells Can Be Encapsulated Without Loss of Viability or Function In Vitro

    PubMed Central

    Vaghjiani, Vijesh; Vaithilingam, Vijayaganapathy; Saraswati, Indah; Sali, Adnan; Murthi, Padma; Kalionis, Bill; Tuch, Bernard E.

    2014-01-01

    Placenta derived human amniotic epithelial cells (hAEC) are an attractive source of stem cells for the generation of hepatocyte-like cells (HLC) for therapeutic applications to treat liver diseases. During hAEC differentiation into HLC, they become increasingly immunogenic, which may result in immune cell-mediated rejection upon transplantation into allogeneic recipients. Placing cells within devices such as alginate microcapsules can prevent immune cell-mediated rejection. The aim of this study was to investigate the characteristics of HLC generated from hAEC and to examine the effects of encapsulation on HLC viability, gene expression, and function. hAEC were differentiated for 4 weeks and evaluated for hepatocyte-specific gene expression and function. Differentiated cells were encapsulated in barium alginate microcapsules and cultured for 7 days and the effect of encapsulation on cell viability, function, and hepatocyte related gene expression was determined. Differentiated cells performed key functions of hepatocytes including urea synthesis, drug-metabolizing cytochrome P450 (CYP)3A4 activity, indocyanine green (ICG) uptake, low-density lipoprotein (LDL) uptake, and exhibited glutathione antioxidant capacity. A number of hepatocyte-related genes involved in fat, cholesterol, bile acid synthesis, and xenobiotic metabolism were also expressed showing that the hAEC had differentiated into HLC. Upon encapsulation, the HLC remained viable for at least 7 days in culture, continued to express genes involved in fat, cholesterol, bile acid, and xenobiotic metabolism and had glutathione antioxidant capacity. CYP3A4 activity and urea synthesis by the encapsulated HLC were higher than that of monolayer HLC cultures. Functional HLC can be derived from hAEC, and HLC can be encapsulated within alginate microcapsules without losing viability or function in vitro. PMID:24295364

  20. Influence of boron addition to Ti-13Zr-13Nb alloy on MG63 osteoblast cell viability and protein adsorption.

    PubMed

    Majumdar, P; Singh, S B; Dhara, S; Chakraborty, M

    2015-01-01

    Cell proliferation, cell morphology and protein adsorption on near β-type Ti-13Zr-13Nb (TZN) alloy and Ti-13Zr-13Nb-0.5B (TZNB) composite have been investigated and compared to evaluate the effect of boron addition which has been added to the Ti alloy to improve their poor tribological properties by forming in situ TiB precipitates. MG63 cell proliferation on substrates with different chemistry but the same topography was compared. The MTT assay test showed that the cell viability on the TZN alloy was higher than the boron containing TZNB composite after 36 h of incubation and the difference was pronounced after 7 days. However, both the materials showed substantially higher cell attachment than the control (polystyrene). For the same period of incubation in fetal bovine serum (FBS), the amount of protein adsorbed on the surface of boron free TZN samples was higher than that in the case of boron containing TZNB composite. The presence of boron in the TZN alloy influenced protein adsorption and cell response and they are lower in TZNB than in TZN as a result of the associated difference in chemical characteristics.

  1. Physical parameters affecting living cells in space

    NASA Astrophysics Data System (ADS)

    Langbein, Dieter

    The question is posed: Why does a living cell react to the absence of gravity? What sensors may it have? Does it note pressure, sedimentation, convection, or other parameters? If somewhere in a liquid volume sodium ions are replaced by potassium ions, the density of the liquid changes locally: the heavier regions sink, the lighter regions rise. This may contribute to species transport, to the metabolism. Under microgravity this mechanism is strongly reduced. On the other hand, other reasons for convection like thermal and solutal interface convection are left. Do they affect species transport? Another important effect of gravity is the hydrostatic pressure. On the macroscopic side, the pressure between our head and feet changes by 0.35 atmospheres. On the microscopic level the hydrostatic pressure on the upper half of a cell membrane is lower than on the lower half. This, by affecting the ion transport through the membrane, may change the surrounding electric potential. It has been suggested to be one of the reasons for graviperception. Following the discussion of these and other effects possibly important in life sciences in space, an order of magnitude analysis of the residual accelerations tolerable during experiments in materials sciences is outlined. In the field of life sciences only rough estimates are available at present.

  2. Cleavage of BLOC1S1 mRNA by IRE1 Is Sequence Specific, Temporally Separate from XBP1 Splicing, and Dispensable for Cell Viability under Acute Endoplasmic Reticulum Stress

    PubMed Central

    Itzhak, Daniel N.; Wardell, Christopher P.; Morgan, Gareth J.; Davies, Faith E.

    2015-01-01

    The unfolded protein response (UPR) remediates endoplasmic reticulum (ER) stress. IRE1, a component of the UPR, senses misfolded protein and cleaves XBP1 mRNA, which is ligated to code for the prosurvival transcription factor. IRE1 also cleaves other mRNAs preceding their degradation, termed regulated IRE1-dependent mRNA decay (RIDD). It has been reported that RIDD may be involved in cell viability under stress and therefore may contribute to cancer cell viability. To investigate RIDD targets that may have functional relevance in cell survival, we identified conserved RIDD targets containing stringent IRE1 RNase target sequences. Using a systematic bioinformatics approach with quantitative-PCR (qPCR) validation, we show that only BLOC1S1 is consistently a RIDD target in all systems tested. Using cancer cell lines, we show that BLOC1S1 is specifically cleaved by IRE1 at guanine 444, but only under conditions of IRE1 hyperactivation. BLOC1S1 cleavage is temporally separate from XBP1 splicing, occurring after depletion of unspliced XBP1. Expression of an uncleavable BLOC1S1 mutant or inhibition of RIDD using an IRE1 RNase inhibitor did not affect cellular recovery from acute ER stress. These data demonstrate that although hyperactivated IRE1 specifically cleaves BLOC1S1, this cleavage event and RIDD as a whole are dispensable for cell viability under acute stress. PMID:25870107

  3. Differential concentration-specific effects of caffeine on cell viability, oxidative stress, and cell cycle in pulmonary oxygen toxicity in vitro

    SciTech Connect

    Tiwari, Kirti Kumar; Chu, Chun; Couroucli, Xanthi; Moorthy, Bhagavatula; Lingappan, Krithika

    2014-08-08

    Highlights: • Caffeine at 0.05 mM decreases oxidative stress in hyperoxia. • Caffeine at 1 mM decreases cell viability, increases oxidative stress in hyperoxia. • Caffeine at 1 but not 0.05 mM, abrogates hyperoxia-induced G2/M arrest. - Abstract: Caffeine is used to prevent bronchopulmonary dysplasia (BPD) in premature neonates. Hyperoxia contributes to the development of BPD, inhibits cell proliferation and decreases cell survival. The mechanisms responsible for the protective effect of caffeine in pulmonary oxygen toxicity remain largely unknown. A549 and MLE 12 pulmonary epithelial cells were exposed to hyperoxia or maintained in room air, in the presence of different concentrations (0, 0.05, 0.1 and 1 mM) of caffeine. Caffeine had a differential concentration-specific effect on cell cycle progression, oxidative stress and viability, with 1 mM concentration being deleterious and 0.05 mM being protective. Reactive oxygen species (ROS) generation during hyperoxia was modulated by caffeine in a similar concentration-specific manner. Caffeine at 1 mM, but not at the 0.05 mM concentration decreased the G2 arrest in these cells. Taken together this study shows the novel funding that caffeine has a concentration-specific effect on cell cycle regulation, ROS generation, and cell survival in hyperoxic conditions.

  4. Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies.

    PubMed

    Lu, Yujie; Darne, Chinmay D; Tan, I-Chih; Zhu, Banghe; Hall, Mary A; Lazard, Zawaunyka W; Davis, Alan R; Simpson, Lashan; Sevick-Muraca, Eva M; Olmsted-Davis, Elizabeth A

    2013-10-01

    Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models.

  5. In vitro-in vivo evaluation of supercritical processed solid dispersions: permeability and viability assessment in Caco-2 cells.

    PubMed

    Sethia, Sundeep; Squillante, Emilio

    2004-12-01

    In this study improvement in the bioavailability of carbamazepine (CBZ) prepared as solid dispersions by conventional solvent evaporation and supercritical fluid (SCF) processing methods was assessed, along with the elucidation of the mechanism of improved absorption. Solid dispersions of CBZ in polyethylene glycol (PEG) with either Gelucire 44/14 or vitamin E-TPGS (TPGS) were evaluated by intrinsic dissolution. Directional transport through Caco-2 cell monolayers was determined in the presence and absence of TPGS. Cell viability in presence of various concentrations of amphiphilic carriers was seen. In vivo oral bioavailability was determined in rats. The apparent intrinsic dissolution rates (IDR) of both conventional- and SCF-CBZ/PEG 8000/TPGS solid dispersions were increased by 13- and 10.6-fold, respectively, relative to neat CBZ. CBZ was not a substrate of P-glycoprotein. Higher CBZ permeability was seen in presence of 0.1% TPGS. Cell viability studies showed significant cytotoxicity only at or above 0.1% amphiphilic carrier. Supercritical treated formulation (without amphiphilic carrier) displayed oral bioavailability on par with those conventional solid dispersions augmented with amphiphilic carriers. An in vitro-in vivo correlation was seen between IDR and the AUC of the various CBZ solid dispersions. Bioavailability of CBZ was more a function of dissolution as opposed to membrane effects. Although bioavailability from SCF processed dispersions was better than conventionally processed counterparts (except for one formulation containing Gelucire 44/14), an interaction of processing method and inclusion of an amphiphilic carrier, rather by one factor alone contributed to optimal absorption, thus giving contradictory results for Gelucire 44/14 and TPGS formulations.

  6. Viability evaluation of culture cells patterned by femtosecond laser-induced impulsive force

    NASA Astrophysics Data System (ADS)

    Takizawa, Noriko; Okano, Kazunori; Uwada, Takayuki; Hosokawa, Yoichiroh; Masuhara, Hiroshi

    2008-02-01

    PC12 cells, which are derived from a rat pheochromocytoma, were independently patterned utilizing an impulsive force resulting in impulsive shockwave and cavitation bubble generation by focused femtosecond laser irradiation. Since the PC12 cells respond reversibly to nerve growth factor by induction of the neuronal phenotype, we can assess an influence that the impulsive force gives to the bioactivity in term of the cell differentiation. The patterned cells were accumulated on an intact dish and cultured for 3 days. The behavior of appearance and cell differentiation was observed by multipoint time-lapse system. On bases of these results, it was proved that the biological activity of the cell is unaffected by the femtosecond laser patterning.

  7. Effects of Corroded and Non-Corroded Biodegradable Mg and Mg Alloys on Viability, Morphology and Differentiation of MC3T3-E1 Cells Elicited by Direct Cell/Material Interaction

    PubMed Central

    Mostofi, Sepideh; Bonyadi Rad, Ehsan; Wiltsche, Helmar; Fasching, Ulrike; Szakacs, Gabor; Ramskogler, Claudia; Srinivasaiah, Sriveena; Ueçal, Muammer; Willumeit, Regine; Weinberg, Annelie-Martina; Schaefer, Ute

    2016-01-01

    This study investigated the effect of biodegradable Mg and Mg alloys on selected properties of MC3T3-E1 cells elicited by direct cell/material interaction. The chemical composition and morphology of the surface of Mg and Mg based alloys (Mg2Ag and Mg10Gd) were analysed by scanning electron microscopy (SEM) and EDX, following corrosion in cell culture medium for 1, 2, 3 and 8 days. The most pronounced difference in surface morphology, namely crystal formation, was observed when Pure Mg and Mg2Ag were immersed in cell medium for 8 days, and was associated with an increase in atomic % of oxygen and a decrease of surface calcium and phosphorous. Crystal formation on the surface of Mg10Gd was, in contrast, negligible at all time points. Time-dependent changes in oxygen, calcium and phosphorous surface content were furthermore not observed for Mg10Gd. MC3T3-E1 cell viability was reduced by culture on the surfaces of corroded Mg, Mg2Ag and Mg10Gd in a corrosion time-independent manner. Cells did not survive when cultured on 3 day pre-corroded Pure Mg and Mg2Ag, indicating crystal formation to be particular detrimental in this regard. Cell viability was not affected when cells were cultured on non-corroded Mg and Mg alloys for up to 12 days. These results suggest that corrosion associated changes in surface morphology and chemical composition significantly hamper cell viability and, thus, that non-corroded surfaces are more conducive to cell survival. An analysis of the differentiation potential of MC3T3-E1 cells cultured on non-corroded samples based on measurement of Collagen I and Runx2 expression, revealed a down-regulation of these markers within the first 6 days following cell seeding on all samples, despite persistent survival and proliferation. Cells cultured on Mg10Gd, however, exhibited a pronounced upregulation of collagen I and Runx2 between days 8 and 12, indicating an enhancement of osteointegration by this alloy that could be valuable for in vivo orthopedic

  8. Effects of Corroded and Non-Corroded Biodegradable Mg and Mg Alloys on Viability, Morphology and Differentiation of MC3T3-E1 Cells Elicited by Direct Cell/Material Interaction.

    PubMed

    Mostofi, Sepideh; Bonyadi Rad, Ehsan; Wiltsche, Helmar; Fasching, Ulrike; Szakacs, Gabor; Ramskogler, Claudia; Srinivasaiah, Sriveena; Ueçal, Muammer; Willumeit, Regine; Weinberg, Annelie-Martina; Schaefer, Ute

    2016-01-01

    This study investigated the effect of biodegradable Mg and Mg alloys on selected properties of MC3T3-E1 cells elicited by direct cell/material interaction. The chemical composition and morphology of the surface of Mg and Mg based alloys (Mg2Ag and Mg10Gd) were analysed by scanning electron microscopy (SEM) and EDX, following corrosion in cell culture medium for 1, 2, 3 and 8 days. The most pronounced difference in surface morphology, namely crystal formation, was observed when Pure Mg and Mg2Ag were immersed in cell medium for 8 days, and was associated with an increase in atomic % of oxygen and a decrease of surface calcium and phosphorous. Crystal formation on the surface of Mg10Gd was, in contrast, negligible at all time points. Time-dependent changes in oxygen, calcium and phosphorous surface content were furthermore not observed for Mg10Gd. MC3T3-E1 cell viability was reduced by culture on the surfaces of corroded Mg, Mg2Ag and Mg10Gd in a corrosion time-independent manner. Cells did not survive when cultured on 3 day pre-corroded Pure Mg and Mg2Ag, indicating crystal formation to be particular detrimental in this regard. Cell viability was not affected when cells were cultured on non-corroded Mg and Mg alloys for up to 12 days. These results suggest that corrosion associated changes in surface morphology and chemical composition significantly hamper cell viability and, thus, that non-corroded surfaces are more conducive to cell survival. An analysis of the differentiation potential of MC3T3-E1 cells cultured on non-corroded samples based on measurement of Collagen I and Runx2 expression, revealed a down-regulation of these markers within the first 6 days following cell seeding on all samples, despite persistent survival and proliferation. Cells cultured on Mg10Gd, however, exhibited a pronounced upregulation of collagen I and Runx2 between days 8 and 12, indicating an enhancement of osteointegration by this alloy that could be valuable for in vivo orthopedic

  9. 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.

  10. Inhibitory effects of multiwall carbon nanotubes with high iron impurity on viability and neuronal differentiation in cultured PC12 cells.

    PubMed

    Meng, Li; Jiang, Aihua; Chen, Rui; Li, Chen-zhong; Wang, Liming; Qu, Ying; Wang, Peng; Zhao, Yuliang; Chen, Chunying

    2013-11-01

    The increasing use of carbon nanotubes (CNTs) in biomedical applications has garnered a great concern on their potential negative effects to human health. CNTs have been reported to potentially disrupt normal neuronal function and they were speculated to accumulate and cause brain damage, although a lot of distinct and exceptional properties and potential wide applications have been associated with this material in neurobiology. Fe impurities strapped inside the CNTs may be partially responsible for neurotoxicity generation. In the present study, we selected rat pheochromocytoma (PC12) cells to investigate and compare the effects of two kinds of multiwall carbon nanotubes (MWCNTs) with different concentrations of Fe impurities which usually come from the massive production of CNTs by chemical vapor deposition. Exposure to Fe-high MWCNTs can reduce cell viability and increase cytoskeletal disruption of undifferentiated PC12 cells, diminish the ability to form mature neurites, and then adversely influence the neuronal dopaminergic phenotype in NGF-treated PC-12 cells. The present results highlight the critical role of iron residue in the adverse response to MWCNTs exposure in neural cells. These findings provide useful information for understanding the toxicity and safe application of carbon nanotubes.

  11. Acetylsalicylic acid and salicylic acid decrease tumor cell viability and glucose metabolism modulating 6-phosphofructo-1-kinase structure and activity.

    PubMed

    Spitz, Guilherme A; Furtado, Cristiane M; Sola-Penna, Mauro; Zancan, Patricia

    2009-01-01

    The common observation that cancer cells present higher glycolytic rates when compared to control cells leads to the proposal of glycolysis as a potential target for the development of anti-tumoral agents. Anti-inflammatory drugs, such as acetylsalicylic acid (ASA) and salicylic acid (SA), present anti-tumoral properties, inducing apoptosis and altering tumor glucose utilization. The present work aims at evaluating whether ASA could directly decrease cell glycolysis through inhibition of the major regulatory enzyme within this pathway, 6-phosphofructo-1-kinase (PFK). We show that ASA and SA inhibit purified PFK in a dose-dependent manner, and that this inhibition occurs due to the modulation of the enzyme quaternary structure. ASA and SA promote the dissociation of the enzyme active tetramers into quite inactive dimers, a common regulatory mechanism of this enzyme. The inhibitory effects of ASA and SA on PFK are fully reversible and can be prevented or reverted by the binding of the enzyme to the actin filaments. Both drugs are also able to decrease glucose consumption by human breast cancer cell line MCF-7, as well as its viability, which decrease parallelly to the inhibition of PFK on these cells. In the end, we demonstrate the ability of ASA and SA to directly modulate an important regulatory intracellular enzyme, and propose that this is one of their mechanisms promoting anti-tumoral effects.

  12. Biased Type 1 Cannabinoid Receptor Signaling Influences Neuronal Viability in a Cell Culture Model of Huntington Disease.

    PubMed

    Laprairie, Robert B; Bagher, Amina M; Kelly, Melanie E M; Denovan-Wright, Eileen M

    2016-03-01

    Huntington disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder with limited treatment options. Prior to motor symptom onset or neuronal cell loss in HD, levels of the type 1 cannabinoid receptor (CB1) decrease in the basal ganglia. Decreasing CB1 levels are strongly correlated with chorea and cognitive deficit. CB1 agonists are functionally selective (biased) for divergent signaling pathways. In this study, six cannabinoids were tested for signaling bias in in vitro models of medium spiny projection neurons expressing wild-type (STHdh(Q7/Q7)) or mutant huntingtin protein (STHdh(Q111/Q111)). Signaling bias was assessed using the Black and Leff operational model. Relative activity [ΔlogR (τ/KA)] and system bias (ΔΔlogR) were calculated relative to the reference compound WIN55,212-2 for Gαi/o, Gαs, Gαq, Gβγ, and β-arrestin1 signaling following treatment with 2-arachidonoylglycerol (2-AG), anandamide (AEA), CP55,940, Δ(9)-tetrahydrocannabinol (THC), cannabidiol (CBD), and THC+CBD (1:1), and compared between wild-type and HD cells. The Emax of Gαi/o-dependent extracellular signal-regulated kinase (ERK) signaling was 50% lower in HD cells compared with wild-type cells. 2-AG and AEA displayed Gαi/o/Gβγ bias and normalized CB1 protein levels and improved cell viability, whereas CP55,940 and THC displayed β-arrestin1 bias and reduced CB1 protein levels and cell viability in HD cells. CBD was not a CB1 agonist but inhibited THC-dependent signaling (THC+CBD). Therefore, enhancing Gαi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD. In contrast, cannabinoids that are β-arrestin-biased--such as THC found at high levels in modern varieties of marijuana--may be detrimental to CB1 signaling, particularly in HD where CB1 levels are already reduced.

  13. Viability of adhered bacterial cells: tracking MinD protein oscillations

    NASA Astrophysics Data System (ADS)

    Barrett, Matt; Colville, Keegan; Schultz-Nielsen, Chris; Jericho, Manfred; Dutcher, John

    2010-03-01

    To study bacterial cells using atomic force microscopy, it is necessary to immobilize the cells on a substrate. Because bacterial cells and common substrates such as glass and mica have a net negative charge, positively charged polymers such as poly-L-lysine (PLL) and polyethyleneimine (PEI) are commonly used as adhesion layers. However, the use of adhesion polymers could stress the cell and even render it inviable. Viable E. coli cells use oscillations of Min proteins along the axis of the rod-shaped cells to ensure accurate cell division. By tagging MinD proteins with GFP, oscillations can be observed using fluorescence microscopy. For a healthy cell in an ideal environment, the oscillation period is measured to be ˜40 s. Prior experiments have shown that PLL increases the oscillation period significantly (up to 80%). In the present study, we have used epifluorescence and total internal reflection fluorescence (TIRF) to track MinD protein oscillations in E. coli bacteria adhered to a variety of positively charged polymers on mica as a function of polymer surface coverage.

  14. Tissue-specific mechanical and geometrical control of cell viability and actin cytoskeleton alignment

    NASA Astrophysics Data System (ADS)

    Wang, Dong; Zheng, Wenfu; Xie, Yunyan; Gong, Peiyuan; Zhao, Fang; Yuan, Bo; Ma, Wanshun; Cui, Yan; Liu, Wenwen; Sun, Yi; Piel, Matthieu; Zhang, Wei; Jiang, Xingyu

    2014-08-01

    Different tissues have specific mechanical properties and cells of different geometries, such as elongated muscle cells and polygonal endothelial cells, which are precisely regulated during embryo development. However, the mechanisms that underlie these processes are not clear. Here, we built an in vitro model to mimic the cellular microenvironment of muscle by combining both mechanical stretch and geometrical control. We found that mechanical stretch was a key factor that determined the optimal geometry of myoblast C2C12 cells under stretch, whereas vascular endothelial cells and fibroblasts had no such dependency. We presented the first experimental evidence that can explain why myoblasts are destined to take the elongated geometry so as to survive and maintain parallel actin filaments along the stretching direction. The study is not only meaningful for the research on myogenesis but also has potential application in regenerative medicine.

  15. Tissue-specific mechanical and geometrical control of cell viability and actin cytoskeleton alignment.

    PubMed

    Wang, Dong; Zheng, Wenfu; Xie, Yunyan; Gong, Peiyuan; Zhao, Fang; Yuan, Bo; Ma, Wanshun; Cui, Yan; Liu, Wenwen; Sun, Yi; Piel, Matthieu; Zhang, Wei; Jiang, Xingyu

    2014-08-22

    Different tissues have specific mechanical properties and cells of different geometries, such as elongated muscle cells and polygonal endothelial cells, which are precisely regulated during embryo development. However, the mechanisms that underlie these processes are not clear. Here, we built an in vitro model to mimic the cellular microenvironment of muscle by combining both mechanical stretch and geometrical control. We found that mechanical stretch was a key factor that determined the optimal geometry of myoblast C2C12 cells under stretch, whereas vascular endothelial cells and fibroblasts had no such dependency. We presented the first experimental evidence that can explain why myoblasts are destined to take the elongated geometry so as to survive and maintain parallel actin filaments along the stretching direction. The study is not only meaningful for the research on myogenesis but also has potential application in regenerative medicine.

  16. Injuries to cultivated BJA-B cells by ajoene, a garlic-derived natural compound: cell viability, glutathione metabolism, and pools of acidic amino acids.

    PubMed

    Scharfenberg, K; Ryll, T; Wagner, R; Wagner, K G

    1994-01-01

    Ajoene (4,5,9-trithiadodeca-1,6,11-triene-9-oxide), a garlic-derived natural compound, which had been shown to have cytostatic/cytotoxic properties, was tested with a B cell lymphoma-derived cell line (BJA-B cells) in order to elucidate its mechanism of cytotoxic action. Viability of the cells was determined by the Trypan blue exclusion test and the colorimetric tetrazolium (MTT) assay, whereas metabolic disturbance was evaluated by measuring the pools of reduced (GSH), oxidized glutathione (GSSG) and the acidic amino acids, Glu and Asp. Fast uptake of ajoene was accompanied by an immediate reduction of the GSH and increase in the GSSG levels. The extent of these changes, as well as the further development of the metabolite pools, depended on the ajoene dose per cell. At a sublethal ajoene dose the GSH and GSSG pools rose at the later stages to levels much higher than in the control experiment. Bleb formation at the cytoplasmic membrane was a further rapid phenomenon, although injuries detected by Trypan blue exclusion developed only at a later stage. The MTT assay, performed in a parallel experiment (48 h after ajoene addition), showed, however, that reduction of cell viability was established at the very beginning of ajoene exposure. Altogether, the action of ajoene strongly resembled oxidative stress (i.e., interference with SH homeostasis and its pleiotropic consequences to cell physiology and metabolism.

  17. Effects of hydroxyapatite nanostructure on channel surface of porcine acellular dermal matrix scaffold on cell viability and osteogenic differentiation of human periodontal ligament stem cells.

    PubMed

    Ge, Shaohua; Zhao, Ning; Wang, Lu; Liu, Hong; Yang, Pishan

    2013-01-01

    A new nanostructured hydroxyapatite-coated porcine acellular dermal matrix (HAp-PADM) was fabricated by a biomimetic mineralization method. Human periodontal ligament stem cells were seeded on HAp-PADM and the effects of this scaffold on cell shape, cytoskeleton organization, cell viability, and osteogenic differentiation were examined. Periodontal ligament stem cells cultured on HAp-PADM exhibited different cell shape when compared with those on pure PADM. Moreover, HAp-PADM promoted cell viability and alkaline phosphatase activity significantly. Based on quantitative real-time polymerase chain reaction, the expression of bone-related markers runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) upregulated in the HAp-PADM scaffold. The enhancement of osteogenic differentiation of periodontal ligament stem cells on the HAp-PADM scaffold was proposed based on the research results. The results of this study highlight the micro-nano, two-level, three-dimensional HAp-PADM composite as a promising scaffold for periodontal tissue engineering.

  18. Effect of estradiol and bisphenol A on human hepatoblastoma cell viability and telomerase activity

    PubMed Central

    Xu, B.L.; Zhao, Q.Z.; Gao, X.Y.; Hou, G.J.

    2015-01-01

    Sex hormones from environmental and physiological sources might play a major role in the pathogenesis of hepatoblastoma in children. This study investigated the effects of estradiol and bisphenol A on the proliferation and telomerase activity of human hepatoblastoma HepG2 cells. The cells were divided into 6 treatment groups: control, bisphenol A, estradiol, anti-estrogen ICI 182,780 (hereinafter ICI), bisphenol A+ICI, and estradiol+ICI. Cell proliferation was measured based on average absorbance using the Cell Counting-8 assay. The cell cycle distribution and apoptotic index were determined by flow cytometry. Telomerase activity was detected by polymerase chain reaction and a telomeric repeat amplification protocol assay. A higher cell density was observed in bisphenol A (P<0.01) and estradiol (P<0.05) groups compared with the control group. Cell numbers in S and G2/M phases after treatment for 48 h were higher (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h (P<0.05) were higher in these groups than in the control group. The cell density was also higher in bisphenol A+ICI (P<0.01) and estradiol+ICI (P<0.05) groups compared with the ICI group. Furthermore, cell numbers were increased in S and G2/M phases (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h were higher (P<0.05) in these groups than in the ICI group. Therefore, bisphenol A and estradiol promote HepG2 cell proliferation in vitro by inhibition of apoptosis and stimulation of telomerase activity via an estrogen receptor-dependent pathway. PMID:26397976

  19. Effect of estradiol and bisphenol A on human hepatoblastoma cell viability and telomerase activity.

    PubMed

    Xu, B L; Zhao, Q Z; Gao, X Y; Hou, G J

    2015-11-01

    Sex hormones from environmental and physiological sources might play a major role in the pathogenesis of hepatoblastoma in children. This study investigated the effects of estradiol and bisphenol A on the proliferation and telomerase activity of human hepatoblastoma HepG2 cells. The cells were divided into 6 treatment groups: control, bisphenol A, estradiol, anti-estrogen ICI 182,780 (hereinafter ICI), bisphenol A+ICI, and estradiol+ICI. Cell proliferation was measured based on average absorbance using the Cell Counting-8 assay. The cell cycle distribution and apoptotic index were determined by flow cytometry. Telomerase activity was detected by polymerase chain reaction and a telomeric repeat amplification protocol assay. A higher cell density was observed in bisphenol A (P<0.01) and estradiol (P<0.05) groups compared with the control group. Cell numbers in S and G2/M phases after treatment for 48 h were higher (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h (P<0.05) were higher in these groups than in the control group. The cell density was also higher in bisphenol A+ICI (P<0.01) and estradiol+ICI (P<0.05) groups compared with the ICI group. Furthermore, cell numbers were increased in S and G2/M phases (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h were higher (P<0.05) in these groups than in the ICI group. Therefore, bisphenol A and estradiol promote HepG2 cell proliferation in vitro by inhibition of apoptosis and stimulation of telomerase activity via an estrogen receptor-dependent pathway.

  20. 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

    2016-07-01

    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. PMID:27267805

  1. Treatment of leptothrix cells with ultrapure water poses a threat to their viability.

    PubMed

    Kunoh, Tatsuki; Suzuki, Tomoko; Shiraishi, Tomonori; Kunoh, Hitoshi; Takada, Jun

    2015-01-01

    The genus Leptothrix, a type of Fe/Mn-oxidizing bacteria, is characterized by its formation of an extracellular and microtubular sheath. Although almost all sheaths harvested from natural aquatic environments are hollow, a few chained bacterial cells are occasionally seen within some sheaths of young stage. We previously reported that sheaths of Leptothrix sp. strain OUMS1 cultured in artificial media became hollow with aging due to spontaneous autolysis within the sheaths. In this study, we investigated environmental conditions that lead the OUMS1 cells to die. Treatment of the cells with ultrapure water or acidic buffers (pH 6.0) caused autolysis of the cells. Under these conditions, the plasma membrane and cytoplasm of cells were drastically damaged, resulting in leakage of intracellular electrolytes and relaxation of genomic DNA. The autolysis was suppressed by the presence of Ca2+. The hydrolysis of peptidoglycan by the lysozyme treatment similarly caused autolysis of the cells and was suppressed also by the presence of Ca2+. However, it remains unclear whether the acidic pH-dependent autolysis is attributable to damage of peptidoglycan. It was observed that L. discophora strain SP-6 cells also underwent autolysis when suspended in ultrapure water; it is however, uncertain whether this phenomenon is common among other members of the genus Leptothrix. PMID:25634812

  2. Treatment of Leptothrix Cells with Ultrapure Water Poses a Threat to Their Viability

    PubMed Central

    Kunoh, Tatsuki; Suzuki, Tomoko; Shiraishi, Tomonori; Kunoh, Hitoshi; Takada, Jun

    2015-01-01

    The genus Leptothrix, a type of Fe/Mn-oxidizing bacteria, is characterized by its formation of an extracellular and microtubular sheath. Although almost all sheaths harvested from natural aquatic environments are hollow, a few chained bacterial cells are occasionally seen within some sheaths of young stage. We previously reported that sheaths of Leptothrix sp. strain OUMS1 cultured in artificial media became hollow with aging due to spontaneous autolysis within the sheaths. In this study, we investigated environmental conditions that lead the OUMS1 cells to die. Treatment of the cells with ultrapure water or acidic buffers (pH 6.0) caused autolysis of the cells. Under these conditions, the plasma membrane and cytoplasm of cells were drastically damaged, resulting in leakage of intracellular electrolytes and relaxation of genomic DNA. The autolysis was suppressed by the presence of Ca2+. The hydrolysis of peptidoglycan by the lysozyme treatment similarly caused autolysis of the cells and was suppressed also by the presence of Ca2+. However, it remains unclear whether the acidic pH-dependent autolysis is attributable to damage of peptidoglycan. It was observed that L. discophora strain SP-6 cells also underwent autolysis when suspended in ultrapure water; it is however, uncertain whether this phenomenon is common among other members of the genus Leptothrix. PMID:25634812

  3. Effects of Non-Collagenous Proteins, TGF-β1, and PDGF-BB on Viability and Proliferation of Dental Pulp Stem Cells

    PubMed Central

    Tabatabaei, Fahimeh Sadat

    2016-01-01

    ABSTRACT Objectives The dentin matrix servers as a reservoir of growth factors, sequestered during dentinogenesis. The aim of this study was to assess the viability and proliferation of dental pulp stem cells in the presence of dentin matrix-derived non-collagenous proteins and two growth factors; platelet-derived growth factor BB and transforming growth factor beta 1. Material and Methods The dental pulp cells were isolated and cultured. The dentin proteins were extracted and purified. The MTT assay was performed for assessment of cell viability and proliferation in the presence of different concentrations of dentin proteins and growth factors during 24 - 72 h post-treatment. Results The cells treated with 250 ng/mL dentin proteins had the best viability and proliferation ability in comparison with other concentrations (P < 0.05). The MTT assay demonstrated that cells cultured with 5 ng/mL platelet-derived growth factor BB had the highest viability at each time point as compared to other groups (P < 0.05). However, in presence of platelet-derived growth factor BB alone and in combination with transforming growth factor beta 1 and dentin proteins (10 ng/mL), significant higher viability was seen at all time points (P < 0.05). The least viability and proliferation at each growth factor concentration was seen in cells treated with combination of transforming growth factor beta 1 and dentin proteins at 72 h (P < 0.05). Conclusions The results indicated that the triple combination of growth factors and matrix-derived non-collagenous proteins (especially at 10 ng/mL concentration) has mitogenic effect on dental pulp stem cells. PMID:27099698

  4. H2A.Z depletion impairs proliferation and viability but not DNA double-strand breaks repair in human immortalized and tumoral cell lines

    PubMed Central

    Taty-Taty, Gemael-Cedrick; Courilleau, Celine; Quaranta, Muriel; Carayon, Alexandre; Chailleux, Catherine; Aymard, François; Trouche, Didier; Canitrot, Yvan

    2014-01-01

    In mammalian cells, DNA double-strand breaks (DSB) can be repaired by 2 main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). To give access to DNA damage to the repair machinery the chromatin structure needs to be relaxed, and chromatin modifications play major roles in the control of these processes. Among the chromatin modifications, changes in nucleosome composition can influence DNA damage response as observed with the H2A.Z histone variant in yeast. In mammals, p400, an ATPase of the SWI/SNF family able to incorporate H2A.Z in chromatin, was found to be important for histone ubiquitination and BRCA1 recruitment around DSB or for HR in cooperation with Rad51. Recent data with 293T cells showed that mammalian H2A.Z is recruited to DSBs and is important to control DNA resection, therefore participating both in HR and NHEJ. Here we show that depletion of H2A.Z in the osteosarcoma U2OS cell line and in immortalized human fibroblasts does not change parameters of DNA DSB repair while affecting clonogenic ability and cell cycle distribution. In addition, no recruitment of H2A.Z around DSB can be detected in U2OS cells either after local laser irradiation or by chromatin immunoprecipitation. These data suggest that the role of H2A.Z in DSB repair is not ubiquitous in mammals. In addition, given that important cellular parameters, such as cell viability and cell cycle distribution, are more sensitive to H2A.Z depletion than DNA repair, our results underline the difficulty to investigate the role of versatile factors such as H2A.Z. PMID:24240188

  5. Determination of physical membrane properties of plant cell protoplasts via the electrofusion technique: prediction of optimal fusion yields and protoplast viability.

    PubMed

    Mehrle, W; Naton, B; Hampp, R

    1990-04-01

    By variation of physical parameters (field strength, pulse duration) which result in electrofusion and electroporation, properties of the plasma membrane of different types of plant cell protoplasts were analyzed. The lower threshold for that field pulse intensity at which membrane breakdown occurred (recorded as fusion event) depended on pulse duration, protoplast size, and protoplast type (tobacco, oat; vacuolated, evacuolated). This fusion characteristic of plant protoplasts can also be taken as a measure of the charging process of the membrane and allows thus a non-invasive determination of the time constant and the specific membrane capacitance. Although the fusion yield was comparable at pulse duration/field strength couples of, e.g., 10 μs/1.5 kV*cm(-1) and 200 μs/0.5 kV*cm(-1), hybrid viability was not. Rates of cell wall regeneration and cell division of tobacco mesophyll protoplasts were not affected but may have been increased at short pulse duration/high field strength. Plating efficiency, in contrast, was significantly decreased with longer pulse duration at low field strengths. PMID:24232787

  6. Role of surface-electrical properties on the cell-viability of carbon thin films grown in nanodomain morphology

    NASA Astrophysics Data System (ADS)

    Javid, Amjed; Kumar, Manish; Yoon, Seokyoung; Lee, Jung Heon; Tajima, Satomi; Hori, Masaru; Geon Han, Jeon

    2016-07-01

    Carbon thin films, having a combination of unique physical and chemical properties, exhibit an interesting biocompatibility and biological response to living entities. Here, the carbon films are developed in the morphology form of nano-domains with nanoscale inter-domain separations, tuned by plasma conditions in the facing target magnetron sputtering process. The wettability and surface energy are found to have a close relation to the inter-domain separations. The chemical structure of carbon films exhibited the relative enhancement of sp3 in comparison to sp2 with the increase of domain separations. The cell-viability of these films shows promising results for L929 mouse fibroblast and Saos-2 bone cells, when inter-domain separation is increased. Electrical conductivity and surface energy are identified to play the key role in different time-scales during the cell-proliferation process. The contribution from electrical conductivity is dominant in the beginning of the cultivation, whereas with the passage of time (~3-5 d) the surface energy takes control over conductivity to enhance the cell proliferation.

  7. Role of surface-electrical properties on the cell-viability of carbon thin films grown in nanodomain morphology

    NASA Astrophysics Data System (ADS)

    Javid, Amjed; Kumar, Manish; Yoon, Seokyoung; Lee, Jung Heon; Tajima, Satomi; Hori, Masaru; Geon Han, Jeon

    2016-07-01

    Carbon thin films, having a combination of unique physical and chemical properties, exhibit an interesting biocompatibility and biological response to living entities. Here, the carbon films are developed in the morphology form of nano-domains with nanoscale inter-domain separations, tuned by plasma conditions in the facing target magnetron sputtering process. The wettability and surface energy are found to have a close relation to the inter-domain separations. The chemical structure of carbon films exhibited the relative enhancement of sp3 in comparison to sp2 with the increase of domain separations. The cell-viability of these films shows promising results for L929 mouse fibroblast and Saos-2 bone cells, when inter-domain separation is increased. Electrical conductivity and surface energy are identified to play the key role in different time-scales during the cell-proliferation process. The contribution from electrical conductivity is dominant in the beginning of the cultivation, whereas with the passage of time (~3–5 d) the surface energy takes control over conductivity to enhance the cell proliferation.

  8. Arctigenin Inhibits Lung Metastasis of Colorectal Cancer by Regulating Cell Viability and Metastatic Phenotypes.

    PubMed

    Han, Yo-Han; Kee, Ji-Ye; Kim, Dae-Seung; Mun, Jeong-Geon; Jeong, Mi-Young; Park, Sang-Hyun; Choi, Byung-Min; Park, Sung-Joo; Kim, Hyun-Jung; Um, Jae-Young; Hong, Seung-Heon

    2016-08-27

    Arctigenin (ARC) has been shown to have an anti-cancer effect in various cell types and tissues. However, there have been no studies concerning metastatic colorectal cancer (CRC). In this study, we investigated the anti-metastatic properties of ARC on colorectal metastasis and present a potential candidate drug. ARC induced cell cycle arrest and apoptosis in CT26 cells through the intrinsic apoptotic pathway via MAPKs signaling. In several metastatic phenotypes, ARC controlled epithelial-mesenchymal transition (EMT) through increasing the expression of epithelial marker E-cadherin and decreasing the expressions of mesenchymal markers; N-cadherin, vimentin, β-catenin, and Snail. Moreover, ARC inhibited migration and invasion through reducing of matrix metalloproteinase-2 (MMP-2) and MMP-9 expressions. In an experimental metastasis model, ARC significantly inhibited lung metastasis of CT26 cells. Taken together, our study demonstrates the inhibitory effects of ARC on colorectal metastasis.

  9. Arctigenin Inhibits Lung Metastasis of Colorectal Cancer by Regulating Cell Viability and Metastatic Phenotypes.

    PubMed

    Han, Yo-Han; Kee, Ji-Ye; Kim, Dae-Seung; Mun, Jeong-Geon; Jeong, Mi-Young; Park, Sang-Hyun; Choi, Byung-Min; Park, Sung-Joo; Kim, Hyun-Jung; Um, Jae-Young; Hong, Seung-Heon

    2016-01-01

    Arctigenin (ARC) has been shown to have an anti-cancer effect in various cell types and tissues. However, there have been no studies concerning metastatic colorectal cancer (CRC). In this study, we investigated the anti-metastatic properties of ARC on colorectal metastasis and present a potential candidate drug. ARC induced cell cycle arrest and apoptosis in CT26 cells through the intrinsic apoptotic pathway via MAPKs signaling. In several metastatic phenotypes, ARC controlled epithelial-mesenchymal transition (EMT) through increasing the expression of epithelial marker E-cadherin and decreasing the expressions of mesenchymal markers; N-cadherin, vimentin, β-catenin, and Snail. Moreover, ARC inhibited migration and invasion through reducing of matrix metalloproteinase-2 (MMP-2) and MMP-9 expressions. In an experimental metastasis model, ARC significantly inhibited lung metastasis of CT26 cells. Taken together, our study demonstrates the inhibitory effects of ARC on colorectal metastasis. PMID:27618887

  10. Effect of low-dimensional alumina structures on viability of L 929 cells

    SciTech Connect

    Fomenko, Alla N. Korovin, Matvey S. Bakina, Olga V. Kazantsev, Sergey O. Glazkova, Elena A. Svarovskaya, Natalia V. Lozhkomoev, Aleksandr S.

    2015-10-27

    In the study, we estimated the cytotoxicity of alumina nanoparticles differing in shape (nanofibers, nanoplates, nanosheets, agglomerates of nanosheets) and close in physicochemical properties (particle size, specific surface area, phase composition, and zeta potential). The alumina structures were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD) data, low-temperature nitrogen adsorption, and dynamic light scattering (DLS). The cytotoxicity was estimated on fibroblast cells of the L929 line. It was found that a more adverse effect on the cells was exerted by alumina nanofibers and nanosheets. The action of nanosheets on the cells was inhibitory and was of about the same level, irrespective of the observation period. The effect of alumina nanosheet agglomerates and nanoplates on the cell proliferation was weak even at an exposure time of 72 h.

  11. The NAD(+) salvage pathway modulates cancer cell viability via p73.

    PubMed

    Sharif, T; Ahn, D-G; Liu, R-Z; Pringle, E; Martell, E; Dai, C; Nunokawa, A; Kwak, M; Clements, D; Murphy, J P; Dean, C; Marcato, P; McCormick, C; Godbout, R; Gujar, S A; Lee, P W K

    2016-04-01

    The involvement of the nicotinamide adenine dinucleotide (NAD(+)) salvage pathway in cancer cell survival is poorly understood. Here we show that the NAD(+) salvage pathway modulates cancer cell survival through the rarely mutated tumour suppressor p73. Our data show that pharmacological inhibition or knockdown of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD(+) salvage pathway, enhances autophagy and decreases survival of cancer cells in a p53-independent manner. Such NAMPT inhibition stabilizes p73 independently of p53 through increased acetylation and decreased ubiquitination, resulting in enhanced autophagy and cell death. These effects of NAMPT inhibition can be effectively reversed using nicotinamide mononucleotide (NMN), the enzymatic product of NAMPT. Similarly, knockdown of p73 also decreases NAMPT inhibition-induced autophagy and cell death, whereas overexpression of p73 alone enhances these effects. We show that the breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) harbour significantly higher levels of NAMPT and lower levels of p73 than does the normal cell line (MCF-10A), and that NAMPT inhibition is cytotoxic exclusively to the cancer cells. Furthermore, data from 176 breast cancer patients demonstrate that higher levels of NAMPT and lower levels of p73 correlate with poorer patient survival, and that high-grade tumours have significantly higher NAMPT/p73 mRNA ratios. Therefore, the inverse relationship between NAMPT and p73 demonstrable in vitro is also reflected from the clinical data. Taken together, our studies reveal a new NAMPT-p73 nexus that likely has important implications for cancer diagnosis, prognosis and treatment.

  12. L-DOPA modulates cell viability through the ERK-c-Jun system in PC12 and dopaminergic neuronal cells.

    PubMed

    Park, Keun Hong; Shin, Keon Sung; Zhao, Ting Ting; Park, Hyun Jin; Lee, Kyung Eun; Lee, Myung Koo

    2016-02-01

    L-DOPA causes neurotoxicity by modulating the Epac-ERK system in PC12 cells. This study investigated the effects of a single treatment with L-DOPA and multiple treatments with L-DOPA (MT-LD) on ERK1/2 and JNK1/2-c-Jun systems. In PC12 cells, a toxic L-DOPA concentration (200 μM) induced sustained ERK1/2 and JNK1/2 phosphorylation that was inhibited by the Epac inhibitor brefeldin A, but not by the PKA inhibitor H89. This ERK1/2 and JNK1/2 phosphorylation was also inhibited by ERK1/2 (U0126) and JNK1/2 (SP600125) inhibitors, respectively, but sustained ERK1/2 phosphorylation was not affected by JNK1/2 phosphorylation. A non-toxic L-DOPA concentration (20 μM) induced c-Jun phosphorylation (Ser73) via transient ERK1/2 phosphorylation, whereas the toxic L-DOPA concentration induced c-Jun phosphorylation (Ser63) and c-Jun expression via Epac-sustained ERK1/2-JNK1/2 phosphorylation, which then enhanced cleaved caspase-3 expression. MT-LD (20 μM) initially enhanced c-Jun phosphorylation (Ser73) (for 1-4 days), but later (5-6 days) induced c-Jun phosphorylation (Ser63) and c-Jun expression. In the 6-hydroxydopamine-lesioned rat model of Parkinson's disease, L-DOPA administration (10 mg/kg) protected against neurotoxicity through c-Jun phosphorylation (Ser73) for 1-2 weeks. However, L-DOPA administration (10 or 30 mg/kg) showed neurotoxicity through c-Jun phosphorylation (Ser63) and c-Jun expression via ERK1/2 phosphorylation for 3-4 weeks. Thus, in PC12 cells, non-toxic L-DOPA treatment maintained cell survival through c-Jun phosphorylation (Ser73). By contrast, toxic L-DOPA treatment or MT-LD (20 μM) induced c-Jun phosphorylation (Ser63) and c-Jun expression via Epac-dependent sustained ERK1/2 and JNK1/2 phosphorylation, which subsequently led to cell death. These results were validated by those obtained after long-term L-DOPA administration in a rat model of Parkinson's disease. Our data indicate that L-DOPA causes neurotoxicity via the ERK1/2-c-Jun system in

  13. Dexamethasone and Azathioprine Promote Cytoskeletal Changes and Affect Mesenchymal Stem Cell Migratory Behavior

    PubMed Central

    Schneider, Natália; Gonçalves, Fabiany da Costa; Pinto, Fernanda Otesbelgue; Lopez, Patrícia Luciana da Costa; Araújo, Anelise Bergmann; Pfaffenseller, Bianca; Passos, Eduardo Pandolfi; Cirne-Lima, Elizabeth Obino; Meurer, Luíse; Lamers, Marcelo Lazzaron; Paz, Ana Helena

    2015-01-01

    Glucocorticoids and immunosuppressive drugs are commonly used to treat inflammatory disorders, such as inflammatory bowel disease (IBD), and despite a few improvements, the remission of IBD is still difficult to maintain. Due to their immunomodulatory properties, mesenchymal stem cells (MSCs) have emerged as regulators of the immune response, and their viability and activation of their migratory properties are essential for successful cell therapy. However, little is known about the effects of immunosuppressant drugs used in IBD treatment on MSC behavior. The aim of this study was to evaluate MSC viability, nuclear morphometry, cell polarity, F-actin and focal adhesion kinase (FAK) distribution, and cell migratory properties in the presence of the immunosuppressive drugs azathioprine (AZA) and dexamethasone (DEX). After an initial characterization, MSCs were treated with DEX (10 μM) or AZA (1 μM) for 24 hrs or 7 days. Neither drug had an effect on cell viability or nuclear morphometry. However, AZA treatment induced a more elongated cell shape, while DEX was associated with a more rounded cell shape (P < 0.05) with a higher presence of ventral actin stress fibers (P < 0.05) and a decrease in protrusion stability. After 7 days of treatment, AZA improved the cell spatial trajectory (ST) and increased the migration speed (24.35%, P < 0.05, n = 4), while DEX impaired ST and migration speed after 24 hrs and 7 days of treatment (-28.69% and -25.37%, respectively; P < 0.05, n = 4). In conclusion, our data suggest that these immunosuppressive drugs each affect MSC morphology and migratory capacity differently, possibly impacting the success of cell therapy. PMID:25756665

  14. Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

    SciTech Connect

    Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung; Yoo, Eun-Sun; Chan Ra, Jeong; Ryu, Kyung-Ha

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.

  15. 14-3-3γ regulates cell viability and milk fat synthesis in lipopolysaccharide-induced dairy cow mammary epithelial cells

    PubMed Central

    LIU, LIXIN; ZHANG, LI; LIN, YE; BIAN, YANJIE; GAO, XUEJUN; QU, BO; LI, QINGZHANG

    2016-01-01

    Our previous study demonstrated that 14-3-3γ overexpression was able to inhibit the production of lipopolysaccharide (LPS)-induced cytokines in dairy cow mammary epithelial cells (DCMECs) by inhibiting the activation of nuclear factor-κB (NF-κB) signaling pathways. However, the association between 14-3-3γ overexpression and milk fat synthesis in LPS-induced DCMECs remains unclear. Therefore, the present study investigated the effect of 14-3-3γ on cell viability and milk fat synthesis in LPS-induced DCMECs. The results of the MTT assay and lactate dehydrogenase activity assay demonstrated that 14-3-3γ overexpression was able to attenuate LPS-induced cytotoxicity in DCMECs, and increase the viability of the cells. In addition, the results of reverse transcription-quantitative polymerase chain reaction suggested that mRNA expression levels of genes associated with milk fat synthesis, including sterol regulatory element binding protein (SREBP1), peroxisome proliferator-activated receptor-γ (PPARG), cluster of differentiation 36, acetyl-coA carboxylase (ACC), fatty acid synthase (FAS) and fatty acid binding protein-3, were significantly upregulated in cells overexpressing the 14-3-3γ protein. In addition, as compared with the LPS-treated group, the activities of FAS and ACC were significantly increased. Furthermore, western blotting demonstrated that 14-3-3γ overexpression enhanced the protein expression levels of phosphorylated SREBP1 and PPARG. These results suggested that high levels of 14-3-3γ protein were able to attenuate LPS-induced cell damage and promote milk fat synthesis in LPS-induced DCMECs by increasing the cell viability and upregulating the expression levels of transcription factors associated with milk fat synthesis. PMID:27073437

  16. Development of sulfadiazine-decorated PLGA nanoparticles loaded with 5-fluorouracil and cell viability.

    PubMed

    Guimarães, Pedro Pires Goulart; Oliveira, Sheila Rodrigues; de Castro Rodrigues, Gabrielle; Gontijo, Savio Morato Lacerda; Lula, Ivana Silva; Cortés, Maria Esperanza; Denadai, Ângelo Márcio Leite; Sinisterra, Rubén Dario

    2015-01-01

    The aim of this work was to synthesize sulfadiazine-poly(lactide-co-glycolide) (SUL-PLGA) nanoparticles (NPs) for the efficient delivery of 5-fluorouracil to cancer cells. The SUL-PLGA conjugation was assessed using FTIR, 1H-NMR, 13C-NMR, elemental analysis and TG and DTA analysis. The SUL-PLGA NPs were characterized using transmission and scanning electron microscopy and dynamic light scattering. Additionally, the zeta potential, drug content, and in vitro 5-FU release were evaluated. We found that for the SUL-PLGA NPs, Dh = 114.0 nm, ZP = -32.1 mV and the encapsulation efficiency was 49%. The 5-FU was released for up to 7 days from the NPs. Cytotoxicity evaluations of 5-FU-loaded NPs (5-FU-SUL-PLGA and 5-FU-PLGA) on two cancer cell lines (Caco-2, A431) and two normal cell lines (fibroblast, osteoblast) were compared. Higher cytotoxicity of 5-FU-SUL-PLGA NPs were found to both cancer cell lines when compared to normal cell lines, demonstrating that the presence of SUL could significantly enhance the cytotoxicity of the 5-FU-SUL-PLGA NPs when compared with 5-FU-PLGA NPs. Thus, the development of 5-FU-SUL-PLGA NPs to cancer cells is a promising strategy for the 5-FU antitumor formulation in the future. PMID:25580685

  17. Intracellular reactive oxygen species are essential for PI3K/Akt/mTOR-dependent IL-7-mediated viability of T-cell acute lymphoblastic leukemia cells.

    PubMed

    Silva, A; Gírio, A; Cebola, I; Santos, C I; Antunes, F; Barata, J T

    2011-06-01

    Interleukin-7 (IL-7) activates phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, thereby mediating viability, proliferation and growth of T-cell acute lymphoblastic leukemia (T-ALL) cells. Reactive oxygen species (ROS) can be upregulated by growth factors and are known to regulate proliferation and viability. Here, we show that IL-7 upregulates ROS in T-ALL cells in a manner that is dependent on PI3K/Akt/mTOR pathway activity and that relies on both NADPH oxidase and mitochondrial respiratory chain. Conversely, IL-7-induced activation of PI3K signaling pathway requires mitochondrial respiration and ROS. We have previously shown that IL-7-mediated activation of PI3K pathway drives the upregulation of the glucose transporter Glut1, promoting glucose uptake in T-ALL cells. Using phloretin to inhibit Glut function, we demonstrate that glucose uptake is mandatory for ROS upregulation in IL-7-treated T-ALL cells, suggesting that IL-7 stimulation leads to increased ROS via PI3K pathway activation and consequent upregulation of Glut1 and glucose uptake. Overall, our data reveal the existence of a critical crosstalk between PI3K/Akt signaling pathway and ROS that is essential for IL-7-mediated T-ALL cell survival, and that may constitute a novel target for therapeutic intervention. PMID:21455214

  18. Specific combinations of the chromatin-modifying enzyme modulators significantly attenuate glioblastoma cell proliferation and viability while exerting minimal effect on normal adult stem cells growth.

    PubMed

    Alexanian, Arshak R; Huang, Yi-Wen

    2015-11-01

    The discoveries of recent decade showed that all critical changes in cancer cells, such as silencing of tumor-suppressor genes and activation of oncogenes, are caused not only by genetic but also by epigenetic mechanisms. Although epigenetic changes are somatically heritable, in contrast to genetic changes, they are potentially reversible, making them good targets for therapeutic intervention. Covalent modifications of chromatin such as methylation and acetylation of histones and methylation of DNA are the important components of epigenetic machinery. In this study, we investigated the effect of different modulators of DNA and histone covalent-modifying enzymes on the proliferation and viability of normal adult stem cells, such as human bone marrow mesenchymal stem cells (hMSCs), and on malignant tumor cells, such as glioblastoma (GB) D54 cells. Results demonstrated that specific combinations of histone methyltransferases and deacetylases inhibitors significantly attenuated D54 cells viability but having only a small effect on hMSCs growth. Taken together, these studies suggest that specific combinations of histone covalent modifiers could be an effective treatment option for the most aggressive type of primary brain tumors such as glioblastoma multiforme.

  19. In vitro organotin administration alters guinea pig cochlear outer hair cell shape and viability.

    PubMed

    Clerici, W J; Chertoff, M E; Brownell, W E; Fechter, L D

    1993-06-01

    Trimethyltin (TMT) and triethyltin (TET) disrupt auditory function at doses far below those shown to be neurotoxic. In vivo studies suggest that the initial effect of TMT on hearing occurs at the inner hair cell/spiral ganglion cell synapse, while later, the outer hair cell (OHC) undergoes structural and functional damage. TET produces acute effects upon afferent neurotransmission similar to those observed following TMT, but TET's effects on OHC structure and function have not been examined. OHCs are motile elements within the cochlea, believed to modulate the sensitivity and tuning within the inner ear. Changes in OHC length may alter hearing function, and length changes have been reported following exposure to various ototoxic agents in vitro. In the present study, 77 OHCs from 45 pigmented male guinea pigs were isolated in primary culture and exposed for 90 min to concentrations between 30 microM and 1.0 mM of TMT or TET and then to bathing medium for 30 min to remove the toxicant. Significant shortening of the OHC cell body occurred at all doses to both organotins, with a mean reduction in length of 15.1 and 20.2% for 1.0 mM TMT and TET, respectively, at the end of testing; control cells were only 3.4% shorter at the end of 90 min of perfusion with bathing medium. The effect of organotin exposure on OHC volume was not consistently related to either TMT or TET concentration or altered cell length. In addition, disruption of the plasma membrane characterized by bleb formation, the forceful ejection of cytoplasm, or bursting was seen in 80% of cells exposed to 1.0 mM TET, although not TMT; lower concentrations of both organotins disrupted the cell membrane in 10-30% of cells. Membrane rupture was not reliably associated with either increased cell volume or decreased length, implicating a weakening of the plasma membrane or cortical lattice as the basis for this effect. Consistent with the irreversible structural weakening of the lateral wall, resorption of

  20. Increased viability of odontoblast-like cells subjected to low-level laser irradiation

    NASA Astrophysics Data System (ADS)

    Oliveira, C. F.; Basso, F. G.; Lins, E. C.; Kurachi, C.; Hebling, J.; Bagnato, V. S.; de Souza Costa, C. A.

    2010-07-01

    Studies have shown that the increase of cell metabolism depends on the low level laser therapy (LLLT) parameters used to irradiate the cells. However, the optimal laser dose to up-regulate pulp cell activity remains unknown. Consequently, the aim of this study was to evaluate the metabolic response of odontoblast-like cells (MDPC-23) exposed to different LLLT doses. Cells at 20000 cells/cm2 were seeded in 24-well plates using plain culture medium (DMEM) and were incubated in a humidified incubator with 5% CO2 at 37°C. After 24 h, the culture medium was replaced by fresh DMEM supplemented with 5% (stress by nutritional deficit) or 10% fetal bovine serum (FBS). The cells were exposed to different laser doses from a near infrared diode laser prototype designed to provide a uniform irradiation of the wells. The experimental groups were: G1: 1.5 J/cm2 + 5% FBS; G2: 1.5 J/cm2 + 10% FBS; G3: 5 J/cm2 + 5% FBS; G4: 5 J/cm2 + 10% FBS; G5: 19 J/cm2 + 5% FBS; G6: 19 J/cm2 + 10% FBS. LLLT was performed in 3 consecutive irradiation cycles with a 24-hour interval. Non-irradiated cells cultured in DMEM supplemented with either 5 or 10% FBS served as control groups. The analysis of the metabolic response was performed by the MTT assay 3 h after the last irradiation. G1 presented an increase in SDH enzyme activity and differed significantly (Mann-Whitney test, p < 0.05) from the other groups. Analysis by scanning electron microscopy showed normal cell morphology in all groups. Under the tested conditions, LLLT stimulated the metabolic activity of MDPC-23 cultured in DMEM supplemented with 5% FBS and exposed to a laser dose of 1.5 J/cm2. These findings are relevant for further studies on the action of near infrared lasers on cells with odontoblast phenotype.

  1. The role of ribosylated-BSA in regulating PC12 cell viability.

    PubMed

    Kuo, Tsun-Yung; Huang, Chuen-Lin; Yang, Jung-Mou; Huang, Wei-Jung; Huang, Nai-Kuei; Chen, Yue-Wen; Lin, Ren-Jye; Yang, Ying-Chen

    2012-08-01

    Glycation, one of the post-translational modifications, is known to influence protein structure and biological function. Advanced glycation end products (AGEs) have been shown to cause pathologies of diabetes. Glycation levels in patients with Alzheimer's disease (AD) are higher than in normal people. However, whether the glycation of susceptible proteins is a triggering event for cell damage or simply a result remains to be elucidated. In this study, we demonstrated that ribose-conjugated BSA (Rib-BSA) directly induces PC12 cell death in a dose- and time-dependent manner. The IC(50) is 4.6 μM. Unlike glucose-incubated BSA, Rib-BSA rapidly forms cytotoxic AGEs. PC12 is vulnerable to Rib-BSA. However, fructose can induce AGE formation, although no effect on cell survival was observed. This effect of Rib-BSA is reversed by pretreatment of pioglitazone and rosiglitazone, which belongs to thiazolidinediones (TZDs) and are peroxisome proliferator-activated receptor (PPAR-γ) ligands. Moreover, Rib-BSA upregulates inducible nitric oxide synthase (iNOS), cycloxygenase 2 (COX-2) expression, and p-38 phosphorylation and leaves extracellular regulated protein1/2 (ERK1/2) phosphorylation unchanged. The Rib-BSA-induced signaling changes are blocked by rosiglitazone and confirmed by PPAR-γ small-interfering RNA transfection. The reduction of cell survival by Rib-BSA is blocked by the iNOS inhibitor and p38 inhibitor. No effect on cell survival was observed using the COX-2 inhibitor. Consequently, these results show that Rib-BSA directly inducing PC12 cell death is a triggering event and TZDs protect PC12 cell from Rib-BSA damage. Signaling molecules, such as PPAR-γ, P38, and iNOS, are involved in Rib-BSA-mediated cytotoxicity.

  2. Quantitative proteomic analysis of the inhibitory effects of CIL-102 on viability and invasiveness in human glioma cells

    SciTech Connect

    Teng, Chih-Chuan; Kuo, Hsing-Chun; Sze, Chun-I

    2013-11-01

    CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone), the major active agent of the alkaloid derivative, has been demonstrated to exert anticancer effects. Herein, we present an investigation focused on the identification of the target(s) of CIL-102's action and the mechanism of its action in apoptotic and anti-invasive pathways. Proteomic approaches were used to purify and identify the protein substrates using 2D difference gel electrophoresis (2D SDS-PAGE) to assess changes in the expression of relevant protein treatment with CIL-102 that resulted in the inhibition of viability and invasion. Our results demonstrate that CIL-102 treatment of U87 cells decreased cell proliferation and invasiveness. CIL-102 dose-dependent induction of apoptosis and inhibitory invasiveness were accompanied by sustained phosphorylation of JNK1/2 and p70S6K as well as generation of the reactive oxygen species. In addition, differential proteins displayed between CIL-102-treated and untreated U87 were determined and validated. There were 11 differentially expressed proteins between the CIL-102-treated and untreated groups. Furthermore, we demonstrated that CIL-102 inhibited cancer cell proliferation and reduced anti-invasion properties by up-regulating the levels of FUMH (Fumarate hydratase). The investigation demonstrated that there was an increase in the cellular levels of FUMH in the CIL-102 reduction in viability and invasion via the activation of JNK1/2 and mTOR signaling modules. NAC administration and shRNA FUMH conferred resistance to CIL-102-inhibited HIF1α and MMP-2 levels via inhibition of JNK1/2 and mTOR activation. We concluded that CIL-102-induced an apoptosis cascade and decreased aggressiveness in astrocytoma cells by modulation of mitochondria function, providing a new mechanism for CIL-102 treatment. - Highlights: • We found the effect of CIL-102 on neuroblastoma cells. • Fumarate hydratase as a CIL-102's target by proteomic differential displays. • CIL

  3. The effect of CO2 laser beam welded AISI 316L austenitic stainless steel on the viability of fibroblast cells, in vitro.

    PubMed

    Köse, Ceyhun; Kaçar, Ramazan; Zorba, Aslı Pınar; Bağırova, Melahat; Allahverdiyev, Adil M

    2016-03-01

    It has been determined by the literature research that there is no clinical study on the in vivo and in vitro interaction of the cells with the laser beam welded joints of AISI 316L biomaterial. It is used as a prosthesis and implant material and that has adequate mechanical properties and corrosion resistance characteristics. Therefore, the interaction of the CO2 laser beam welded samples and samples of the base metal of AISI 316L austenitic stainless steel with L929 fibroblast cells as an element of connective tissue under in vitro conditions has been studied. To study the effect of the base metal and the laser welded test specimens on the viability of the fibroblast cells that act as an element of connective tissues in the body, they were kept in DMEMF-12 medium for 7, 14, 28 days and 18 months. The viability study was experimentally studied using the MTT method for 7, 14, 28 days. In addition, the direct interaction of the fibroblast cells seeded on 6 different plates with the samples was examined with an inverted microscope. The MTT cell viability experiment was repeated on the cells that were in contact with the samples. The statistical relationship was analyzed using a Tukey test for the variance with the GraphPad statistics software. The data regarding metallic ion release were identified with the ICP-MS method after the laser welded and main material samples were kept in cell culture medium for 18 months. The cell viability of the laser welded sample has been detected to be higher than that of the base metal and the control based on 7th day data. However, the laser welded sample's viability of the fibroblast cells has diminished by time during the test period of 14 and 28 days and base metal shows better viability when compared to the laser welded samples. On the other hand, the base metal and the laser welded sample show better cell viability effect when compared to the control group. According to the ICP-MS results of the main material and laser welded

  4. Bisphosphonates modulate vital functions of human osteoblasts and affect their interactions with breast cancer cells.

    PubMed

    Kaiser, Tatjana; Teufel, Ingrid; Geiger, Konstanze; Vater, Yvonne; Aicher, Wilhelm K; Klein, Gerd; Fehm, Tanja

    2013-07-01

    Bisphosphonates (BPs) are in clinical use for the treatment of breast cancer patients with bone metastases. Their anti-resorptive effect is mainly explained by inhibition of osteoclast activity, but recent evidence also points to a direct action of BPs on bone-forming osteoblasts. However, the mechanisms how BPs influence osteoblasts and their interactions with breast cancer cells are still poorly characterized. Human osteoblasts isolated from bone specimens were characterized in depth by their expression of osteogenic marker genes. The influence of the nitrogen-containing BPs zoledronate (Zol), ibandronate (Iban), and pamidronate (Pam) on molecular and cellular functions of osteoblasts was assessed focusing on cell proliferation and viability, apoptosis, cytokine secretion, and osteogenic-associated genes. Furthermore, effects of BPs on osteoblast-breast tumor cell interactions were examined in an established in vitro model system. The BPs Zol and Pam inhibited cell viability of osteoblasts. This effect was mediated by an induction of caspase-dependent apoptosis in osteoblasts. By interfering with the mevalonate pathway, Zol also reduces the proliferation of osteoblasts. The expression of phenotypic markers of osteogenic differentiation was altered by Zol and Pam. In addition, both BPs strongly influenced the secretion of the chemokine CCL2 by osteoblasts. Breast cancer cells also responded to Zol and Pam with a reduced cell adhesion to osteoblast-derived extracellular matrix molecules and with a decreased migration in response to osteoblast-secreted factors. BPs revealed prominent effects on human osteoblasts. Zol and Pam as the most potent BPs affected not only the expression of osteogenic markers, osteoblast viability, and proliferation but also important osteoblast-tumor cell interactions. Changing the osteoblast metabolism by BPs modulates migration and adhesion of breast cancer cells as well. PMID:23807419

  5. Overexpression of Midkine promotes the viability of BA/F3 cells

    SciTech Connect

    Wang, Yang; Xing, Haiyan; Tian, Zheng; Tang, Kejing; Wang, Jiying; Xu, Zhifang; Rao, Qing; Wang, Min; Wang, Jianxiang

    2009-07-03

    Midkine (MK), a heparin-binding growth factor, has been reported to be overexpressed in a variety of human solid tumors. In the previous study, we found that MK was overexpressed in bone marrow samples derived from acute leukemia (AL) patients. To elucidate the role of MK, we stably transfected MK in IL-3-dependent BA/F3 cells. The results indicated that the capacity of proliferation and colony formation was significantly increased in the MK-transfected subclones than in the empty vector-transfected subclones. MK potentiated proliferation of BA/F3 cells by promoting cell cycle progression. Apoptosis assays showed a remarkable reduction of apoptosis in MK expressing subclones. Exogenous MK could induce the phosphorylation of Raf-1, and inhibit the expression of Bax in BA/F3 cells. These results indicate that MK might be involved in the pathogenesis of leukemia and could be taken as an ideal diagnostic marker and molecular target for the treatment of acute leukemia.

  6. Enhancement of cell viability by fabrication of macroscopic 3D hydrogel scaffolds using an innovative cell-dispensing technique supplemented by preosteoblast-laden micro-beads.

    PubMed

    Lee, Hyeongjin; Ahn, Seunghyun; Chun, Wook; Kim, Geunhyung

    2014-04-15

    We propose a new cell-encapsulated dispensing method consisting of hydrogel struts, embedded with cell-laden micro-beads. To develop the scaffolds, we accommodated a three-axis robot dispensing system and aerosol spraying of a cross-linking agent to effect tentative surface gelation of hydrogel alginate struts. To show the feasibility of the method, we used pre-osteoblast (MC3T3-E1) cells. Using this technique, we obtained a reasonable cell viability (>90% after several culture periods) relative to that of a scaffold onto which cells were dispensed in the conventional manner, and successfully fabricated a realistic macroscopic pore-size in a controlled manner with 100% pore-interconnected 3D alginate hydrogel scaffolds of 20 mm × 20 mm × 6 mm.

  7. Cry1Ab Treatment Has No Effects on Viability of Cultured Porcine Intestinal Cells, but Triggers Hsp70 Expression

    PubMed Central

    Bondzio, Angelika; Lodemann, Ulrike; Weise, Christoph; Einspanier, Ralf

    2013-01-01

    In vitro testing can contribute to reduce the risk that the use of genetically modified (GM) crops and their proteins show unintended toxic effects. Here we introduce a porcine intestinal cell culture (IPEC-J2) as appropriate in vitro model and tested the possible toxic potential of Cry1Ab protein, commonly expressed in GM-maize. For comprehensive risk assessment we used WST-1 conversion and ATP content as metabolic markers for proliferation, lactate dehydrogenase release as indicator for cells with compromised membrane and transepithelial electrical resistance as parameter indicating membrane barrier function. The results were compared to the effects of valinomycin, a potassium ionophore, known to induce cytotoxic effects in most mammalian cell types. Whereas no toxicity was observed after Cry1Ab treatment, valinomycin induced a decrease in IPEC-J2 viability. This was confirmed by dynamic monitoring of cellular responses. Additionally, two dimensional differential in-gel electrophoresis was performed. Only three proteins were differentially expressed. The functions of these proteins were associated with responses to stress. The up-regulation of heat shock protein Hsp70 was verified by Western blotting as well as by enzyme-linked immunosorbent assay and may be related to a protective function. These findings suggest that the combination of in vitro testing and proteomic analysis may serve as a promising tool for mechanism based safety assessment. PMID:23861753

  8. Effects of the polycyclic ketone tonalide (AHTN) on some cell viability parameters and transcription of P450 and immunoregulatory genes in rainbow trout RTG-2 cells.

    PubMed

    Randelli, Elisa; Rossini, Valerio; Corsi, Ilaria; Focardi, Silvano; Fausto, Anna M; Buonocore, Francesco; Scapigliati, Giuseppe

    2011-12-01

    To assess the potential effects of environmental pollutants belonging to the musk fragrances group in the physiology of aquatic animal species, in this work we treated rainbow trout RTG-2 cells with the polycyclic ketone tonalide (AHTN) at dilutions ranging from 3.5 to 500 ng/ml. The following parameters were monitored: intracellular ATP concentration (energy production), mitochondrial membrane potential (early apoptosis marker), cell viability (vital staining with DFP), quantitative expression of genes coding for the cytochrome P450 detoxifying enzymes CYP1A1 and CYP3A27, and of genes coding for the immunoregulatory peptides IL-1β, IL-8, TNFα, Cox-2 and TGF-β. Obtained results showed that incubation with tonalide induced in RTG-2 cells no effects on cell viability, a slight increase of mitochondrial membrane potential activity, and a significant increase in intracellular ATP concentration. However, dramatic effects were observed in transcription levels of some tested genes, with upregulation levels of 300 and 600 times measured for TGF-β and TNFα, respectively and of 150 times for the CYP3A27 gene. Our results show for the first time the potent effects exerted by tonalide on immunoregulatory genes of RTG-2 cells and also indicate that the measured sensitivity of RTG-2 towards tonalide was in the same range of that currently available using chemical methods. A possible use of the panel of genes we employed as a tool for the monitoring of musk fragrances in biological samples is discussed.

  9. In vitro cytotoxicity of superparamagnetic iron oxide nanoparticles on neuronal and glial cells. Evaluation of nanoparticle interference with viability tests.

    PubMed

    Costa, Carla; Brandão, Fátima; Bessa, Maria João; Costa, Solange; Valdiglesias, Vanessa; Kiliç, Gözde; Fernández-Bertólez, Natalia; Quaresma, Pedro; Pereira, Eulália; Pásaro, Eduardo; Laffon, Blanca; Teixeira, João Paulo

    2016-03-01

    Superparamagnetic iron oxide nanoparticles (ION) have attracted great interest for use in several biomedical fields. In general, they are considered biocompatible, but little is known of their effects on the human nervous system. The main objective of this work was to evaluate the cytotoxicity of two ION (magnetite), coated with silica and oleic acid, previously determining the possible interference of the ION with the methodological procedures to assure the reliability of the results obtained. Human neuroblastoma SHSY5Y and glioblastoma A172 cells were exposed to different concentrations of ION (5-300 µg ml(-1)), prepared in complete and serum-free cell culture medium for three exposure times (3, 6 and 24 h). Cytotoxicity was evaluated by means of the MTT, neutral red uptake and alamar blue assays. Characterization of the main physical-chemical properties of the ION tested was also performed. Results demonstrated that both ION could significantly alter absorbance readings. To reduce these interferences, protocols were modified by introducing additional washing steps and cell-free systems. Significant decreases in cell viability were observed for both cell lines in specific conditions by all assays. In general, oleic acid-coated ION were less cytotoxic than silica-coated ION; besides, a serum-protective effect was observed for both ION studied and cell lines. These results contribute to increase the knowledge of the potential harmful effects of ION on the human nervous system. Understanding these effects is essential to establish satisfactory regulatory policies on the safe use of magnetite nanoparticles in biomedical applications.

  10. 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

  11. Caffeic acid improves cell viability and protects against DNA damage: involvement of reactive oxygen species and extracellular signal-regulated kinase

    PubMed Central

    Li, Y.; Chen, L.J.; Jiang, F.; Yang, Y.; Wang, X.X.; Zhang, Z.; Li, Z.; Li, L.

    2015-01-01

    Hormesis is an adaptive response to a variety of oxidative stresses that renders cells resistant to harmful doses of stressing agents. Caffeic acid (CaA) is an important antioxidant that has protective effects against DNA damage caused by reactive oxygen species (ROS). However, whether CaA-induced protection is a hormetic effect remains unknown, as is the molecular mechanism that is involved. We found that a low concentration (10 μM) of CaA increased human liver L-02 cell viability, attenuated hydrogen peroxide (H2O2)-mediated decreases in cell viability, and decreased the extent of H2O2-induced DNA double-strand breaks (DSBs). In L-02 cells exposed to H2O2, CaA treatment reduced ROS levels, which might have played a protective role. CaA also activated the extracellular signal-regulated kinase (ERK) signal pathway in a time-dependent manner. Inhibition of ERK by its inhibitor U0126 or by its specific small interfering RNA (siRNA) blocked the CaA-induced improvement in cell viability and the protective effects against H2O2-mediated DNA damage. This study adds to the understanding of the antioxidant effects of CaA by identifying a novel molecular mechanism of enhanced cell viability and protection against DNA damage. PMID:25831202

  12. Differences in body temperature, cell viability, and HSP-70 concentrations between Pelibuey and Suffolk sheep under heat stress.

    PubMed

    Romero, Rosita Denny; Montero Pardo, Arnulfo; Montaldo, Hugo Horacio; Rodríguez, Ana Delia; Hernández Cerón, Joel

    2013-11-01

    Pelibuey and Suffolk sheep were compared as to their capacity to regulate body temperature under environmental hyperthermia by measuring their differences in cellular response to heat stress (HS). In a first experiment, seven Pelibuey and seven Suffolk ewes were kept in a climatic chamber for 6 h daily during 10 days (temperatures within the 18 to 39.5 °C range). As chamber temperature rose, sheep rectal temperature increased in both groups, but to a lesser extent in Pelibuey (0.3 °C) than in Suffolk sheep (0.7 °C) (P < 0.05). In a second experiment, cellular viability was assessed using cultured blood mononuclear cells from 15 Pelibuey and 15 Suffolk sheep. They were incubated at 37 °C for 24 h (control) or 43 °C for 6 h followed by 18 h at 37 °C (HS). In a third experiment, another blood mononuclear cells culture from eight Pelibuey and eight Suffolk sheep was kept at 37 °C for 15 h; these were subsequently cultured for 6 h at 37 °C (controls) or 43 °C (HS). Next, HSP-70 concentration was determined. HS reduced the percentage of viable cells to a greater extent in Suffolk [37 °C (73.7 %) vs. 43 °C (61.9 %); P < 0.05] than in Pelibuey sheep [37 °C (74.9 %) vs. 43 °C (66.7 %); P > 0.05]. HS significantly increased HSP-70 average concentrations for both breeds at 43 °C. A significant effect was observed for the breed by temperature interaction (P < 0.05) caused by a greater difference between Pelibuey and Suffolk at 43 °C (2.85 vs. 0.53 ng/mL, respectively; P < 0.05) than at 37 °C (0.05 vs. 0.03 ng/mL, respectively; P > 0.05). In conclusion, Pelibuey sheep show more effective body temperature regulation under conditions of environmental hyperthermia. Also, cell viability after HS was higher in Pelibuey than in Suffolk, an effect that could be mediated by an HSP-70-related mechanism.

  13. STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability

    PubMed Central

    Luo, Tuoping; Masson, Kristina; Jaffe, Jacob D.; Silkworth, Whitney; Ross, Nathan T.; Scherer, Christina A.; Scholl, Claudia; Fröhling, Stefan; Carr, Steven A.; Stern, Andrew M.; Schreiber, Stuart L.; Golub, Todd R.

    2012-01-01

    Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33’s kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33’s kinase activity does not represent a promising anti-KRAS therapeutic strategy. PMID:22323609

  14. Human cells and cell membrane molecular models are affected in vitro by chlorpromazine.

    PubMed

    Suwalsky, Mario; Villena, Fernando; Sotomayor, Carlos P; Bolognin, Silvia; Zatta, Paolo

    2008-06-01

    This study presents evidence that chlorpromazine (CPZ) affects human cells and cell membrane molecular models. Human SH-SY5Y neuroblastoma cells incubated with 0.1 mM CPZ suffered a decrease of cell viability. On the other hand, phase contrast microscopy observations of human erythrocytes indicated that they underwent a morphological alteration as 1 microM CPZ changed their discoid normal shape to stomatocytes, and to hemolysis with 1 mM CPZ. X-ray diffraction experiments performed on dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of the major phospholipids present in the outer and inner sides of the erythrocyte membrane, respectively showed that CPZ disordered the polar head and acyl chain regions of both DMPC and DMPE, where these interactions were stronger with DMPC bilayers. Fluorescence spectroscopy on DMPC LUV at 18 degrees C confirmed these results. In fact, the assays showed that CPZ induced a significant reduction of their generalized polarization (GP) and anisotropy (r) values, indicative of enhanced disorder at the polar head and acyl chain regions of the DMPC lipid bilayer. PMID:18372093

  15. Effects of depsidones from Hypogymnia physodes on HeLa cell viability and growth.

    PubMed

    Stojanović, I Z; Najman, S; Jovanović, O; Petrović, G; Najdanović, J; Vasiljević, P; Smelcerović, A

    2014-01-01

    The anti-proliferative activitiy of Hypogymnia physodes methanol extracts (ME) and its main constituents, physodalic acid (P1), physodic acid (P2), and 3-hydroxy physodic acid (P3), was tested on human cancer HeLa cell lines. Three lichen depsidones, P1, P2 and P3, were isolated from H. physodes ME using column chromatography and their structures were determined by UV, ESI TOF MS, 1H and 13C NMR. The content of P1, P2 and P3 in ME was determined using reversed-phase highperformance liquid chromatography with photodiode array detection. P1-3 represented even 70 % of the studied extract. The HeLa cells were incubated during 24 and 72 h in the presence of ME and depsidones P1, P2 and P3, at concentrations of 10-1000 μg/ml. Compounds P2 and P3 showed higher activity than compound P1. Half maximal inhibitory concentrations (IC50, μg/ml) of P1, P2, P3 and ME for 24-h incubation were 964, 171, 97 and 254 μg/ml, respectively, while for 72-h incubation they were 283, 66, 63 and 68 μg/ml. As far as we know, this is the first report on the effect of H. physodes ME and their depsidones on HeLa cells. PMID:24785112

  16. Phytochemicals isolated from leaves of Chromolaena odorata: impact on viability and clonogenicity of cancer cell lines.

    PubMed

    Kouamé, Prevost Bi-Koffi; Jacques, Camille; Bedi, Gustave; Silvestre, Virginie; Loquet, Denis; Barillé-Nion, Sophie; Robins, Richard J; Tea, Illa

    2013-06-01

    The leaves of Chromolaena odorata (Asteraceae) are exploited extensively in West and Central African ethnopharmacy for the treatment of a wide range of conditions, despite this being a non-native species established in the last 50 years. With the objective of seeking bioactive principles, the nonvolatile compounds, an ethanolic (80% v/v) extract was made and fractionated. From the hexane-soluble fraction, three compounds were isolated. Two of these, 5-hydroxy-7,4'-dimethoxyflavanone and 2'-hydroxy-4,4',5',6'-tetramethoxychalcone, have previously been identified in C. odorata leaves. The third was fully characterised spectroscopically and found to be 1,6-dimethyl-4-(1-methylethyl)naphthalene (cadalene), not previously isolated from the Asteraceae. All three compounds were tested for their cytotoxicity and anticancer properties. 2'-Hydroxy-4,4',5',6'-tetramethoxychalcone was found to be both cytotoxic and anticlonogenic at 20 µm in cell lines Cal51, MCF7 and MDAMB-468, and to act synergistically with the Bcl2 inhibitor ABT737 to enhance apoptosis in Cal51 breast cancer cells.

  17. Inactivation of E. Coli cell viability and DNA Photo-breakage by Pulsed Nitrogen Laser Radiation

    SciTech Connect

    Cheba, Ben Amar; Alzaag, Ali; Tilfah, Nafie A.

    2005-03-17

    The mutagenic and lethal effect of nitrogen laser radiation: 337.1 nm wave length, 1.5 millijoul pulse energy, 10 nanosecond pulse with and pulse repetition rate range from 1 to 50 Pulse/ second was evaluated on E. Coli cells. Results indicated that irradiation of E. coli JMP39 with pulse repetition of 8 , 16 , 32 pulse/sec, for 1, 5 , 10, 25 min respectively led to a significant decrease in cell count proportional to irradiation dose with significant increase in lacmutation frequency accompanied with some mutations in pattern of antibiotic resistance. The effect of nitrogen laser on the genomic content of the strain JMP39 was also studied by irradiating the total DNA with 30 pulse/second for 1 ,5, 15 , 30 min then subjected to both agarose gel electrophoresis and scanning spectrophotometry. The first technique revealed to DNA photo breakage and significant decrease in DNA absorbency was noticed by scanning spectrophotometry. This could be attributed to photo-decomposition resulted from multi-photo-excitation of UV-Laser pulses.

  18. Increase in cell viability by polyamines through stimulation of the synthesis of ppGpp regulatory protein and ω protein of RNA polymerase in Escherichia coli.

    PubMed

    Terui, Yusuke; Akiyama, Mariko; Sakamoto, Akihiko; Tomitori, Hideyuki; Yamamoto, Kaneyoshi; Ishihama, Akira; Igarashi, Kazuei; Kashiwagi, Keiko

    2012-02-01

    It is known that polyamines increase cell growth through stimulation of the synthesis of several kinds of proteins encoded by the so-called "polyamine modulon". We recently reported that polyamines also increase cell viability at the stationary phase of cell growth through stimulation of the synthesis of ribosome modulation factor, a component of the polyamine modulon. Accordingly, we looked for other proteins involved in cell viability whose synthesis is stimulated by polyamines. It was found that the synthesis of ppGpp regulatory protein (SpoT) and ω protein of RNA polymerase (RpoZ) was stimulated by polyamines at the level of translation. Stimulation of the synthesis of SpoT and RpoZ by polyamines was due to an inefficient initiation codon UUG in spoT mRNA and an unusual location of a Shine-Dalgarno (SD) sequence in rpoZ mRNA. Accordingly, the spoT and rpoZ genes are components of the polyamine modulon involved in cell viability. Reduced cell viability caused by polyamine deficiency was prevented by modified spoT and rpoZ genes whose synthesis was not influenced by polyamines. Under these conditions, the level of ppGpp increased in parallel with increase of SpoT protein. The results indicate that polyamine stimulation of synthesis of SpoT and RpoZ plays important roles for cell viability through stimulation of ppGpp synthesis by SpoT and modulation of RNA synthesis by ppGpp-RpoZ complex.

  19. Preparation, physicochemical characterization, and cell viability evaluation of long-circulating and pH-sensitive liposomes containing ursolic acid.

    PubMed

    Caldeira de Araújo Lopes, Sávia; Vinícius Melo Novais, Marcus; Salviano Teixeira, Cláudia; Honorato-Sampaio, Kinulpe; Tadeu Pereira, Márcio; Ferreira, Lucas Antônio Miranda; Braga, Fernão Castro; Cristina Oliveira, Mônica

    2013-01-01

    Cancer is one of the leading causes of death worldwide. Although several drugs are used clinically, some tumors either do not respond or are resistant to the existing pharmacotherapy, thus justifying the search for new drugs. Ursolic acid (UA) is a triterpene found in different plant species that has been shown to possess significant antitumor activity. However, UA presents a low solubility in aqueous medium, which presents a barrier to its biological applications. In this context, the use of liposomes presents a promising strategy to deliver UA and allow for its intravenous administration. In this work, long-circulating and pH-sensitive liposomes containing UA (SpHL-UA) were developed, and their chemical and physicochemical properties were evaluated. SpHL-UA presented adequate properties, including a mean diameter of 191.1 ± 6.4 nm, a zeta potential of 1.2 ± 1.4 mV, and a UA entrapment of 0.77 ± 0.01 mg/mL. Moreover, this formulation showed a good stability after having been stored for 2 months at 4 °C. The viability studies on breast (MDA-MB-231) and prostate (LNCaP) cancer cell lines demonstrated that SpHL-UA treatment significantly inhibited cancer cell proliferation. Therefore, the results of the present work suggest the applicability of SpHL-UA as a new and promising anticancer formulation. PMID:23984367

  20. Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability[W

    PubMed Central

    Schapire, Arnaldo L.; Voigt, Boris; Jasik, Jan; Rosado, Abel; Lopez-Cobollo, Rosa; Menzel, Diedrik; Salinas, Julio; Mancuso, Stefano; Valpuesta, Victoriano; Baluska, Frantisek; Botella, Miguel A.

    2008-01-01

    Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca2+-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca2+-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness. PMID:19088329

  1. Substrate properties affect collective cell motion

    NASA Astrophysics Data System (ADS)

    Pegoraro, Adrian; Guo, Ming; Ehrlicher, Allen; Weitz, David

    2013-03-01

    When cells move collectively, cooperative motion, which is characterized by long range correlations in cell movement, is necessary for migration. This collective cell motion is influenced by cell-cell interactions as well as by cell-substrate coupling. Furthermore, on soft substrates it is possible for cells to mechanically couple over long distances through the substrate itself. By changing the properties of the substrate, it is possible to decouple some of these contributions and better understand the role they play in collective cell motion. We vary both the substrate stiffness and adhesion protein concentration and find changes in the collective cell motion of the cells despite only small differences in total cell density and average cell size in the confluent layers. We test these changes on polyacrylamide and PDMS substrates as well as on structured substrates made of PDMS posts that prevent mechanical coupling through the substrate while still allowing stiffness to be varied.

  2. Viability of fibroblasts in cell culture after treatment with different chemical retraction agents.

    PubMed

    Kopac, I; Batista, U; Cvetko, E; Marion, L

    2002-01-01

    Prior to fixed prosthodontic impression procedures, temporary horizontal retraction of the free gingival tissue should be accomplished apically to the preparation finishing line. The mechanical-chemical method using cotton retraction cords of various sizes impregnated with various retraction chemicals is the most commonly employed retraction technique. Most retraction agents have pH values from 0.8 to 0.3, and are therefore hazardous to the cut dentine and periodontal tissues. Sympathomimetic vasoconstrictors introduced recently have a pH of 5.6, and are free of systemic side-effects. The present study using the dye exclusion test, colony forming ability test and colorimetric assay was undertaken to evaluate cytotoxic effects of four chemical retraction agents on cultured V-79 fibroblasts, and the dependence of cytotoxicity on the agent concentration and time of exposure. Original concentrations of retraction agents produced stronger cytotoxic effects than dilutions of 1:1 and 1:10. The most aggressive agent, 25% aluminium chloride, took only 1 min to damage all cell cultures. The proportion of cells damaged after 10 min of exposure to tetrahydrozoline was 60%, which was significantly less compared with other chemicals tested. With the colony forming ability test using retraction agents diluted to 1:10 the greatest number of colonies emerged in samples treated with tetrahydrozoline (statistical significance: P < 0.01). The colorimetric assay showed equal cytotoxic effects for 25% aluminium sulphate and tetrahydrozoline. The colorimetric test used in the study has proved an ergonomic, accurate and reliable test for cytotoxicity determination. PMID:11844038

  3. Viability of fibroblasts in cell culture after treatment with different chemical retraction agents.

    PubMed

    Kopac, I; Batista, U; Cvetko, E; Marion, L

    2002-01-01

    Prior to fixed prosthodontic impression procedures, temporary horizontal retraction of the free gingival tissue should be accomplished apically to the preparation finishing line. The mechanical-chemical method using cotton retraction cords of various sizes impregnated with various retraction chemicals is the most commonly employed retraction technique. Most retraction agents have pH values from 0.8 to 0.3, and are therefore hazardous to the cut dentine and periodontal tissues. Sympathomimetic vasoconstrictors introduced recently have a pH of 5.6, and are free of systemic side-effects. The present study using the dye exclusion test, colony forming ability test and colorimetric assay was undertaken to evaluate cytotoxic effects of four chemical retraction agents on cultured V-79 fibroblasts, and the dependence of cytotoxicity on the agent concentration and time of exposure. Original concentrations of retraction agents produced stronger cytotoxic effects than dilutions of 1:1 and 1:10. The most aggressive agent, 25% aluminium chloride, took only 1 min to damage all cell cultures. The proportion of cells damaged after 10 min of exposure to tetrahydrozoline was 60%, which was significantly less compared with other chemicals tested. With the colony forming ability test using retraction agents diluted to 1:10 the greatest number of colonies emerged in samples treated with tetrahydrozoline (statistical significance: P < 0.01). The colorimetric assay showed equal cytotoxic effects for 25% aluminium sulphate and tetrahydrozoline. The colorimetric test used in the study has proved an ergonomic, accurate and reliable test for cytotoxicity determination.

  4. Trehalose reserve in Saccharomyces cerevisiae: phenomenon of transport, accumulation and role in cell viability.

    PubMed

    Plourde-Owobi, L; Durner, S; Goma, G; François, J

    2000-04-10

    Strains of Saccharomyces cerevisiae deleted for TPS1 encoding trehalose-6-phosphate synthase still accumulate trehalose when harbouring a functional MAL locus. We demonstrate that this accumulation results from an active uptake of trehalose present in the 'yeast extract' used to make the enriched culture media and that no accumulation is observed in mineral media. The uptake of trehalose was shown to be mediated by the alpha-glucoside transporter encoded by AGT1, the expression of which is linked to the presence of a functional MAL locus. Deletion of this gene in a MAL+ tps1 mutant abolished trehalose accumulation on a maltose or galactose mineral medium. However, small amounts of disaccharide were still detected in a agt1 tps1 double mutant when the medium was supplemented with 10 g trehalose l(-1), indicating the existence of a non-concentrative low-affinity sugar transporter. The presence of the high-affinity trehalose permease allowed us to investigate the effect of increasing exogenous trehalose from 0 to 10 g(-1) on intracellular accumulation. A maximum of ca. 10% (wt/wt dry cells) trehalose was attained in the presence of only 1 g l(-1) of disaccharide in the medium. The capability to monitor the intracellular content of trehalose by varying its extracellular concentration, independent of genetic alterations of the trehalose metabolic machinery, allowed the remarkable contribution of this molecule in stress tolerance to be demonstrated, as the higher the trehalose content, the longer the cell survival to a severe heat shock and to glucose starvation.

  5. PTEN regulates AMPA receptor-mediated cell viability in iPS-derived motor neurons.

    PubMed

    Yang, D-J; Wang, X-L; Ismail, A; Ashman, C J; Valori, C F; Wang, G; Gao, S; Higginbottom, A; Ince, P G; Azzouz, M; Xu, J; Shaw, P J; Ning, K

    2014-02-27

    Excitatory transmission in the brain is commonly mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In amyotrophic lateral sclerosis (ALS), AMPA receptors allow cytotoxic levels of calcium into neurons, contributing to motor neuron injury. We have previously shown that oculomotor neurons resistant to the disease process in ALS show reduced AMPA-mediated inward calcium currents compared with vulnerable spinal motor neurons. We have also shown that PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown via siRNA promotes motor neuron survival in models of spinal muscular atrophy (SMA) and ALS. It has been reported that inhibition of PTEN attenuates the death of hippocampal neurons post injury by decreasing the effective translocation of the GluR2 subunit into the membrane. In addition, leptin can regulate AMPA receptor trafficking via PTEN inhibition. Thus, we speculate that manipulation of AMPA receptors by PTEN may represent a potential therapeutic strategy for neuroprotective intervention in ALS and other neurodegenerative disorders. To this end, the first step is to establish a fibroblast-iPS-motor neuron in vitro cell model to study AMPA receptor manipulation. Here we report that iPS-derived motor neurons from human fibroblasts express AMPA receptors. PTEN depletion decreases AMPA receptor expression and AMPA-mediated whole-cell currents, resulting in inhibition of AMPA-induced neuronal death in primary cultured and iPS-derived motor neurons. Taken together, our results imply that PTEN depletion may protect motor neurons by inhibition of excitatory transmission that represents a therapeutic strategy of potential benefit for the amelioration of excitotoxicity in ALS and other neurodegenerative disorders.

  6. Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability

    PubMed Central

    Yanagisawa, Tatsuo; Takahashi, Hideyuki; Suzuki, Takehiro; Masuda, Akiko; Dohmae, Naoshi; Yokoyama, Shigeyuki

    2016-01-01

    Translation elongation factor P (EF-P), a ubiquitous protein over the entire range of bacterial species, rescues ribosomal stalling at consecutive prolines in proteins. In Escherichia coli and Salmonella enterica, the post-translational β-lysyl modification of Lys34 of EF-P is important for the EF-P activity. The β-lysyl EF-P modification pathway is conserved among only 26–28% of bacteria. Recently, it was found that the Shewanella oneidensis and Pseudomonas aeruginosa EF-P proteins, containing an Arg residue at position 32, are modified with rhamnose, which is a novel post-translational modification. In these bacteria, EF-P and its Arg modification are both dispensable for cell viability, similar to the E. coli and S. enterica EF-P proteins and their Lys34 modification. However, in the present study, we found that EF-P and Arg32 are essential for the viability of the human pathogen, Neisseria meningitidis. We therefore analyzed the modification of Arg32 in the N. meningitidis EF-P protein, and identified the same rhamnosyl modification as in the S. oneidensis and P. aeruginosa EF-P proteins. N. meningitidis also has the orthologue of the rhamnosyl modification enzyme (EarP) from S. oneidensis and P. aeruginosa. Therefore, EarP should be a promising target for antibacterial drug development specifically against N. meningitidis. The pair of genes encoding N. meningitidis EF-P and EarP suppressed the slow-growth phenotype of the EF-P-deficient mutant of E. coli, indicating that the activity of N. meningitidis rhamnosyl–EF-P for rescuing the stalled ribosomes at proline stretches is similar to that of E. coli β-lysyl–EF-P. The possible reasons for the unique requirement of rhamnosyl–EF-P for N. meningitidis cells are that more proline stretch-containing proteins are essential and/or the basal ribosomal activity to synthesize proline stretch-containing proteins in the absence of EF-P is lower in this bacterium than in others. PMID:26840407

  7. Biocorrosion behavior and cell viability of adhesive polymer coated magnesium based alloys for medical implants

    NASA Astrophysics Data System (ADS)

    Abdal-hay, Abdalla; Dewidar, Montasser; Lim, Jae Kyoo

    2012-11-01

    The present study was ultimately aimed to design novel adhesive biodegradable polymer, poly(vinyl acetate) (PVAc), coatings onto Mg based alloys by the dip-coating technique in order to control the degradation rate and enhance the biocompatibility of magnesium alloys. The influence of various solvents on PVAc surface topography and their protection of Mg alloys were dramatically studied in vitro. Electrochemical polarization, degradation, and PVAc film cytocompatibility were also tested. Our results showed that the solvent had a significant effect on coating quality. PVAc/dichloromethane solution showed a porous structure and solution concentration could control the porous size. The coatings prepared using tetrahydrofuran and dimethylformamide solvents are exceptional in their ability to generate porous morphology even at low polymer concentration. In general, the corrosion performance appears to be different on different PVAc-solvent system. Immersion tests illustrated that the porous morphology on PVAc stabilized corrosion rates. A uniform corrosion attack in artificial simulation body fluid was also exhibited. The cytocompatibility of osteoblast cells (MC3T3) revealed high adherence, proliferation, and survival on the porous structure of PVAc coated Mg alloy, which was not observed for the uncoated samples. This novel PVAc coating is a promising candidate for biodegradable implant materials, which might widen the use of Mg based implants.

  8. Potential irritation of lysine derivative surfactants by hemolysis and HaCaT cell viability.

    PubMed

    Sanchez, L; Mitjans, M; Infante, M R; Vinardell, M P

    2006-02-01

    Surfactants represent one of the most common constituents in topical pharmaceutical and cosmetic applications or cleansers. Since adverse skin and ocular reactions can be caused by them, it is important to evaluate damaging effects. Amino acid-based surfactants deserve particular attention because of their low toxicity and environmental friendly properties. New lysine derivative surfactants associated with heavy and light counterions were tested. The ocular irritancy was assessed by hemolysis, and photohemolysis was employed to evaluate their phototoxicity. Cytotoxicity on HaCaT cells was determined by neutral red uptake and MTT assay to predict skin irritation. All lysine derivative surfactants were less hemolytic and thus less eye-irritating than the commercial surfactants used as model irritants. No phototoxic effects were found. All surfactants presented cytotoxic effects as demonstrated by decrease of neutral red uptake and reduction of MTT salt, with clear concentration-effect profiles. However, the rates of cytotoxicity on HaCaT for the new surfactants suggested that they were less cytotoxic and then, less skin-irritating than the reference ones; surfactants with heavy counterions were the less cytotoxic. The anionic surfactants investigated in the present work may constitute a promising class of surfactants given their low irritancy potential for pharmaceutical and cosmetic preparations. PMID:16135402

  9. Effects of nickel-smelting fumes on the regulation of NIH/3T3 cell viability, necrosis, and expression of hMLH1 and RASSF1A.

    PubMed

    Wang, Jun; Yu, Cui-Ping; Hu, Xue-Ying; Wu, Yong-Hui

    2014-01-01

    Nickel is widely used and distributed in various industries. This study investigated the effect of nickel-smelting fumes on the regulation of NIH/3T3 cell viability, apoptosis, and necrosis and the expression of the tumor suppressor genes hMLH1 and RASSF1A. Cell viability was determined using a methylthiazolyl tetrazolium colorimetric assay. NIH/3T3 cell viability was reduced after exposure to different concentrations of nickel-smelting fumes, but cell apoptosis and necrosis were induced. Moreover, cell morphology changed significantly after exposure to different concentrations of nickel-smelting fumes, as determined using an inverted microscope or transmission electron microscope. Real-time RT-PCR and Western blot analyses showed that exposure of cells to concentrations of ≥100 µg/mL of nickel-smelting fumes upregulated the expression of hMLH1 and RASSF1A compared to the negative controls. These data suggest that nickel-smelting fumes could be toxic to cells, upregulating the expression of hMLH1 and RASSF1A and in turn inducing cell apoptosis and necrosis. PMID:24579805

  10. Effects of nickel-smelting fumes on the regulation of NIH/3T3 cell viability, necrosis, and expression of hMLH1 and RASSF1A.

    PubMed

    Wang, Jun; Yu, Cui-Ping; Hu, Xue-Ying; Wu, Yong-Hui

    2014-01-01

    Nickel is widely used and distributed in various industries. This study investigated the effect of nickel-smelting fumes on the regulation of NIH/3T3 cell viability, apoptosis, and necrosis and the expression of the tumor suppressor genes hMLH1 and RASSF1A.