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Sample records for affects cell viability

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-05-01

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

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

    PubMed

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

    2011-01-01

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

  4. Cell viability assays: introduction.

    PubMed

    Stoddart, Martin J

    2011-01-01

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

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

    PubMed

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

    2001-03-01

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

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

    PubMed Central

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

    2017-01-01

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

  7. 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

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

    PubMed

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

    2011-08-10

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

  9. 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.

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

    PubMed Central

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

    2017-01-01

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

  11. 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

  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. Bioluminescence assay for cell viability.

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed

    Bulati, Matteo; Longo, Alessandra; Masullo, Tiziana; Vlah, Sara; Bennici, Carmelo; Bonura, Angela; Salamone, Monica; Tagliavia, Marcello; Nicosia, Aldo; Mazzola, Salvatore; Colombo, Paolo; Cuttitta, Angela

    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.

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

    PubMed

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

    2015-09-01

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

  18. 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

  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. 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.

  2. Cell viability test after laser guidance

    NASA Astrophysics Data System (ADS)

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

    2006-02-01

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

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

    PubMed

    Kraskiewicz, Honorata; FitzGerald, Una

    2011-05-01

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

  4. [Nicotinamide influence on pancreatic cells viability].

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2011-02-02

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

  6. 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.

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

    PubMed

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

    2013-10-01

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

  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

    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.

  9. 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

  10. 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

  11. 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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  15. 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.

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

    PubMed

    Luan, X; Diekwisch, T G H

    2002-08-01

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

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

    PubMed

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

    2014-10-05

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

  18. Dominance of mutations affecting viability in Drosophila melanogaster.

    PubMed Central

    Fry, James D; Nuzhdin, Sergey V

    2003-01-01

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

  19. 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

  20. Fermented red ginseng extract inhibits cancer cell proliferation and viability.

    PubMed

    Oh, Jisun; Jeon, Seong Bin; Lee, Yuri; Lee, Hyeji; Kim, Ju; Kwon, Bo Ra; Yu, Kang-Yeol; Cha, Jeong-Dan; Hwang, Seung-Mi; Choi, Kyung-Min; Jeong, Yong-Seob

    2015-04-01

    Red ginseng (Panax ginseng C.A. Meyer) is the most widely recognized medicinal herb due to its remedial effects in various disorders, such as cancers, diabetes, and heart problems. In this study, we investigated the anticancer effect of fermented red ginseng extract (f-RGE; provided by Jeonju Biomaterials Institute, Jeonju, South Korea) in a parallel comparison with the effect of nonfermented red ginseng extract (nf-RGE; control) on several cancer cell lines--MCF-7 breast cancer cells, HepG2 hepatocellular carcinoma cells, and reprogrammed MCF-7 cells (mimicking cancer stem cells). Cells were cultured at various concentrations of RGE (from 0.5 up to 5 mg/mL) and their viabilities and proliferative properties were examined. Our data demonstrate the following: (1) nf-RGE inhibited cell viability at ≥1 mg/mL for MCF-7 cells and ≥2 mg/mL for HepG2 cells, (2) in the presence of a carcinogenic agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), nf-RGE treatment in combination with paclitaxel synergistically decreased MCF-7 as well as HepG2 cell viability, (3) f-RGE (which contained a greater level of Rg3 content) more effectively decreased the viability of MCF-7 and HepG2 cells compared to nf-RGE, and (4) f-RGE appeared more potent for inhibiting cancerous differentiation of reprogrammed MCF-7 cells in a synergistic fashion with paclitaxel, especially in the presence of TPA, compared to nf-RGE. These findings suggest that f-RGE treatment may be more effective for decreasing cancer cell survival by inducing apoptotic cell death and also presumably for preventing cancer stem cell differentiation compared to nf-RGE.

  1. 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

  2. Single cell viability and impact of heating by laser absorption.

    PubMed

    Wetzel, Franziska; Rönicke, Susanne; Müller, Karla; Gyger, Markus; Rose, Daniel; Zink, Mareike; Käs, Josef

    2011-09-01

    Optical traps such as tweezers and stretchers are widely used to probe the mechanical properties of cells. Beyond their large range of applications, the use of infrared laser light in optical traps causes significant heating effects in the cell. This study investigated the effect of laser-induced heating on cell viability. Common viability assays are not very sensitive to damages caused in short periods of time or are not practicable for single cell analysis. We used cell spreading, a vital ability of cells, as a new sensitive viability marker. The optical stretcher, a two beam laser trap, was used to simulate heat shocks that cells typically experience during measurements in optical traps. The results show that about 60% of the cells survived heat shocks without vital damage at temperatures of up to 58 ± 2°C for 0.5 s. By varying the duration of the heat shocks, it was shown that 60% of the cells stayed viable when exposed to 48 ± 2°C for 5 s.

  3. Influence of electroporation on chicken blastoderm cell viability in vitro.

    PubMed

    Wawrzynska, Magdalena; Bednarczyk, Marek; Łakota, Pawel; Lubiszewska, Marta

    2008-01-01

    The aim of this study was to compare two types of devices used for blastoderm cell (BC) transfection: the Nucleofector (Amaxa, Biosystems) and the Multiporator (Eppendorf). To assess the influence of electric current on BCs, different conditions of both nucleofection and electroporation were used. Next, the viability of cells was assessed. The highest number of cells (90.8%) was viable after nucleofection in the G10 program. After transfection in the presence of pmaxGFP, the A23 program was found to be most advantageous. The elecroporation experiment with the Multiporator (Eppendorf) showed a significant influence of osmotic pressure and voltage on BC viability. Namely, in the isoosmolar buffer BC viability was statistically higher (P < or = 0.05) in comparison to the hypoosmolar buffer. The, viability of cells was statistically higher (P < or = 0.05) after application of 25V as compared to 50V. The efficiency of transfection in the presence of EGFP-C 1 after electroporation in 2 pulses, 25V, 500 micros in the isoosmolar buffer was better than in the recommended conditions in the Amaxa Biosystems A23 program.

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

    PubMed

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

    2010-05-07

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

  5. 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.

  6. Assessment of cell viability in primary neuronal cultures.

    PubMed

    Aras, Mandar A; Hartnett, Karen A; Aizenman, Elias

    2008-07-01

    This unit contains five protocols for assaying cell viability in vitro using primary neuronal cultures, including a novel method for use with transfected neurons. Three of the assays are based on the principle that cell death cascades alter membrane permeability. The lactate dehydrogenase (LDH) release assay measures the amount of the cytoplasmic enzyme released into the bathing medium, while the trypan blue and propidium iodide assays measure the ability of cells to exclude dye from their cytoplasm. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay measures the mitochondrial activity of viable cells by quantifying the conversion of the tetrazolium salt to its formazan product. Finally, the fifth assay details the measurement of luciferase expression as an indication of neuronal viability within a relatively small population of transfected neurons.

  7. Phenylboronic acid selectively inhibits human prostate and breast cancer cell migration and decreases viability.

    PubMed

    Bradke, Tiffany M; Hall, Casey; Carper, Stephen W; Plopper, George E

    2008-01-01

    We compared the in vitro effect of boric acid (BA) versus phenylboronic acid (PBA) on the migration of prostate and breast cancer cell lines and non-tumorigenic cells from the same tissues. Treatment at 24 hours with BA (< or =500 microM) did not inhibit chemotaxis on fibronectin in any cell line. However, treatment over the same time course with concentrations of PBA as low as 1 muM significantly inhibited cancer cell migration without effecting non-tumorigenic cell lines. The compounds did not affect cell adhesion or viability at 24 hours but did alter morphology; both decreased cancer cell viability at eight days. These results suggest that PBA is more potent than BA in targeting the metastatic and proliferative properties of cancer cells.

  8. 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...

  9. Mitotane reduces human and mouse ACTH-secreting pituitary cell viability and function.

    PubMed

    Gentilin, Erica; Tagliati, Federico; Terzolo, Massimo; Zoli, Matteo; Lapparelli, Marcello; Minoia, Mariella; Ambrosio, Maria Rosaria; Degli Uberti, Ettore C; Zatelli, Maria Chiara

    2013-09-01

    Medical therapy for Cushing's disease (CD) is currently based on agents mainly targeting adrenocortical function. Lately, pituitary-directed drugs have been developed, with limited efficacy. Mitotane, a potent adrenolytic drug, has been recently investigated for the treatment of CD, but the direct pituitary effects have not been clarified so far. The aim of our study was to investigate whether mitotane may affect corticotroph function and cell survival in the mouse pituitary cell line AtT20/D16v-F2 and in the primary cultures of human ACTH-secreting pituitary adenomas, as an in vitro model of pituitary corticotrophs. We found that in the AtT20/D16v-F2 cell line and in primary cultures, mitotane reduces cell viability by inducing caspase-mediated apoptosis and reduces ACTH secretion. In the AtT20/D16v-F2 cell line, mitotane reduces Pomc expression and blocks the stimulatory effects of corticotropin-releasing hormone on cell viability, ACTH secretion, and Pomc expression. These effects were apparent at mitotane doses greater than those usually necessary for reducing cortisol secretion in Cushing's syndrome, but still in the therapeutic window for adrenocortical carcinoma treatment. In conclusion, our results demonstrate that mitotane affects cell viability and function of human and mouse ACTH-secreting pituitary adenoma cells. These data indicate that mitotane could have direct pituitary effects on corticotroph cells.

  10. The in vitro impact of toothpaste extracts on cell viability.

    PubMed

    Cvikl, Barbara; Lussi, Adrian; Gruber, Reinhard

    2015-06-01

    Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity.

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

    PubMed

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

    2016-11-09

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

  12. 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.

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

    PubMed

    Duarte, Cristina; Gudiña, Eduardo J; Lima, Cristovao F; Rodrigues, Ligia R

    2014-01-01

    Biosurfactants are molecules with surface activity produced by microorganisms that can be used in many biomedical applications. The anti-tumour potential of these molecules is being studied, although results are still scarce and few data are available regarding the mechanisms underlying such activity. In this work, the anti-tumour activity of a surfactin produced by Bacillus subtilis 573 and a glycoprotein (BioEG) produced by Lactobacillus paracasei subsp. paracasei A20 was evaluated. Both biosurfactants were tested against two breast cancer cell lines, T47D and MDA-MB-231, and a non-tumour fibroblast cell line (MC-3 T3-E1), specifically regarding cell viability and proliferation. Surfactin was found to decrease viability of both breast cancer cell lines studied. A 24 h exposure to 0.05 g l(-1) surfactin led to inhibition of cell proliferation as shown by cell cycle arrest at G1 phase. Similarly, exposure of cells to 0.15 g l(-1) BioEG for 48 h decreased cancer cells' viability, without affecting normal fibroblasts. Moreover, BioEG induced the cell cycle arrest at G1 for both breast cancer cell lines. The biosurfactant BioEG was shown to be more active than surfactin against the studied breast cancer cells. The results gathered in this work are very promising regarding the biosurfactants potential for breast cancer treatment and encourage further work with the BioEG glycoprotein.

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

    PubMed

    Krasnow, Mark; Matthews, Mark; Shackel, Ken

    2008-01-01

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

  15. The mouse dead-end gene isoform α is necessary for germ cell and embryonic viability

    PubMed Central

    Bhattacharya, Chitralekha; Aggarwal, Sita; Zhu, Rui; Kumar, Madhu; Zhao, Ming; Meistrich, Marvin L.; Matin, Angabin

    2007-01-01

    Inactivation of the dead-end (Dnd1) gene in the Ter mouse strain results in depletion of primordial germ cells (PGCs) so that mice become sterile. However, on the 129 mouse strain background, loss of Dnd1 also increases testicular germ cell tumor incidence in parallel to PGC depletion. We report that inactivation of Dnd1 also affects embryonic viability in the 129 strain. Mouse Dnd1 encodes two protein isoforms, DND1-isoform α (DND1- α) and DND1-isoform β (DND1-β). Using isoform specific antibodies, we determined DND1-α is expressed in embryos and embryonic gonads whereas DND1-β expression is restricted to germ cells of the adult testis. Our data implicates DND1-α isoform to be necessary for germ cell viability and therefore its loss in Ter mice results in PGC depletion, germ cell tumor development and partial embryonic lethality in the 129 strain. PMID:17291453

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2008-11-01

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

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

    PubMed

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

    2010-04-01

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

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

    PubMed

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

    2014-04-24

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

  1. Enhanced cell viability and cell adhesion using low conductivity medium for negative dielectrophoretic cell patterning.

    PubMed

    Puttaswamy, Srinivasu Valagerahally; Sivashankar, Shilpa; Chen, Rong-Jhe; Chin, Chung-Kuang; Chang, Hwan-You; Liu, Cheng Hsien

    2010-10-01

    Negative dielectrophoretic (n-DEP) cell manipulation is an efficient way to pattern human liver cells on micro-electrode arrays. Maintaining cell viability is an important objective for this approach. This study investigates the effect of low conductivity medium and the optimally designed microchip on cell viability and cell adhesion. To explore the influence of conductivity on cell viability and cell adhesion, we have used earlier reported dielectrophoresis (DEP) buffer with a conductivity of 10.2 mS/m and three formulated media with conductivity of 9.02 (M1), 8.14 (M2), 9.55 (M3) mS/m. The earlier reported isotonic sucrose/dextrose buffer (DEP buffer) used for DEP manipulation has the drawback of poor cell adhesion and cell viability. A microchip prototype with well-defined positioning of titanium electrode arrays was designed and fabricated on a glass substrate. The gap between the radial electrodes was accurately determined to achieve good cell patterning performance. Parameters such as dimension of positioning electrode, amplitude, and frequency of voltage signal were investigated to optimize the performance of the microchip.

  2. Nanodiamonds on tetrahedral amorphous carbon significantly enhance dopamine detection and cell viability.

    PubMed

    Peltola, Emilia; Wester, Niklas; Holt, Katherine B; Johansson, Leena-Sisko; Koskinen, Jari; Myllymäki, Vesa; Laurila, Tomi

    2017-02-15

    We hypothesize that by using integrated carbon nanostructures on tetrahedral amorphous carbon (ta-C), it is possible to take the performance and characteristics of these bioelectrodes to a completely new level. The integrated carbon electrodes were realized by combining nanodiamonds (NDs) with ta-C thin films coated on Ti-coated Si-substrates. NDs were functionalized with mixture of carboxyl and amine groups NDandante or amine NDamine, carboxyl NDvox or hydroxyl groups NDH and drop-casted or spray-coated onto substrate. By utilizing these novel structures we show that (i) the detection limit for dopamine can be improved by two orders of magnitude [from 10µM to 50nM] in comparison to ta-C thin film electrodes and (ii) the coating method significantly affects electrochemical properties of NDs and (iii) the ND coatings selectively promote cell viability. NDandante and NDH showed most promising electrochemical properties. The viability of human mesenchymal stem cells and osteoblastic SaOS-2 cells was increased on all ND surfaces, whereas the viability of mouse neural stem cells and rat neuroblastic cells was improved on NDandante and NDH and reduced on NDamine and NDvox. The viability of C6 cells remained unchanged, indicating that these surfaces will not cause excess gliosis. In summary, we demonstrated here that by using functionalized NDs on ta-C thin films we can significantly improve sensitivity towards dopamine as well as selectively promote cell viability. Thus, these novel carbon nanostructures provide an interesting concept for development of various in vivo targeted sensor solutions.

  3. 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.

  4. Effect of fluoride on the cell viability, cell organelle potential, and photosynthetic capacity of freshwater and soil algae.

    PubMed

    Chae, Yooeun; Kim, Dokyung; An, Youn-Joo

    2016-12-01

    Although fluoride occurs naturally in the environment, excessive amounts of fluoride in freshwater and terrestrial ecosystems can be harmful. We evaluated the toxicity of fluoride compounds on the growth, viability, and photosynthetic capacity of freshwater (Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata) and terrestrial (Chlorococcum infusionum) algae. To measure algal growth inhibition, a flow cytometric method was adopted (i.e., cell size, granularity, and auto-fluorescence measurements), and algal yield was calculated to assess cell viability. Rhodamine123 and fluorescein diacetate were used to evaluate mitochondrial membrane potential (MMA, ΔΨm) and cell permeability. Nine parameters related to the photosynthetic capacity of algae were also evaluated. The results indicated that high concentrations of fluoride compounds affected cell viability, cell organelle potential, and photosynthetic functions. The cell viability measurements of the three algal species decreased, but apoptosis was only observed in C. infusionum. The MMA (ΔΨm) of cells exposed to fluoride varied among species, and the cell permeability of the three species generally decreased. The decrease in the photosynthetic activity of algae may be attributable to the combination of fluoride ions (F(-)) with magnesium ions (Mg(2+)) in chlorophyll. Our results therefore provide strong evidence for the potential risks of fluoride compounds to microflora and microfauna in freshwater and terrestrial ecosystems.

  5. Cytoglobin modulates myogenic progenitor cell viability and muscle regeneration.

    PubMed

    Singh, Sarvjeet; Canseco, Diana C; Manda, Shilpa M; Shelton, John M; Chirumamilla, Rajendra R; Goetsch, Sean C; Ye, Qiu; Gerard, Robert D; Schneider, Jay W; Richardson, James A; Rothermel, Beverly A; Mammen, Pradeep P A

    2014-01-07

    Mammalian skeletal muscle can remodel, repair, and regenerate itself by mobilizing satellite cells, a resident population of myogenic progenitor cells. Muscle injury and subsequent activation of myogenic progenitor cells is associated with oxidative stress. Cytoglobin is a hemoprotein expressed in response to oxidative stress in a variety of tissues, including striated muscle. In this study, we demonstrate that cytoglobin is up-regulated in activated myogenic progenitor cells, where it localizes to the nucleus and contributes to cell viability. siRNA-mediated depletion of cytoglobin from C2C12 myoblasts increased levels of reactive oxygen species and apoptotic cell death both at baseline and in response to stress stimuli. Conversely, overexpression of cytoglobin reduced reactive oxygen species levels, caspase activity, and cell death. Mice in which cytoglobin was knocked out specifically in skeletal muscle were generated to examine the role of cytoglobin in vivo. Myogenic progenitor cells isolated from these mice were severely deficient in their ability to form myotubes as compared with myogenic progenitor cells from wild-type littermates. Consistent with this finding, the capacity for muscle regeneration was severely impaired in mice deficient for skeletal-muscle cytoglobin. Collectively, these data demonstrate that cytoglobin serves an important role in muscle repair and regeneration.

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

    PubMed Central

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

    2014-01-01

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

  7. The Impaired Viability of Prostate Cancer Cell Lines by the Recombinant Plant Kallikrein Inhibitor*

    PubMed Central

    Ferreira, Joana Gasperazzo; Diniz, Paula Malloy Motta; de Paula, Cláudia Alessandra Andrade; Lobo, Yara Aparecida; Paredes-Gamero, Edgar Julian; Paschoalin, Thaysa; Nogueira-Pedro, Amanda; Maza, Paloma Korehisa; Toledo, Marcos Sergio; Suzuki, Erika; Oliva, Maria Luiza Vilela

    2013-01-01

    Prostate cancer is the most common type of cancer, and kallikreins play an important role in the establishment of this disease. rBbKIm is the recombinant Bauhinia bauhinioides kallikreins inhibitor that was modified to include the RGD/RGE motifs of the inhibitor BrTI from Bauhinia rufa. This work reports the effects of rBbKIm on DU145 and PC3 prostate cancer cell lines. rBbKIm inhibited the cell viability of DU145 and PC3 cells but did not affect the viability of fibroblasts. rBbKIm caused an arrest of the PC3 cell cycle at the G0/G1 and G2/M phases but did not affect the DU145 cell cycle, although rBbKIm triggers apoptosis and cytochrome c release into the cytosol of both cell types. The differences in caspase activation were observed because rBbKIm treatment promoted activation of caspase-3 in DU145 cells, whereas caspase-9 but not caspase-3 was activated in PC3 cells. Because angiogenesis is important to the development of a tumor, the effect of rBbKIm in this process was also analyzed, and an inhibition of 49% was observed in in vitro endothelial cell capillary-like tube network formation. In summary, we demonstrated that different properties of the protease inhibitor rBbKIm may be explored for investigating the androgen-independent prostate cancer cell lines PC3 and DU145. PMID:23511635

  8. Enhanced resveratrol production in Vitis vinifera cell suspension cultures by heavy metals without loss of cell viability.

    PubMed

    Cai, Zhenzhen; Kastell, Anja; Speiser, Claire; Smetanska, Iryna

    2013-09-01

    The effects of heavy metal ions (Co(2+), Ag(+), Cd(2+)) on cell viability and secondary metabolite production, particularly anthocyanins and phenolic acids in Vitis vinifera cell suspension cultures, were investigated. Of these, Co at all three used concentrations (5.0, 25, and 50 μM), Ag, and Cd at low concentration (5.0 μM) were most effective to stimulate the phenolic acid production, increasing the 3-O-glucosyl-resveratrol up to 1.6-fold of the control level (250.5 versus 152.4 μmol/g), 4 h after the treatments. Meanwhile, the elicitors at effective concentrations did not suppress cell growth, while the cell viability maintained. In contrast, Ag and Cd at high concentrations (25 and 50 μM) remarkably reduced the cell viability, decreasing the cell viability up to about 15 % of the control level, 24 h after the treatments. The heavy metal ions did not affect the anthocyanin production. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, peroxidase activity, medium pH value, and conductivity were only slightly elevated by the heavy metal ions. The results suggest that some of the secondary metabolites production was stimulated by the used elicitors, but there was not a stress response of the cells.

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

    PubMed

    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.

  10. 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

  11. CellViCAM--Cell viability classification for animal cell cultures using dark field micrographs.

    PubMed

    Burgemeister, S; Nattkemper, T W; Noll, T; Hoffrogge, R; Flaschel, E

    2010-09-15

    Online monitoring of cell density and cell viability is a challenging but essential task to control and optimize biotechnical processes and is of particular interest for the growing field of animal cell cultures. For this purpose, we introduce an optical approach for automated cell detection and viability classification of suspended mammalian cells. Our proposed system CellViCAM is capable of evaluating dark field micrographs by means of several image processing and supervised machine learning techniques without the use of any dyes or fluorescent labeling. Using a human cell line as the reference culture, an efficient cell detection procedure has been established also enabling a cell density estimation. Furthermore, a comprehensive but reagent-free viability analysis, based on a semi-automatic training data generation, has been developed. By means of an extensive validation dataset we can show that the CellViCAM approach can be considered as an equivalent to staining-based methods and moreover, how it provides a technical platform for a more differentiated cell state classification into living, necrotic, early and late apoptosis.

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

    PubMed

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

    1991-10-01

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

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

    PubMed Central

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

    2016-01-01

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

  14. Effects of diluents on cell culture viability measured by automated cell counter

    PubMed Central

    Chen, Aaron; Leith, Matthew; Tu, Roger; Tahim, Gurpreet; Sudra, Anish; Bhargava, Swapnil

    2017-01-01

    Commercially available automated cell counters based on trypan blue dye-exclusion are widely used in industrial cell culture process development and manufacturing to increase throughput and eliminate inherent variability in subjective interpretation associated with manual hemocytometers. When using these cell counters, sample dilution is often necessary to stay within the assay measurement range; however, the effect of time and diluents on cell culture is not well understood. This report presents the adverse effect of phosphate buffered saline as a diluent on cell viability when used in combination with an automated cell counter. The reduced cell viability was attributed to shear stress introduced by the automated cell counter. Furthermore, length of time samples were incubated in phosphate buffered saline also contributed to the observed drop in cell viability. Finally, as erroneous viability measurements can severely impact process decisions and product quality, this report identifies several alternative diluents that can maintain cell culture viability over time in order to ensure accurate representation of cell culture conditions. PMID:28264018

  15. 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

  16. 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

  17. Regulation of the viability of Nf1 deficient cells by PKC isoforms.

    PubMed

    Zhou, Xiaodong; Shen, Ling; Parris, Toshima; Huang, Junchi; Yi, Bo; Helou, Khalil; Chen, Changyan

    2014-11-15

    Suppression of protein kinase C (PKC) is known to be synthetically lethal with ras mutations in various types of cancer cells. The studies also showed that blockade of PKC affected the viability of Nf1 deficient cells. Since PKC family consists of more than 10 isoforms, our study aimed at identifying which isoform(s) played the crucial role in sensitizing Nf1 deficient cells to apoptosis. Using genetic and chemical PKC inhibitors, we demonstrated that the concurrent inhibition of PKC α and β induced Nf1 deficient ST or 96.2 cells, but not SNF02.2 cells with a normal Nf1 or ST cells ectopically expressing Nf1 effective domain gene, to apoptosis. In this process, PKC δ in Nf1 deficient cells, but not in ST/Nf1 cells, was upregulated and translocated to the nucleus. Furthermore, caspase 3 was cleaved and cytochrome c was released to the cytosol. Thus, it appeared that PKC δ and α/β are the crucial components for sustaining the aberrant Ras signaling and further viability of Nf1 deficient cells. The abrogation of these two isoforms activated their opponent PKC δ for switching on the caspase 3-governed apoptotic machinery.

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

    PubMed Central

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

    2016-01-01

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

  19. 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

  20. 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.

  1. Influence of Flow Behavior of Alginate-Cell Suspensions on Cell Viability and Proliferation.

    PubMed

    Ning, Liqun; Guillemot, Arthur; Zhao, Jingxuan; Kipouros, Georges; Chen, Xiongbiao

    2016-07-01

    Tissue scaffolds with living cells fabricated by three-dimensional bioprinting/plotting techniques are becoming more prevalent in tissue repair and regeneration. In the bioprinting process, cells are subject to process-induced forces (such as shear force) that can result in cell damage and loss of cell function. The flow behavior of the biomaterial solutions that encapsulate living cells in this process plays an important role. This study used a rheometer to examine the flow behavior of alginate solution and alginate-Schwann cell (RSC96), alginate-fibroblast cell (NIH-3T3), and alginate-skeletal muscle cell (L8) suspensions during shearing with respect to effects on cell viability and proliferation. The flow behavior of all the alginate-cell suspensions varied with alginate concentration and cell density and had a significant influence on the viability and proliferation of the cells once sheared as well as on the recovery of the sheared cells. These findings provide a mean to preserve cell viability and/or retain cell proliferation function in the bioprinting process by regulating the flow behavior of cell-biomaterial suspensions and process parameters.

  2. 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.

  3. FGF1-gold nanoparticle conjugates targeting FGFR efficiently decrease cell viability upon NIR irradiation

    PubMed Central

    Szlachcic, Anna; Pala, Katarzyna; Zakrzewska, Malgorzata; Jakimowicz, Piotr; Wiedlocha, Antoni; Otlewski, Jacek

    2012-01-01

    Fibroblast growth factor receptors (FGFRs) are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we designed, constructed, and characterized FGFR-targeted gold nanoconjugates suitable for infrared-induced thermal ablation (localized heating leading to cancer cell death) based on gold nanoparticles (AuNPs). We showed that a recombinant ligand of all FGFRs, human fibroblast growth factor 1 (FGF1), can be used as an agent targeting covalently bound AuNPs to cancer cells overexpressing FGFRs. To assure thermal stability, protease resistance, and prolonged half-life of the targeting protein, we employed highly stable FGF1 variant that retains the biological activities of the wild type FGF1. Novel FGF1 variant, AuNP conjugates are specifically internalized only by the cells expressing FGFRs, and they significantly reduce their viability after irradiation with near-infrared light (down to 40% of control cell viability), whereas the proliferation potential of cells lacking FGFRs is not affected. These results demonstrate the feasibility of FGF1-coated AuNPs for targeted cancer therapy. PMID:23226697

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

    PubMed

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

    2011-01-01

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

  5. Kaempferol nanoparticles achieve strong and selective inhibition of ovarian cancer cell viability.

    PubMed

    Luo, Haitao; Jiang, Bingbing; Li, Bingyun; Li, Zhaoliang; Jiang, Bing-Hua; Chen, Yi Charlie

    2012-01-01

    Ovarian cancer is one of the leading causes of cancer death for women throughout the Western world. Kaempferol, a natural flavonoid, has shown promise in the chemoprevention of ovarian cancer. A common concern about using dietary supplements for chemoprevention is their bioavailability. Nanoparticles have shown promise in increasing the bioavailability of some chemicals. Here we developed five different types of nanoparticles incorporating kaempferol and tested their efficacy in the inhibition of viability of cancerous and normal ovarian cells. We found that positively charged nanoparticle formulations did not lead to a significant reduction in cancer cell viability, whereas nonionic polymeric nanoparticles resulted in enhanced reduction of cancer cell viability. Among the nonionic polymeric nanoparticles, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nanoparticles incorporating kaempferol led to significant reduction in cell viability of both cancerous and normal cells. Poly(DL-lactic acid-co-glycolic acid) (PLGA) nanoparticles incorporating kaempferol resulted in enhanced reduction of cancer cell viability together with no significant reduction in cell viability of normal cells compared with kaempferol alone. Therefore, both PEO-PPO-PEO and PLGA nanoparticle formulations were effective in reducing cancer cell viability, while PLGA nanoparticles incorporating kaempferol had selective toxicity against cancer cells and normal cells. A PLGA nanoparticle formulation could be advantageous in the prevention and treatment of ovarian cancers. On the other hand, PEO-PPO-PEO nanoparticles incorporating kaempferol were more effective inhibitors of cancer cells, but they also significantly reduced the viability of normal cells. PEO-PPO-PEO nanoparticles incorporating kaempferol may be suitable as a cancer-targeting strategy, which could limit the effects of the nanoparticles on normal cells while retaining their potency against cancer cells. We

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

    PubMed

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

    1991-05-15

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

  7. Radiation-induced glioblastoma signaling cascade regulates viability, apoptosis and differentiation of neural stem cells (NSC).

    PubMed

    Ivanov, Vladimir N; Hei, Tom K

    2014-12-01

    Ionizing radiation alone or in combination with chemotherapy is the main treatment modality for brain tumors including glioblastoma. Adult neurons and astrocytes demonstrate substantial radioresistance; in contrast, human neural stem cells (NSC) are highly sensitive to radiation via induction of apoptosis. Irradiation of tumor cells has the potential risk of affecting the viability and function of NSC. In this study, we have evaluated the effects of irradiated glioblastoma cells on viability, proliferation and differentiation potential of non-irradiated (bystander) NSC through radiation-induced signaling cascades. Using media transfer experiments, we demonstrated significant effects of the U87MG glioblastoma secretome after gamma-irradiation on apoptosis in non-irradiated NSC. Addition of anti-TRAIL antibody to the transferred media partially suppressed apoptosis in NSC. Furthermore, we observed a dramatic increase in the production and secretion of IL8, TGFβ1 and IL6 by irradiated glioblastoma cells, which could promote glioblastoma cell survival and modify the effects of death factors in bystander NSC. While differentiation of NSC into neurons and astrocytes occurred efficiently with the corresponding differentiation media, pretreatment of NSC for 8 h with medium from irradiated glioblastoma cells selectively suppressed the differentiation of NSC into neurons, but not into astrocytes. Exogenous IL8 and TGFβ1 increased NSC/NPC survival, but also suppressed neuronal differentiation. On the other hand, IL6 was known to positively affect survival and differentiation of astrocyte progenitors. We established a U87MG neurosphere culture that was substantially enriched by SOX2(+) and CD133(+) glioma stem-like cells (GSC). Gamma-irradiation up-regulated apoptotic death in GSC via the FasL/Fas pathway. Media transfer experiments from irradiated GSC to non-targeted NSC again demonstrated induction of apoptosis and suppression of neuronal differentiation of NSC. In

  8. 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.

  9. 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.

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

    PubMed Central

    Cui, Xiaoxuan; Zhang, Lu; Magli, Amanda R.; Catera, Rosa; Yan, Xiao-Jie; Griffin, Daniel O.; Rothstein, Thomas L.; Barrientos, Jacqueline; Kolitz, Jonathan E.; Allen, Steven L.; Rai, Kanti R.; Chiorazzi, Nicholas; Chu, Charles C.

    2015-01-01

    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

  11. 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)

  12. 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.

  13. 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.

  14. 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.

  15. Decitabine has a biphasic effect on natural killer cell viability, phenotype, and function under proliferative conditions.

    PubMed

    Kopp, Lisa M; Ray, Anish; Denman, Cecele J; Senyukov, Vladimir S; Somanchi, Srinivas S; Zhu, Shiguo; Lee, Dean A

    2013-07-01

    receptor NKp44, but also causes decreased viability, proliferation, and expression of the activating receptor NKG2D. Decitabine treatment results in a biphasic effect in overall NK cell lytic function, which correlates with a biphasic pattern of global hypomethylation. Decitabine affects the expression of activating and inhibitory receptors in NK cells at low concentrations when exposed during cell proliferation. High doses of decitabine decrease NK cell proliferation and viability, likely through direct inhibition of mRNA transcription. The results of these combined effects leads to a biphasic response in hypomethylation and cytotoxicity. This suggests that optimal immunomodulation with decitabine occurs at low dose ranges and that high doses abrogate this effect through inhibition of proliferation and direct toxicity to NK cells.

  16. High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial Lux reporter.

    PubMed

    Class, Bradley; Thorne, Natasha; Aguisanda, Francis; Southall, Noel; McKew, John C; Zheng, Wei

    2015-04-01

    Cell viability assays are extensively used to determine cell health, evaluate growth conditions, and assess compound cytotoxicity. Most existing assays are endpoint assays, in which data are collected at one time point after termination of the experiment. The time point at which toxicity of a compound is evident, however, depends on the mechanism of that compound. An ideal cell viability assay allows the determination of compound toxicity kinetically without having to terminate the assay prematurely. We optimized and validated a reagent-addition-free cell viability assay using an autoluminescent HEK293 cell line that stably expresses bacterial luciferase and all substrates necessary for bioluminescence. This cell viability assay can be used for real-time, long-term measurement of compound cytotoxicity in live cells with a signal-to-basal ratio of 20- to 200-fold and Z-factors of ~0.6 after 24-, 48- 72-, or 96-h incubation with compound. We also found that the potencies of nine cytotoxic compounds correlated well with those measured by four other commonly used cell viability assays. The results demonstrated that this kinetic cell viability assay using the HEK293(lux) autoluminescent cell line is useful for high-throughput evaluation of compound cytotoxicity.

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

    PubMed Central

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

    2002-01-01

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

  18. Radiolabeled red cell viability. I. Comparison of /sup 51/Cr, /sup 99m/Tc, and /sup 111/In for measuring the viability of autologous stored red cells

    SciTech Connect

    Marcus, C.S.; Myhre, B.A.; Angulo, M.C.; Salk, R.D.; Essex, C.E.; Demianew, S.H.

    1987-09-01

    The simultaneous determination of autologous /sup 99m/Tc red cell (RBC) and /sup 51/Cr RBC viability at 24 hours was measured in 19 normal volunteers whose RBCs had been stored in additive media (Nutracel) for 42 or 49 days. The ratio of the /sup 51/Cr:/sup 99m/Tc value was 1.23. In this experiment we also calculated /sup 51/Cr RBC viability by both the single-isotope method (extrapolation) and the double-isotope method (using /sup 125/I human serum albumin for an independent plasma volume) in the same volunteers. The corresponding viability values were not significantly different. The simultaneous determination of autologous /sup 111/In-oxine RBC and /sup 51/Cr RBC viability at 24 hours was measured in 19 other normal volunteers whose RBCs had been stored in citrate-phosphate-dextrose-adenine (CPDA-1) for 1 or 15 days. The ratio of the /sup 51/Cr:/sup 111/In value was 1.1. Use of these 24-hour viability ratios as conversion factors permits direct comparison of /sup 99m/Tc or /sup 111/In RBC viability with a /sup 51/Cr standard, and therefore expands the application of these newer RBC radiolabels.

  19. Low Dose BCG Infection as a Model for Macrophage Activation Maintaining Cell Viability

    PubMed Central

    Chávez-Galán, Leslie; Vesin, Dominique; Martinvalet, Denis

    2016-01-01

    Mycobacterium bovis BCG, the current vaccine against tuberculosis, is ingested by macrophages promoting the development of effector functions including cell death and microbicidal mechanisms. Despite accumulating reports on M. tuberculosis, mechanisms of BCG/macrophage interaction remain relatively undefined. In vivo, few bacilli are sufficient to establish a mycobacterial infection; however, in vitro studies systematically use high mycobacterium doses. In this study, we analyze macrophage/BCG interactions and microenvironment upon infection with low BCG doses and propose an in vitro model to study cell activation without affecting viability. We show that RAW macrophages infected with BCG at MOI 1 activated higher and sustained levels of proinflammatory cytokines and transcription factors while MOI 0.1 was more efficient for early stimulation of IL-1β, MCP-1, and KC. Both BCG infection doses induced iNOS and NO in a dose-dependent manner and maintained nuclear and mitochondrial structures. Microenvironment generated by MOI 1 induced macrophage proliferation but not MOI 0.1 infection. In conclusion, BCG infection at low dose is an efficient in vitro model to study macrophage/BCG interactions that maintains macrophage viability and mitochondrial structures. This represents a novel model that can be applied to BCG research fields including mycobacterial infections, cancer immunotherapy, and prevention of autoimmunity and allergies. PMID:27833923

  20. Low Dose BCG Infection as a Model for Macrophage Activation Maintaining Cell Viability.

    PubMed

    Chávez-Galán, Leslie; Vesin, Dominique; Martinvalet, Denis; Garcia, Irene

    2016-01-01

    Mycobacterium bovis BCG, the current vaccine against tuberculosis, is ingested by macrophages promoting the development of effector functions including cell death and microbicidal mechanisms. Despite accumulating reports on M. tuberculosis, mechanisms of BCG/macrophage interaction remain relatively undefined. In vivo, few bacilli are sufficient to establish a mycobacterial infection; however, in vitro studies systematically use high mycobacterium doses. In this study, we analyze macrophage/BCG interactions and microenvironment upon infection with low BCG doses and propose an in vitro model to study cell activation without affecting viability. We show that RAW macrophages infected with BCG at MOI 1 activated higher and sustained levels of proinflammatory cytokines and transcription factors while MOI 0.1 was more efficient for early stimulation of IL-1β, MCP-1, and KC. Both BCG infection doses induced iNOS and NO in a dose-dependent manner and maintained nuclear and mitochondrial structures. Microenvironment generated by MOI 1 induced macrophage proliferation but not MOI 0.1 infection. In conclusion, BCG infection at low dose is an efficient in vitro model to study macrophage/BCG interactions that maintains macrophage viability and mitochondrial structures. This represents a novel model that can be applied to BCG research fields including mycobacterial infections, cancer immunotherapy, and prevention of autoimmunity and allergies.

  1. Improvement of Longevity and Viability of Sperm Cells Isolated from Pollen of Zea mays L. 1

    PubMed Central

    Zhang, Guichang; Williams, Connie M.; Campenot, Mary K.; McGann, Locksley E.; Cass, David D.

    1992-01-01

    Our previous studies showed that the common maize (Zea mays L.) sperm isolation medium (Brewbaker and Kwack salts in 0.44 m sucrose without buffering) caused cell lysis in vitro. In an attempt to remedy this situation, 6 sugars, 10 buffers, 5 pH values, and 3 membrane protective agents were screened to improve longevity and viability of isolated Zea mays sperm cells as estimated by hemacytometry and flow cytometry. Use of 0.55 m galactose in the isolation solution increased sperm yield by 2.5-fold compared with sucrose, and suspension of isolated sperm cells in the galactose solution gave the best longevity among the six sugars. Buffering the galactose solution with 2 mm 2-(N-morpholino)ethanesulfonic acid significantly improved longevity, whereas other buffers had no effect or decreased the longevity and/or viability. Among the five pH values tested (5.0, 6.0, 6.7, 7.0, and 8.0), pH 6.7 appeared to be optimal for maintenance of both longevity and viability. Screening of membrane protectants showed that cysteine caused a rapid decrease in cell viability and increased lysis, whereas dithiothreitol increased the cell numbers but lowered their viability. Addition of 0.1% bovine serum albumin increased cell numbers and viability, and about 70% of the cells remained viable after 72 h of suspension. Cell longevity and viability were also improved in 0.44 m sucrose when the solution was conditioned with 2-(N-morpholino)ethanesulfonic acid and bovine serum albumin. Use of 2-(N-morpholino)ethanesulfonic acid and bovine serum albumin inthe isolation and suspension medium significantly improved the viability and longevity of sperm cells isolated from Zea mays pollen. PMID:16652985

  2. 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.

  3. 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

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

    PubMed

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

    2011-12-01

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

  5. Effect of Butyrate on Collagen Expression, Cell Viability, Cell Cycle Progression and Related Proteins Expression of MG-63 Osteoblastic Cells

    PubMed Central

    Chang, Mei-Chi; Tsai, Yi-Ling; Liou, Eric Jein-Wein; Tang, Chia-Mei; Wang, Tong-Mei; Liu, Hsin-Cheng; Liao, Ming-Wei; Yeung, Sin-Yuet; Chan, Chiu-Po; Jeng, Jiiang-Huei

    2016-01-01

    Aims Butyric acid is one major metabolic product generated by anaerobic Gram-negative bacteria of periodontal and root canal infection. Butyric acid affects the activity of periodontal cells such as osteoblasts. The purposes of this study were to investigate the effects of butyrate on MG-63 osteoblasts. Methods MG-63 cells were exposed to butyrate and cell viability was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mRNA and protein expression of type I collagen and cell cycle-related proteins were measured by reverse-transcriptase polymerase chain reaction (RT-PCR), western blotting or immunofluorescent staining. Cellular production of reactive oxygen species (ROS) was analyzed by 2',7'-dichlorofluorescein (DCF) fluorescence flow cytometry. Results Exposure to butyrate suppressed cell proliferation, and induced G2/M (8 and 16 mM) cell cycle arrest of MG-63 cells. Some cell apoptosis was noted. The mRNA expression of cdc2 and cyclin-B1 decreased after exposure to butyrate. The protein expression of type I collagen, cdc2 and cyclin B1 were decreased, whereas the expression of p21, p27 and p57 was stimulated. Under the treatment of butyrate, ROS production in MG-63 cells markedly increased. Conclusions The secretion of butyric acid by periodontal and root canal microorganisms may inhibit bone cell growth and matrix turnover. This is possibly due to induction of cell cycle arrest and ROS generation and inhibition of collagen expression. These results suggest the involvement of butyric acid in the pathogenesis of periodontal and periapical tissue destruction by impairing bone healing responses. PMID:27893752

  6. 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.

  7. Rapid exchange of oil-phase in microencapsulation chip to enhance cell viability.

    PubMed

    Kim, Choong; Lee, Kang Sun; Kim, Young Eun; Lee, Kyu-Jung; Lee, Soo Hyun; Kim, Tae Song; Kang, Ji Yoon

    2009-05-07

    This paper describes a microfluidic device for the microencapsulation of cells in alginate beads to enhance cell viability. The alginate droplet including cells was gelified with calcified oleic acid, using two-phase microfluidics. The on-chip gelation had generated monodisperse spherical alginate beads, which could not be readily obtained via conventional external gelation in a calcium chloride bath. However, the prolonged exposure of encapsulated cells to the toxic oil phase caused serious damage to the cells. Therefore, we proposed the formulation of a rapid oil-exchange chip which transforms the toxic oleic acid to harmless mineral oil. The flushing out of oleic acid after the gelation of alginate beads effected a dramatic increase in the viability of P19 embryonic carcinoma cells, up to 90%. The experimental results demonstrated that the cell viability was proportional to the flow rate of squeezing mineral oil.

  8. The effect of simultaneous exposure of HEMn-DP and HEMn-LP melanocytes to nicotine and UV-radiation on the cell viability and melanogenesis.

    PubMed

    Delijewski, Marcin; Wrześniok, Dorota; Beberok, Artur; Rok, Jakub; Otręba, Michał; Buszman, Ewa

    2016-11-01

    Nicotine is a main compound of tobacco plants and may affect more than a billion people all over the world that are permanently exposed to nicotine from cigarettes, various forms of smoking cessation therapies, electronic cigarettes or second-hand smoke. It is known that nicotine forms complexes with melanin what may lead to accumulation of this alkaloid in tissues of living organisms containing the pigment. This may affect the viability of cells and process of melanin biosynthesis that takes place in melanocytes. Although UV radiation is known to be a particular inductor of melanin biosynthesis, its simultaneous effect with nicotine on this process as well as the viability of human cells containing melanin have not been assessed so far. The aim of this study was to examine the simultaneous impact of nicotine and UV radiation on viability and melanogenesis in cultured normal human melanocytes dark (HEMn-DP) and light (HEMn-LP) pigmented. Nicotine together with UV radiation induced concentration-dependent loss in melanocytes viability. The higher cell loss was observed in dark pigmented melanocytes in comparison to light pigmented cells. Simultaneous exposure of cells to nicotine and UV radiation also caused changes in melanization process in both tested cell lines. The data suggest that simultaneous exposure of melanocytes to nicotine and UV radiation up-regulates melanogenesis and affects cell viability. Observed processes are more pronounced in dark pigmented cells.

  9. 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

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

    PubMed

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

    2014-09-09

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

  11. 3D finite element analysis of nutrient distributions and cell viability in the intervertebral disc: effects of deformation and degeneration.

    PubMed

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

    2011-09-01

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

  12. 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.

  13. 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...

  14. 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.

  15. 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.

  16. Viability-reducing activity of Coryllus avellana L. extracts against human cancer cell lines.

    PubMed

    Gallego, Ana; Metón, Isidoro; Baanante, Isabel V; Ouazzani, Jamal; Adelin, Emilie; Palazon, Javier; Bonfill, Mercedes; Moyano, Elisabeth

    2017-02-28

    The increasing rate of cancer incidence has encouraged the search for novel natural sources of anticancer compounds. The presence of small quantities of taxol and taxanes in Corylus avellana L. has impelled new potential applications for this plant in the field of biomedicine. In the present work, the cell viability-reducing activity of stems and leaves from three different hazel trees was studiedagainst three human-derived cancer cell lines (HeLa, HepG2 and MCF-7). Both leaf and stem extracts significantly reduced viability of the three cell lines either after maceration with methanol or using taxane extraction methods. Since maceration reduced cell viability to a greater extent than taxane extraction methods, we scaled up the maceration extraction process using a method for solid/liquid extraction (Zippertex technology). Methanol leaf extracts promoted a higher reduction in viability of all cell lines assayed than stem extracts. Fractionation of methanol leaf extracts using silica gel chormatography led to the purification and identification of two compounds by HPLC-MS and NMR: (3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl) heptane 3-O-β-d-glucopyranoside and quercetin-3-O-rhamnoside. The isolated compounds decreased viability of HeLa and HepG2 cells to a greater extent than MCF-7 cells. Our results suggest a potential use of C. avellana extracts in the pharmacotherapy of cervical cancer and hepatocarcinoma and, to a lesser extent, breast cancer.

  17. The direct effect of estrogen on cell viability and apoptosis in human gastric cancer cells.

    PubMed

    Qin, Jian; Liu, Min; Ding, Qianshan; Ji, Xiang; Hao, Yarong; Wu, Xiaomin; Xiong, Jie

    2014-10-01

    Epidemiology researches indicated that gastric cancer is a male-predominant disease; both expression level of estrogen and expression pattern of estrogen receptors (ERs) influence its carcinogenesis. But the direct effect of estrogen on gastric cancer cells is still unclear. This study aimed to explore the direct effect of β-estradiol (E2) on gastric cancer cells. SGC7901 and BGC823 were treated with a serial of concentrations of E2. The survival rates of both the cell lines were significantly reduced, and the reduction of viability was due to apoptosis triggered by E2 treatment. Caspase 3 was activated in response to the increasing E2 concentration in both SGC7901 and BGC823. Cleaved Caspase 3 fragments were detected, and the expression levels of Bcl-2 and Bcl-xL were reduced. Apoptosis was further confirmed by flow cytometry. The expression level of PEG10, an androgen receptor target gene, was reduced during E2 treatment. Both ERα and ERβ were expressed in these cell lines, and the result of bioinformatics analysis of gastric cancer from GEO datasets indicated that the expression levels of both ERα and ERβ were significantly higher in noncancerous gastric tissues than in gastric cancer tissues. Our research indicated that estrogen can reduce cell viability and promote apoptosis in gastric cancer cells directly; ERs expression level is associated with gastric cancer. Our research will help to understand the mechanism of gender disparity in gastric cancer.

  18. 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.

  19. Optimizing Photo-Encapsulation Viability of Heart Valve Cell Types in 3D Printable Composite Hydrogels.

    PubMed

    Kang, Laura Hockaday; Armstrong, Patrick A; Lee, Lauren Julia; Duan, Bin; Kang, Kevin Heeyong; Butcher, Jonathan Talbot

    2017-02-01

    Photocrosslinking hydrogel technologies are attractive for the biofabrication of cardiovascular soft tissues, but 3D printing success is dependent on multiple variables. In this study we systematically test variables associated with photocrosslinking hydrogels (photoinitiator type, photoinitiator concentration, and light intensity) for their effects on encapsulated cells in an extrusion 3D printable mixture of methacrylated gelatin/poly-ethylene glycol diacrylate/alginate (MEGEL/PEGDA3350/alginate). The fabrication conditions that produced desired hydrogel mechanical properties were compared against those that optimize aortic valve or mesenchymal stem cell viability. In the 3D hydrogel culture environment and fabrication setting studied, Irgacure can increase hydrogel stiffness with a lower proportional decrease in encapsulated cell viability compared to VA086. Human adipose derived mesenchymal stem cells (HADMSC) survived increasing photoinitiator concentrations in photo-encapsulation conditions better than aortic valve interstitial cells (HAVIC) and aortic valve sinus smooth muscle cells (HASSMC). Within the range of photo-encapsulation fabrication conditions tested with MEGEL/PEGDA/alginate (0.25-1.0% w/v VA086, 0.025-0.1% w/v Irgacure 2959, and 365 nm light intensity 2-136 mW/cm(2)), the highest viabilities achieved were 95, 93, and 93% live for HASSMC, HAVIC, and HADMSC respectively. These results identify parameter combinations that optimize cell viability during 3D printing for multiple cell types. These results also indicate that general oxidative stress is higher in photocrosslinking conditions that induce lower cell viability. However, suppressing this increase in intracellular oxidative stress did not improve cell viability, which suggests that other stress mechanisms also contribute.

  20. p53 induction and cell viability modulation by genotoxic individual chemicals and mixtures.

    PubMed

    Di Paolo, Carolina; Müller, Yvonne; Thalmann, Beat; Hollert, Henner; Seiler, Thomas-Benjamin

    2017-03-16

    The binding of the p53 tumor suppression protein to DNA response elements after genotoxic stress can be quantified by cell-based reporter gene assays as a DNA damage endpoint. Currently, bioassay evaluation of environmental samples requires further knowledge on p53 induction by chemical mixtures and on cytotoxicity interference with p53 induction analysis for proper interpretation of results. We investigated the effects of genotoxic pharmaceuticals (actinomycin D, cyclophosphamide) and nitroaromatic compounds (4-nitroquinoline 1-oxide, 3-nitrobenzanthrone) on p53 induction and cell viability using a reporter gene and a colorimetric assay, respectively. Individual exposures were conducted in the absence or presence of metabolic activation system, while binary and tertiary mixtures were tested in its absence only. Cell viability reduction tended to present direct correlation with p53 induction, and induction peaks occurred mainly at chemical concentrations causing cell viability below 80%. Mixtures presented in general good agreement between predicted and measured p53 induction factors at lower concentrations, while higher chemical concentrations gave lower values than expected. Cytotoxicity evaluation supported the selection of concentration ranges for the p53 assay and the interpretation of its results. The often used 80% viability threshold as a basis to select the maximum test concentration for cell-based assays was not adequate for p53 induction assessment. Instead, concentrations causing up to 50% cell viability reduction should be evaluated in order to identify the lowest observed effect concentration and peak values following meaningful p53 induction.

  1. Effects of YM155 on survivin levels and viability in neuroblastoma cells with acquired drug resistance

    PubMed Central

    Voges, Yvonne; Michaelis, Martin; Rothweiler, Florian; Schaller, Torsten; Schneider, Constanze; Politt, Katharina; Mernberger, Marco; Nist, Andrea; Stiewe, Thorsten; Wass, Mark N; Rödel, Franz; Cinatl, Jindrich

    2016-01-01

    Resistance formation after initial therapy response (acquired resistance) is common in high-risk neuroblastoma patients. YM155 is a drug candidate that was introduced as a survivin suppressant. This mechanism was later challenged, and DNA damage induction and Mcl-1 depletion were suggested instead. Here we investigated the efficacy and mechanism of action of YM155 in neuroblastoma cells with acquired drug resistance. The efficacy of YM155 was determined in neuroblastoma cell lines and their sublines with acquired resistance to clinically relevant drugs. Survivin levels, Mcl-1 levels, and DNA damage formation were determined in response to YM155. RNAi-mediated depletion of survivin, Mcl-1, and p53 was performed to investigate their roles during YM155 treatment. Clinical YM155 concentrations affected the viability of drug-resistant neuroblastoma cells through survivin depletion and p53 activation. MDM2 inhibitor-induced p53 activation further enhanced YM155 activity. Loss of p53 function generally affected anti-neuroblastoma approaches targeting survivin. Upregulation of ABCB1 (causes YM155 efflux) and downregulation of SLC35F2 (causes YM155 uptake) mediated YM155-specific resistance. YM155-adapted cells displayed increased ABCB1 levels, decreased SLC35F2 levels, and a p53 mutation. YM155-adapted neuroblastoma cells were also characterized by decreased sensitivity to RNAi-mediated survivin depletion, further confirming survivin as a critical YM155 target in neuroblastoma. In conclusion, YM155 targets survivin in neuroblastoma. Furthermore, survivin is a promising therapeutic target for p53 wild-type neuroblastomas after resistance acquisition (neuroblastomas are rarely p53-mutated), potentially in combination with p53 activators. In addition, we show that the adaptation of cancer cells to molecular-targeted anticancer drugs is an effective strategy to elucidate a drug's mechanism of action. PMID:27735941

  2. 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.

  3. 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.

  4. 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

  5. Embryonic stem cells from blastomeres maintaining embryo viability.

    PubMed

    Klimanskaya, Irina

    2013-01-01

    A wide variety of cell and tissue types that are sought in regenerative medicine can be generated from embryonic stem cells (ESCs), and currently two derivatives of human embryonic stem cells (hESCs) have entered human clinical trials. However, the ethical controversy surrounding this technology, which uses preimplantation human embryos to generate cell lines, is limiting research and the development of new therapies. Several new technologies such as induced pluripotent cells or parthenogenetically derived pluripotent cells hold great promise, but more research is needed before their derivatives can be proven to be safe and functional for use in human patients. The blastomere biopsy-based technique allows the derivation of human ESClines without sacrificing a human embryo and was shown to be robust and produce safe and functional derivatives of therapeutic value.

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

    PubMed

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

    2004-01-01

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

  7. An implantable electrical bioreactor for enhancement of cell viability.

    PubMed

    Kim, Jung Hoon; Lee, Tae Hyung; Song, Yun Mi; Kim, In Sook; Cho, Tae Hyung; Hwang, Sune Jung; Kim, Sung June

    2011-01-01

    Low survival of injected cells which are prepared by ex-vivo culture is main obstacle in cell-based tissue regeneration. To elevate cell adaptation, we designed an implantable electrical bioreactor where human mesenchymal stromal cells (hMSCs) can be cultured and stimulated electrically. Bioreactor was composed of biocompatible cylindrical Teflon body containing a flexible polyimide electrode and implantable stimulator. The Teflon body has about 300 holes with a diameter of 300 um for effective nutrients supply inside the bioreactor and has a length of 17 mm and a diameter of 8mm for implantation. After hMSCs seeded on the collagen sponge that serves as scaffold to form a bone tissue graft, they are cultured in the bioreactor with biphasic electric current (BEC) stimulation. BEC stimulation with amplitude of 20/40 uA, duration of 100 us and a frequency of 100 Hz was applied for one week in the early stage of cultivation. Subsequently, after hMSCS were cultured for another week without electrical stimulation, cell response such as cell proliferation, cell attachment and gene expression are evaluated. In vitro and In vivo culture of hMSCs showed 19% and 22% increase in cell proliferation at stimulated groups, compared to unstimulated control. The expression of type I collagen increased significantly at stimulated group. These results suggest that the usage of implantable electrical bioreactor can be a good strategy to enhance the efficiency of stem cell-based tissue engineering.

  8. Comparison of various techniques for determining viability of Paracoccidioides brasiliensis yeast-form cells.

    PubMed Central

    Restrepo, A; Cano, L E; de Bedout, C; Brummer, E; Stevens, D A

    1982-01-01

    The viability of Paracoccidioides brasiliensis yeast-form cells was determined by colony-forming units, direct fluorescent staining, and production of germ tubes in slide culture. The first procedure was unreliable and time consuming; the latter two showed better correlation with hemacytometer total cell counts and required significantly less time. PMID:7107858

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

    PubMed

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

    2011-01-01

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

  10. 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.

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

    PubMed

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

    2015-01-01

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

  12. Reduced cell viability and apoptosis induction in human thyroid carcinoma and mesothelioma cells exposed to cidofovir.

    PubMed

    Catalani, Simona; Palma, Francesco; Battistelli, Serafina; Nuvoli, Barbara; Galati, Rossella; Benedetti, Serena

    2017-02-20

    Besides its well-recognized antiviral activity, Cidofovir (CDV) has been shown to exert anticancer properties both within in vitro and in vivo models. The aim of this study was to evaluate the effects of CDV on still unexplored cultured cancer cells from human mesothelioma as well as breast, colon, liver, lung, prostate, and thyroid carcinomas. Overall, a dose- and time-dependent inhibition of cell viability was observed after CDV exposure. To clarify the mechanisms underlying CDV action, apoptotic cell death was investigated in two infected cell lines [Ist-Mes1 and Ist-Mes2 mesothelioma cells (SV40+)] and in two uninfected cell lines (NCI-H2425 mesothelioma cells and FTC-133 thyroid cancer cells), which resulted the most sensitive to CDV treatment. Reduced expression of procaspase-3 and increased expression of PARP p85 fragment were observed in both infected and uninfected mesothelioma cells, indicating apoptosis induction by CDV in a virus-independent manner. Similarly, the increase of the pro-apoptotic proteins p53, cytochrome c and caspase-3, the decrease of the survival protein Bcl-x, and the increment of Bax/Bcl-2 ratio revealed the occurrence of apoptosis in CDV-treated FTC-133. The presence of nuclear DNA fragmentation confirmed apoptotic cell death by CDV. Overall, our findings warrant further investigations to explore the therapeutic potential of CDV for human mesothelioma and follicular thyroid carcinoma.

  13. The xCELLigence system for real-time and label-free monitoring of cell viability.

    PubMed

    Ke, Ning; Wang, Xiaobo; Xu, Xiao; Abassi, Yama A

    2011-01-01

    We describe here the use of the xCELLigence system for label-free and real-time monitoring of cell -viability. The xCELLigence system uses specially designed microtiter plates containing interdigitated gold microelectrodes to noninvasively monitor the viability of cultured cells using electrical impedance as the readout. The continuous monitoring of cell viability by the xCELLigence system makes it possible to distinguish between different perturbations of cell viability, such as senescence, cell toxicity (cell death), and reduced proliferation (cell cycle arrest). In addition, the time resolution of the xCELLigence system allows for the determination of optimal time points to perform standard cell viability assays as well as other end-point assays to understand the mode of action. We have used the WST-1 assay (end-point viability readout), the cell index determination (continuous monitoring of viability by xCELLigence), and the DNA fragmentation assay (end-point apoptosis assay) to systematically examine cytotoxic effects triggered by two cytotoxic compounds with different cell-killing kinetics. Good correlation was observed for viability readouts between WST-1 and cell index. The significance of time resolution by xCELLigence readout is exemplified by its ability to pinpoint the optimal time points for conducting end point viability and apoptosis assays.

  14. Differential regulation of autophagy and cell viability by ceramide species.

    PubMed

    Cruickshanks, Nichola; Roberts, Jane L; Bareford, M Danielle; Tavallai, Mehrad; Poklepovic, Andrew; Booth, Laurence; Spiegel, Sarah; Dent, Paul

    2015-01-01

    The present studies sought to determine whether the anti-folate pemetrexed (Alimta) and the sphingosine-1-phosphate receptor modulator FTY720 (Fingolimod, Gilenya) interacted to kill tumor cells. FTY720 and pemetrexed interacted in a greater than additive fashion to kill breast, brain and colorectal cancer cells. Loss of p53 function weakly enhanced the toxicity of FTY720 whereas deletion of activated RAS strongly or expression of catalytically inactive AKT facilitated killing. Combined drug exposure reduced the activity of AKT, p70 S6K and mTOR and activated JNK and p38 MAPK. Expression of activated forms of AKT, p70 S6K and mTOR or inhibition of JNK and p38 MAPK suppressed the interaction between FTY720 and pemetrexed. Treatment of cells with FTY720 and pemetrexed increased the numbers of early autophagosomes but not autolysosomes, which correlated with increased LC3II processing and increased p62 levels, suggestive of stalled autophagic flux. Knock down of ATG5 or Beclin1 suppressed autophagosome formation and cell killing. Knock down of ceramide synthase 6 suppressed autophagosome production and cell killing whereas knock down of ceramide synthase 2 enhanced vesicle formation and facilitated death. Collectively our findings argue that pemetrexed and FTY720 could be a novel adjunct modality for breast cancer treatment.

  15. Differential regulation of autophagy and cell viability by ceramide species

    PubMed Central

    Cruickshanks, Nichola; Roberts, Jane L; Bareford, M Danielle; Tavallai, Mehrad; Poklepovic, Andrew; Booth, Laurence; Spiegel, Sarah; Dent, Paul

    2015-01-01

    The present studies sought to determine whether the anti-folate pemetrexed (Alimta) and the sphingosine-1-phosphate receptor modulator FTY720 (Fingolimod, Gilenya) interacted to kill tumor cells. FTY720 and pemetrexed interacted in a greater than additive fashion to kill breast, brain and colorectal cancer cells. Loss of p53 function weakly enhanced the toxicity of FTY720 whereas deletion of activated RAS strongly or expression of catalytically inactive AKT facilitated killing. Combined drug exposure reduced the activity of AKT, p70 S6K and mTOR and activated JNK and p38 MAPK. Expression of activated forms of AKT, p70 S6K and mTOR or inhibition of JNK and p38 MAPK suppressed the interaction between FTY720 and pemetrexed. Treatment of cells with FTY720 and pemetrexed increased the numbers of early autophagosomes but not autolysosomes, which correlated with increased LC3II processing and increased p62 levels, suggestive of stalled autophagic flux. Knock down of ATG5 or Beclin1 suppressed autophagosome formation and cell killing. Knock down of ceramide synthase 6 suppressed autophagosome production and cell killing whereas knock down of ceramide synthase 2 enhanced vesicle formation and facilitated death. Collectively our findings argue that pemetrexed and FTY720 could be a novel adjunct modality for breast cancer treatment. PMID:25803131

  16. 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.

  17. Verification of cell viability in bioengineered tissues and organs before clinical transplantation.

    PubMed

    Jungebluth, Philipp; Haag, Johannes C; Lim, Mei L; Lemon, Greg; Sjöqvist, Sebastian; Gustafsson, Ylva; Ajalloueian, Fatemeh; Gilevich, Irina; Simonson, Oscar E; Grinnemo, Karl H; Corbascio, Matthias; Baiguera, Silvia; Del Gaudio, Costantino; Strömblad, Staffan; Macchiarini, Paolo

    2013-05-01

    The clinical outcome of transplantations of bioartificial tissues and organs depends on the presence of living cells. There are still no standard operative protocols that are simple, fast and reliable for confirming the presence of viable cells on bioartificial scaffolds prior to transplantation. By using mathematical modeling, we have developed a colorimetric-based system (colorimetric scale bar) to predict the cell viability and density for sufficient surface coverage. First, we refined a method which can provide information about cell viability and numbers in an in vitro setting: i) immunohistological staining by Phalloidin/DAPI and ii) a modified colorimetric cell viability assay. These laboratory-based methods and the developed colorimetric-based system were then validated in rat transplantation studies of unseeded and seeded tracheal grafts. This was done to provide critical information on whether the graft would be suitable for transplantation or if additional cell seeding was necessary. The potential clinical impact of the colorimetric scale bar was confirmed using patient samples. In conclusion, we have developed a robust, fast and reproducible colorimetric tool that can verify and warrant viability and integrity of an engineered tissue/organ prior to transplantation. This should facilitate a successful transplantation outcome and ensure patient safety.

  18. Adipose Derived-Mesenchymal Stem Cells Viability and Differentiating Features for Orthopaedic Reparative Applications: Banking of Adipose Tissue

    PubMed Central

    Alotto, Daniela; Belisario, Dimas Carolina; Casarin, Stefania; Fumagalli, Mara; Cambieri, Irene; Piana, Raimondo; Stella, Maurizio; Ferracini, Riccardo; Castagnoli, Carlotta

    2016-01-01

    Osteoarthritis is characterized by loss of articular cartilage also due to reduced chondrogenic activity of mesenchymal stem cells (MSCs) from patients. Adipose tissue is an attractive source of MSCs (ATD-MSCs), representing an effective tool for reparative medicine, particularly for treatment of osteoarthritis, due to their chondrogenic and osteogenic differentiation capability. The treatment of symptomatic knee arthritis with ATD-MSCs proved effective with a single infusion, but multiple infusions could be also more efficacious. Here we studied some crucial aspects of adipose tissue banking procedures, evaluating ATD-MSCs viability, and differentiation capability after cryopreservation, to guarantee the quality of the tissue for multiple infusions. We reported that the presence of local anesthetic during lipoaspiration negatively affects cell viability of cryopreserved adipose tissue and cell growth of ATD-MSCs in culture. We observed that DMSO guarantees a faster growth of ATD-MSCs in culture than trehalose. At last, ATD-MSCs derived from fresh and cryopreserved samples at −80°C and −196°C showed viability and differentiation ability comparable to fresh samples. These data indicate that cryopreservation of adipose tissue at −80°C and −196°C is equivalent and preserves the content of ATD-MSCs in Stromal Vascular Fraction (SVF), guaranteeing the differentiation ability of ATD-MSCs. PMID:28018432

  19. Effects of cigarette smoke on cell viability, linoleic acid metabolism and cholesterol synthesis, in THP-1 cells.

    PubMed

    Ghezzi, Silvia; Risé, Patrizia; Ceruti, Stefania; Galli, Claudio

    2007-07-01

    Cigarette smoke (CS) contains thousands of substances, mainly free radicals that have as a target the polyunsaturated fatty acids (PUFA). Long chain PUFA are produced through elongation and desaturation reactions from their precursors; the desaturation reactions are catalyzed by different enzymes: the conversion of 18:2n-6 (linoleic acid, LA) to 18:3n-6 by Delta6 desaturase, while that of 20:3n-6 to 20:4n-6 by Delta5 desaturase. The aim of this work is to evaluate the effect of serum exposed to cigarette smoke (SE-FBS) on (1) cell viability and proliferation, (2) [1-(14)C] LA conversion and desaturase activities in THP-1 cells, a monocytic cell line. In THP-1, CS inhibits cell proliferation dose-dependently, by producing a modification in the cell cycle with a reduced number of cells in synthesis and mitosis phases at higher concentrations. CS also decreases [1-(14)C] LA conversion to its derivatives in a concentration-dependent manner, inhibiting the activities of Delta6 and mainly Delta5 desaturase. In addition, CS does not modify the incorporation of LA into various lipid classes but it reduces cholesterol synthesis from radiolabelled acetate, and increases free fatty acid, TG and CE levels. In conclusion, CS affects lipid metabolism, inhibiting LA conversion and desaturase activities. CS also shifts the "de novo" lipid synthesis from free cholesterol to TG and CE, where LA is preferentially esterified.

  20. Inhibitory effects of mitotane on viability and secretory activity in mouse gonadotroph cell lines.

    PubMed

    Gentilin, Erica; Molè, Daniela; Gagliano, Teresa; Minoia, Mariella; Ambrosio, Maria Rosaria; Degli Uberti, Ettore C; Zatelli, Maria Chiara

    2014-06-01

    Mitotane represents the mainstay medical treatment for metastatic, inoperable or recurrent adrenocortical carcinoma. Besides the well-known adverse events, mitotane therapy is associated also with endocrinological effects, including sexual and reproductive dysfunction. The majority of male patients undergoing adjuvant mitotane therapy show a picture of hypogonadism, characterized by low free testosterone and high sex hormone binding globulin levels and unmodified LH concentrations. Since mitotane has been shown to have direct pituitary effects, we investigated whether mitotane may influence both cell viability and function of gonadotroph cells in the settings of two pituitary cell lines. We found that mitotane reduces cell viability, induces apoptosis, modifies cell cycle phase distribution and secretion of gonadotroph cells. The present data strengthen previous evidence showing a direct mitotane effect at pituitary level and represent a possible explanation of the lack of LH increase following decrease in free testosterone in patients undergoing adjuvant mitotane therapy.

  1. 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.

  2. Cell viability score (CVS) as a good indicator of critical concentration of benzalkonium chloride for toxicity in cultured ocular surface cell lines.

    PubMed

    Iwasawa, Atsuo; Ayaki, Masahiko; Niwano, Yoshimi

    2013-07-01

    Cytotoxicity of benzalkonium chloride (BAK) is a major factor affecting drug cytotoxicity. This study aimed to determine the critical concentration of BAK for cultured ocular cells, using SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells). Cell viability was determined following the exposure of cells to 11 concentrations of BAK for 10, 30, or 60 min using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and neutral red assays, and the cell viability score (CVS) was used to evaluate comprehensively the toxicity of BAK. The CVS system consists of two values. The CVS50 was determined by the number of measurements for viability ≥50% of control. The CVS40/80 was calculated as follows: CVS40/80=(number of measurements for viability values >80%)-(number of measurements for viability values <40%). Both %CVS50 and %CVS40/80 decreased with concentrations of BAK. When BAK concentrations were 0.01% or higher, %CVS50 and %CVS40/80 became 0 and less than -90, respectively. Meanwhile, when BAK concentrations were 0.001% or lower, %CVS50 became 100. In the case of %CVS40/80, when the BAK concentrations were 0.002% or lower, the values reached 75 or more, and when 0.0005% or lower, the %CVS40/80 value reached 100. Accordingly, BAK induced very low cytotoxicity in the cultured ocular cell lines at concentrations of 0.002% or lower. The concentration-dependency confirmed that the CVS score is useful for expressing drug cytotoxicity in a simple and comprehensive manner.

  3. Effect of culture age, protectants, and initial cell concentration on viability of freeze-dried cells of Metschnikowia pulcherrima.

    PubMed

    Spadaro, Davide; Ciavorella, Annalisa Alessandra; Lopez-Reyes, Jorge Giovanny; Garibaldi, Angelo; Gullino, Maria Lodovica

    2010-10-01

    The effect of freeze-drying using different lyoprotectants at different concentrations on the viability and biocontrol efficacy of Metschnikowia pulcherrima was evaluated. The effects of initial yeast cell concentration and culture age on viability were also considered. Yeast cells grown for 36 h were more resistant to freeze-drying than were 48 h cells. An initial concentration of 10⁸ cells·mL⁻¹ favoured the highest survival after freeze-drying. When maltose (25%, m/v) was used as protectant, a high cell viability was obtained (64.2%). Cells maintained a high viability after 6 months of storage at 4 °C. The biocontrol efficacy of freeze-dried cells was similar to the activity of fresh cells on 'Gala' apples and was slightly lower on 'Golden Delicious' apples. After optimizing freeze-drying conditions, the viability of M. pulcherrima cells was similar to that obtained in other studies. The results constitute a first step towards the commercial development of M. pulcherrima as a biocontrol agent.

  4. β-escin selectively targets the glioblastoma-initiating cell population and reduces cell viability

    PubMed Central

    Harford-Wright, Elizabeth; Bidère, Nicolas; Gavard, Julie

    2016-01-01

    Glioblastoma multiforme (GBM) is a highly aggressive tumour of the central nervous system and is associated with an extremely poor prognosis. Within GBM exists a subpopulation of cells, glioblastoma-initiating cells (GIC), which possess the characteristics of progenitor cells, have the ability to initiate tumour growth and resist to current treatment strategies. We aimed at identifying novel specific inhibitors of GIC expansion through use of a large-scale chemical screen of approved small molecules. Here, we report the identification of the natural compound β-escin as a selective inhibitor of GIC viability. Indeed, β-escin was significantly cytotoxic in nine patient-derived GIC, whilst exhibiting no substantial effect on the other human cancer or control cell lines tested. In addition, β-escin was more effective at reducing GIC growth than current clinically used cytotoxic agents. We further show that β-escin triggers caspase-dependent cell death combined with a loss of stemness properties. However, blocking apoptosis could not rescue the β-escin-induced reduction in sphere formation or stemness marker activity, indicating that β-escin directly modifies the stem identity of GIC, independent of the induction of cell death. Thus, this study has repositioned β-escin as a promising potential candidate to selectively target the aggressive population of initiating cells within GBM. PMID:27589691

  5. Influence of lycopene on cell viability, cell cycle, and apoptosis of human prostate cancer and benign hyperplastic cells.

    PubMed

    Soares, Nathalia da Costa Pereira; Teodoro, Anderson Junger; Oliveira, Felipe Leite; Santos, Carlos Antonio do Nascimento; Takiya, Christina Maeda; Junior, Oswaldo Saback; Bianco, Mario; Junior, Antonio Palumbo; Nasciutti, Luiz Eurico; Ferreira, Luciana Bueno; Gimba, Etel Rodrigues Pereira; Borojevic, Radovan

    2013-01-01

    Prostate cancer is the most common malignancy in men and the second leading cause of cancer-related mortality in men of the Western world. Lycopene has received attention because of its expcted potential to prevent cancer. In the present study, we evaluated the influence of lycopene on cell viability, cell cycle, and apoptosis of human prostate cancer cells and benign prostate hyperplastic cells. Using MTT assay, we observed a decrease of cell viability in all cancer cell lines after treatment with lycopene, which decreased the percentage of cells in G0/G1 phase and increased in S and G2/M phases after 96 h of treatment in metastatic prostate cancer cell lineages. Flow citometry analysis of cell cycle revealed lycopene promoted cell cycle arrest in G0/G1 phase after 48 and 96 h of treatment in a primary cancer cell line. Using real time PCR assay, lycopene also induced apoptosis in prostate cancer cells with altered gene expression of Bax and Bcl-2. No effect was observed in benign prostate hyperplasia cells. These results suggest an effect of lycopene on activity of human prostate cancer cells.

  6. A microfluidic-based cell encapsulation platform to achieve high long-term cell viability in photopolymerized PEGNB hydrogel microspheres.

    PubMed

    Jiang, Zhongliang; Xia, Bingzhao; McBride, Ralph; Oakey, John

    2017-01-07

    Cell encapsulation within photopolymerized polyethylene glycol (PEG)-based hydrogel scaffolds has been demonstrated as a robust strategy for cell delivery, tissue engineering, regenerative medicine, and developing in vitro platforms to study cellular behavior and fate. Strategies to achieve spatial and temporal control over PEG hydrogel mechanical properties, chemical functionalization, and cytocompatibility have advanced considerably in recent years. Recent microfluidic technologies have enabled the miniaturization of PEG hydrogels, thus enabling the fabrication of miniaturized cell-laden vehicles. However, rapid oxygen diffusive transport times on the microscale dramatically inhibit chain growth photopolymerization of polyethylene glycol diacrylate (PEGDA), thus decreasing the viability of cells encapsulated within these microstructures. Another promising PEG-based scaffold material, PEG norbornene (PEGNB), is formed by a step-growth photopolymerization and is not inhibited by oxygen. PEGNB has also been shown to be more cytocompatible than PEGDA and allows for orthogonal addition reactions. The step-growth kinetics, however, are slow and therefore challenging to fully polymerize within droplets flowing through microfluidic devices. Here, we describe a microfluidic-based droplet fabrication platform that generates consistently monodisperse cell-laden water-in-oil emulsions. Microfluidically generated PEGNB droplets are collected and photopolymerized under UV exposure in bulk emulsions. In this work, we compare this microfluidic-based cell encapsulation platform with a vortex-based method on the basis of microgel size, uniformity, post-encapsulation cell viability and long-term cell viability. Several factors that influence post-encapsulation cell viability were identified. Finally, long-term cell viability achieved by this platform was compared to a similar cell encapsulation platform using PEGDA. We show that this PEGNB microencapsulation platform is capable of

  7. 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.

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

    PubMed

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

    2014-08-01

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

  9. Evaluation of cell viability and functionality in vessel-like bioprintable cell-laden tubular channels.

    PubMed

    Yu, Yin; Zhang, Yahui; Martin, James A; Ozbolat, Ibrahim T

    2013-09-01

    Organ printing is a novel concept recently introduced in developing artificial three-dimensional organs to bridge the gap between transplantation needs and organ shortage. One of the major challenges is inclusion of blood-vessellike channels between layers to support cell viability, postprinting functionality in terms of nutrient transport, and waste removal. In this research, we developed a novel and effective method to print tubular channels encapsulating cells in alginate to mimic the natural vascular system. An experimental investigation into the influence on cartilage progenitor cell (CPCs) survival, and the function of printing parameters during and after the printing process were presented. CPC functionality was evaluated by checking tissue-specific genetic marker expression and extracellular matrix production. Our results demonstrated the capability of direct fabrication of cell-laden tubular channels by our newly designed coaxial nozzle assembly and revealed that the bioprinting process could induce quantifiable cell death due to changes in dispensing pressure, coaxial nozzle geometry, and biomaterial concentration. Cells were able to recover during incubation, as well as to undergo differentiation with high-level cartilage-associated gene expression. These findings may not only help optimize our system but also can be applied to biomanufacturing of 3D functional cellular tissue engineering constructs for various organ systems.

  10. 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.

  11. 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.

  12. Luteolin inhibits lung metastasis, cell migration, and viability of triple-negative breast cancer cells

    PubMed Central

    Cook, Matthew T; Liang, Yayun; Besch-Williford, Cynthia; Hyder, Salman M

    2017-01-01

    Most breast cancer-related deaths from triple-negative breast cancer (TNBC) occur following metastasis of cancer cells and development of tumors at secondary sites. Because TNBCs lack the three receptors targeted by current chemotherapeutic regimens, they are typically treated with extremely aggressive and highly toxic non-targeted treatment strategies. Women with TNBC frequently develop metastatic lesions originating from drug-resistant residual cells and have poor prognosis. For this reason, novel therapeutic strategies that are safer and more effective are sought. Luteolin (LU) is a naturally occurring, non-toxic plant compound that has proven effective against several types of cancer. With this in mind, we conducted in vivo and in vitro studies to determine whether LU might suppress metastasis of TNBC. In an in vivo mouse metastasis model, LU suppressed metastasis of human MDA-MB-435 and MDA-MB-231 (4175) LM2 TNBC cells to the lungs. In in vitro assays, LU inhibited cell migration and viability of MDA-MB-435 and MDA-MB-231 (4175) LM2 cells. Further, LU induced apoptosis in MDA-MB-231 (4175) LM2 cells. Relatively low levels (10 µM) of LU significantly inhibited vascular endothelial growth factor (VEGF) secretion in MDA-MB-231 (4175) LM2 cells, suggesting that it has the ability to suppress a potent angiogenic and cell survival factor. In addition, migration of MDA-MB-231 (4175) LM2 cells was inhibited upon exposure to an antibody against the VEGF receptor, KDR, but not by exposure to a VEGF165 antibody. Collectively, these data suggest that the anti-metastatic properties of LU may, in part, be due to its ability to block VEGF production and KDR-mediated activity, thereby inhibiting tumor cell migration. These studies suggest that LU deserves further investigation as a potential treatment option for women with TNBC. PMID:28096694

  13. 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.

  14. 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

  15. 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.

  16. Monocarboxylate transporter 8 modulates the viability and invasive capacity of human placental cells and fetoplacental growth in mice.

    PubMed

    Vasilopoulou, Elisavet; Loubière, Laurence S; Heuer, Heike; Trajkovic-Arsic, Marija; Darras, Veerle M; Visser, Theo J; Lash, Gendie E; Whitley, Guy S; McCabe, Christopher J; Franklyn, Jayne A; Kilby, Mark D; Chan, Shiao Y

    2013-01-01

    Monocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation, with increased expression in trophoblast cells from growth-restricted pregnancies. We postulate that MCT8 plays an important role in placental development and transplacental TH transport. We investigated the effect of altering MCT8 expression in human trophoblast in vitro and in a Mct8 knockout mouse model. Silencing of endogenous MCT8 reduced T3 uptake into human extravillous trophoblast-like cells (SGHPL-4; 40%, P<0.05) and primary cytotrophoblast (15%, P<0.05). MCT8 over-expression transiently increased T3 uptake (SGHPL-4∶30%, P<0.05; cytotrophoblast: 15%, P<0.05). Silencing MCT8 did not significantly affect SGHPL-4 invasion, but with MCT8 over-expression T3 treatment promoted invasion compared with no T3 (3.3-fold; P<0.05). Furthermore, MCT8 silencing increased cytotrophoblast viability (∼20%, P<0.05) and MCT8 over-expression reduced cytotrophoblast viability independently of T3 (∼20%, P<0.05). In vivo, Mct8 knockout reduced fetal:placental weight ratios compared with wild-type controls at gestational day 18 (25%, P<0.05) but absolute fetal and placental weights were not significantly different. The volume fraction of the labyrinthine zone of the placenta, which facilitates maternal-fetal exchange, was reduced in Mct8 knockout placentae (10%, P<0.05). However, there was no effect on mouse placental cell proliferation in vivo. We conclude that MCT8 makes a significant contribution to T3 uptake into human trophoblast cells and has a role in modulating human trophoblast cell invasion and viability. In mice, Mct8 knockout has subtle effects upon fetoplacental growth and does not significantly affect placental cell viability probably due to compensatory mechanisms in vivo.

  17. Monocarboxylate Transporter 8 Modulates the Viability and Invasive Capacity of Human Placental Cells and Fetoplacental Growth in Mice

    PubMed Central

    Vasilopoulou, Elisavet; Loubière, Laurence S.; Heuer, Heike; Trajkovic-Arsic, Marija; Darras, Veerle M.; Visser, Theo J.; Lash, Gendie E.; Whitley, Guy S.; McCabe, Christopher J.; Franklyn, Jayne A.; Kilby, Mark D.; Chan, Shiao Y.

    2013-01-01

    Monocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation, with increased expression in trophoblast cells from growth-restricted pregnancies. We postulate that MCT8 plays an important role in placental development and transplacental TH transport. We investigated the effect of altering MCT8 expression in human trophoblast in vitro and in a Mct8 knockout mouse model. Silencing of endogenous MCT8 reduced T3 uptake into human extravillous trophoblast-like cells (SGHPL-4; 40%, P<0.05) and primary cytotrophoblast (15%, P<0.05). MCT8 over-expression transiently increased T3 uptake (SGHPL-4∶30%, P<0.05; cytotrophoblast: 15%, P<0.05). Silencing MCT8 did not significantly affect SGHPL-4 invasion, but with MCT8 over-expression T3 treatment promoted invasion compared with no T3 (3.3-fold; P<0.05). Furthermore, MCT8 silencing increased cytotrophoblast viability (∼20%, P<0.05) and MCT8 over-expression reduced cytotrophoblast viability independently of T3 (∼20%, P<0.05). In vivo, Mct8 knockout reduced fetal:placental weight ratios compared with wild-type controls at gestational day 18 (25%, P<0.05) but absolute fetal and placental weights were not significantly different. The volume fraction of the labyrinthine zone of the placenta, which facilitates maternal-fetal exchange, was reduced in Mct8 knockout placentae (10%, P<0.05). However, there was no effect on mouse placental cell proliferation in vivo. We conclude that MCT8 makes a significant contribution to T3 uptake into human trophoblast cells and has a role in modulating human trophoblast cell invasion and viability. In mice, Mct8 knockout has subtle effects upon fetoplacental growth and does not significantly affect placental cell viability probably due to compensatory mechanisms in vivo. PMID

  18. Post-thaw viability of cryopreserved hematopoietic progenitor cell grafts: does it matter?

    PubMed

    Vrhovac, Radovan; Perić, Zinaida; Jurenec, Silvana; Kardum-Skelin, Ika; Jelić-Puskarić, Biljana; Jaksić, Branimir

    2010-03-01

    Cell viability in peripheral blood progenitor cell (PBPC) grafts and its influence on the clinical course following transplantation was evaluated in 81 consecutive transplantations (72 autologous, 9 allogeneic) performed in patients with hematological diseases. Viability of cells in PBPC grafts immediately upon collection was 98.6 +/- 3.5%, after addition of dimethyl sulfoxide (DMSO) 73.3 +/- 21.8%, and post-thaw 65.2 +/- 16.1%. It did not differ significantly between patients with different diagnoses, gender, age, type of priming used, dose of G-CSF administered or number of CD34+ cells collected. However grafts stored for more than 60 days showed lower post-thaw viability compared to the ones thawed in the 60 days following cryopreservation (56.61 +/- 15.2% vs. 67.6 +/- 15.5%, p = 0.04). Post-thaw graft viability did not influence engraftment time, but there was a predisposition towards infectious complications in the post-transplant period in patients receiving grafts with lower percentage of viable cells. They developed febrile neutropenia more often (72.2% vs. 50% of patients, p = 0.05) and had more febrile days (2.4 +/- 2.6 vs. 1.5 +/- 2.3, p = 0.05) following transplantation. We have demonstrated that PBPC grafts are capable of long term engraftment regardless of the graft storage time or percentage of viable cells post-thaw, which confirms the robustness of CD34+ cells during the freeze/thaw procedures carried out in daily clinical practice. Granulocyte concentration in PBPC grafts could have an influence on infectious complications following transplantation and needs to be further investigated on a larger number of patients.

  19. 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.

  20. Effect of bioink properties on printability and cell viability for 3D bioplotting of embryonic stem cells.

    PubMed

    Ouyang, Liliang; Yao, Rui; Zhao, Yu; Sun, Wei

    2016-09-16

    3D cell printing is an emerging technology for fabricating complex cell-laden constructs with precise and pre-designed geometry, structure and composition to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. This technology enables spatial manipulation of cells and biomaterials, also referred to as 'bioink', and thus allows study of cellular interactions in a 3D microenvironment and/or in the formation of functional tissues and organs. Recently, many efforts have been made to develop new bioinks and to apply more cell sources for better biocompatibility and biofunctionality. However, the influences of printing parameters on the shape fidelity of 3D constructs as well as on cell viability after the cell printing process have been poorly characterized. Furthermore, parameter optimization based on a specific cell type might not be suitable for other types of cells, especially cells with high sensibility. In this study, we systematically studied the influence of bioink properties and printing parameters on bioink printability and embryonic stem cell (ESC) viability in the process of extrusion-based cell printing, also known as bioplotting. A novel method was established to determine suitable conditions for bioplotting ESCs to achieve both good printability and high cell viability. The rheological properties of gelatin/alginate bioinks were evaluated to determine the gelation properties under different bioink compositions, printing temperatures and holding times. The bioink printability was characterized by a newly developed semi-quantitative method. The results demonstrated that bioinks with longer gelation times would result in poorer printability. The live/dead assay showed that ESC viability increased with higher printing temperatures and lower gelatin concentrations. Furthermore, an exponential relationship was obtained between ESC viability and induced shear stress. By defining the proper printability and

  1. Relative impact of 3- and 5-hydroxyl groups of cytosporone B on cancer cell viability.

    PubMed

    Xia, Zebin; Cao, Xihua; Rico-Bautista, Elizabeth; Yu, Jinghua; Chen, Liqun; Chen, Jiebo; Bobkov, Andrey; Wolf, Dieter A; Zhang, Xiao-Kun; Dawson, Marcia I

    2013-02-01

    A novel and the shortest route, thus far, for preparing cytosporone B (Csn-B) is reported. Csn-B and two analogs were used to probe the importance of hydroxyl groups at the 3- and 5-positions of the Csn-B benzene ring in inhibiting the viability of human H460 lung cancer and LNCaP prostate cancer cells, inducing H460 cell apoptosis, and interacting with the NR4A1 (TR3) ligand-binding domain (LBD). These studies indicate that Csn-B and 5-Me-Csn-B, having a phenolic hydroxyl at the 3-position of their aromatic rings, had similar activities in inhibiting cancer cell viability and in inducing apoptosis, whereas 3,5-(Me)2-Csn-B was unable to do so. These results are in agreement with ligand-binding experiments showing that the interaction with the NR4A1 LBD required the presence of the 3-hydroxyl group.

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

    PubMed

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

    2015-01-01

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

  3. 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

  4. An exploration of plastic deformation dependence of cell viability and adhesion in metallic implant materials.

    PubMed

    Uzer, B; Toker, S M; Cingoz, A; Bagci-Onder, T; Gerstein, G; Maier, H J; Canadinc, D

    2016-07-01

    The relationship between cell viability and adhesion behavior, and micro-deformation mechanisms was investigated on austenitic 316L stainless steel samples, which were subjected to different amounts of plastic strains (5%, 15%, 25%, 35% and 60%) to promote a variety in the slip and twin activities in the microstructure. Confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FESEM) revealed that cells most favored the samples with the largest plastic deformation, such that they spread more and formed significant filopodial extensions. Specifically, brain tumor cells seeded on the 35% deformed samples exhibited the best adhesion performance, where a significant slip activity was prevalent, accompanied by considerable slip-twin interactions. Furthermore, maximum viability was exhibited by the cells seeded on the 60% deformed samples, which were particularly designed in a specific geometry that could endure greater strain values. Overall, the current findings open a new venue for the production of metallic implants with enhanced biocompatibility, such that the adhesion and viability of the cells surrounding an implant can be optimized by tailoring the surface relief of the material, which is dictated by the micro-deformation mechanism activities facilitated by plastic deformation imposed by machining.

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

    PubMed

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

    2009-06-01

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

  6. Differential effects of single-walled carbon nanotubes on cell viability of human lung and pharynx carcinoma cell lines.

    PubMed

    Hitoshi, Kotaro; Katoh, Miki; Suzuki, Tomoko; Ando, Yoshinori; Nadai, Masayuki

    2011-06-01

    Carbon nanotubes (CNTs) are attracting significant attention as a novel material for future innovations. Many in vitro studies have assessed the cytotoxicity of CNTs, but the effects of CNTs differ depending on the cell lines and the synthetic method adopted for fabricating CNTs. In the present study, the differential effects of single-walled CNTs (SWCNTs) on the cell viability of A549 cells from human lung carcinomas and FaDu cells from human head and neck carcinomas were investigated. The SWCNTs used in the present study were synthesized with nickel and yttrium (SO-SWCNTs), and iron (FH-P-SWCNTs) as catalysts. Cell viability was evaluated on the basis of cell-membrane biomass, adenosine triphosphate (ATP) content, and intracellular metabolic capacity. After 24-hr exposure of A549 and FaDu cells to 1.0 mg/ml SO-SWCNTs, the cell-membrane biomass of A549 cells decreased to 43% as compared to the control cells, whereas that of FaDu cells remained over 90%. After 24-hr exposure of A549 and FaDu cells to 1.0 mg/ml SO-SWCNT, the intracellular metabolic capacity decreased to 24% and 37%, respectively, and the ATP content decreased to 40% and 54%, respectively. SWCNTs had a greater impact on the viability values of A549 cells than on those of FaDu cells. In addition, cells exposed to FH-P-SWCNTs exhibited a higher viability than those exposed to SO-SWCNTs. Caspase 3/7 activity was not increased at maximum concentration of 1.0 mg/ml SO-SWCNTs. It was surmised that sensitivity to SWCNTs differs among the 2 cell lines; additionally, SWCNT characteristics may produce different effects on these cell lines.

  7. 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

  8. Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.

    PubMed

    Duellman, Sarah J; Zhou, Wenhui; Meisenheimer, Poncho; Vidugiris, Gediminas; Cali, James J; Gautam, Prson; Wennerberg, Krister; Vidugiriene, Jolanta

    2015-10-01

    Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z' = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.

  9. Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening

    PubMed Central

    Zhou, Wenhui; Meisenheimer, Poncho; Vidugiris, Gediminas; Cali, James J.; Gautam, Prson; Wennerberg, Krister; Vidugiriene, Jolanta

    2015-01-01

    Abstract Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z′ = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples. PMID:26383544

  10. Priming Equine Bone Marrow-Derived Mesenchymal Stem Cells with Proinflammatory Cytokines: Implications in Immunomodulation-Immunogenicity Balance, Cell Viability, and Differentiation Potential.

    PubMed

    Barrachina, Laura; Remacha, Ana Rosa; Romero, Antonio; Vázquez, Francisco José; Albareda, Jorge; Prades, Marta; Gosálvez, Jaime; Roy, Rosa; Zaragoza, Pilar; Martín-Burriel, Inmaculada; Rodellar, Clementina

    2017-01-01

    Mesenchymal stem cells (MSCs) have a great potential for treating equine musculoskeletal injuries. Although their mechanisms of action are not completely known, their immunomodulatory properties appear to be key in their functions. The expression of immunoregulatory molecules by MSCs is regulated by proinflammatory cytokines; so inflammatory priming of MSCs might improve their therapeutic potential. However, inflammatory environment could also increase MSC immunogenicity and decrease MSC viability and differentiation capacity. The aim of this study was to assess the effect of cytokine priming on equine bone marrow-derived MSC (eBM-MSC) immunoregulation, immunogenicity, viability, and differentiation potential, to enhance MSC immunoregulatory properties, without impairing their immune-evasive status, viability, and plasticity. Equine BM-MSCs (n = 4) were exposed to 5 ng/mL of TNFα and IFNγ for 12 h (CK5-priming). Subsequently, expression of genes coding for immunomodulatory, immunogenic, and apoptosis-related molecules was analyzed by real-time quantitative polymerase chain reaction. Chromatin integrity and proliferation assays were assessed to evaluate cell viability. Trilineage differentiation was evaluated by specific staining and gene expression. Cells were reseeded in a basal medium for additional 7 days post-CK5 to elucidate if priming-induced changes were maintained along the time. CK5-priming led to an upregulation of immunoregulatory genes IDO, iNOS, IL-6, COX-2, and VCAM-1. MHC-II and CD40 were also upregulated, but no change in other costimulatory molecules was observed. These changes were not maintained 7 days after CK5-priming. Viability and differentiation potential were maintained after CK5-priming. These findings suggest that CK5-priming of eBM-MSCs could improve their in vivo effectiveness without affecting other eBM-MSC properties.

  11. Evaluation of Cell Viability Dyes in Antiviral Assays with RNA Viruses that Exhibit Different Cytopathogenic Properties.

    PubMed

    Smee, Donald F; Hurst, Brett L; Evans, W Joseph; Clyde, Nathan; Wright, Sean; Peterson, Christopher; Jung, Kie-Hoon; Day, Craig W

    2017-03-27

    Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic properties. Dyes included alamarBlue(®) measured by absorbance (ALB-A) and fluorescence (ALB-F), neutral red (NR), Viral ToxGlo™ (VTG), and WST-1. Viruses were chikungunya, dengue type 2, and Junin, which generally cause 100, 80-90, and 50% maximal cytopathic effect (CPE), respectively, in Vero 76. Compounds evaluated were 6-azauridine, BCX-4430, 3-deazaguanine, EICAR, favipiravir, infergen, mycophenolic acid (MPA), ribavirin, and tiazofurin. The 50% virus-inhibitory (EC50) values for each inhibitor and virus combination did not vary significantly based on the dye used. However, dyes varied in distinguishing the vitality of virus-infected cultures when not all cells were killed by virus infection. For example, VTG uptake into dengue-infected cells was nearly 50% when visual examination showed only 10-20% cell survival. ALB-A measured infected cell viability differently than ALB-F as follows: 16% versus 32% (dengue-infected), respectively, and 51% versus 72% (Junin-infected), respectively. Cytotoxicity (CC50) assays with dyes in uninfected proliferating cells produced similar CC50 values for EICAR (1.5-8.9μM) and MPA (0.8-2.5μM). 6-Azauridine toxicity was 6.1-17.5μM with NR, VTG, and WST-1, compared to 48-92μM with ALB-A and ALB-F.(P<0.001). Curiously, the CC50 values for 3-deazaguanine were 83-93μM with ALB-F versus 2.4-7.0μM with all other dyes including ALB-A (P<0.001). Overall, ALB minimized the toxicities detected with these two inhibitors. Because the choice of dyes affected CC50 values, this impacted on the resulting in vitro selectivity indexes (calculated as CC50/EC50 ratio).

  12. 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

  13. The influence of micronutrients in cell culture: a reflection on viability and genomic stability.

    PubMed

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

    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.

  14. 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.

  15. 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

  16. Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements.

    PubMed

    Bhatia, Sujata K; Yetter, Ann B

    2008-08-01

    Medical devices and implanted biomaterials are often assessed for biological reactivity using visual scores of cell-material interactions. In such testing, biomaterials are assigned cytotoxicity ratings based on visual evidence of morphological cellular changes, including cell lysis, rounding, spreading, and proliferation. For example, ISO 10993 cytotoxicity testing of medical devices allows the use of a visual grading scale. The present study compared visual in vitro cytotoxicity ratings to quantitative in vitro cytotoxicity measurements for biomaterials to determine the level of correlation between visual scoring and a quantitative cell viability assay. Biomaterials representing a spectrum of biological reactivity levels were evaluated, including organo-tin polyvinylchloride (PVC; a known cytotoxic material), ultra-high molecular weight polyethylene (a known non-cytotoxic material), and implantable tissue adhesives. Each material was incubated in direct contact with mouse 3T3 fibroblast cell cultures for 24 h. Visual scores were assigned to the materials using a 5-point rating scale; the scorer was blinded to the material identities. Quantitative measurements of cell viability were performed using a 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay; again, the assay operator was blinded to material identities. The investigation revealed a high degree of correlation between visual cytotoxicity ratings and quantitative cell viability measurements; a Pearson's correlation gave a correlation coefficient of 0.90 between the visual cytotoxicity score and the percent viable cells. An equation relating the visual cytotoxicity score and the percent viable cells was derived. The results of this study are significant for the design and interpretation of in vitro cytotoxicity studies of novel biomaterials.

  17. Influence of oxidized low-density lipoproteins (LDL) on the viability of osteoblastic cells.

    PubMed

    Brodeur, Mathieu R; Brissette, Louise; Falstrault, Louise; Ouellet, Pascale; Moreau, Robert

    2008-02-15

    Cardiovascular diseases have recently been noted as potential risk factors for osteoporosis development. Although it is poorly understood how these two pathologies are related, it is a known fact that oxidized low-density lipoproteins (OxLDL) constitute potential determinants for both of them. The current study investigated the metabolism of OxLDL by osteoblasts and its effect on osteoblastic viability. The results obtained show that OxLDL are internalized but not degraded by osteoblasts while they can selectively transfer their CE to these cells. It is also demonstrated that OxLDL induce proliferation at low concentrations but cell death at high concentrations. This reduction of osteoblast viability was associated with lysosomal membrane damage caused by OxLDL as demonstrated by acridine orange relocalization. Accordingly, chloroquine, an inhibitor of lysosomal activity, accentuated cell death induced by OxLDL. Finally, we demonstrate that osteoblasts have the capacity to oxidize LDL and thereby potentially increase the local concentration of OxLDL. Overall, the current study confirms the potential role of OxLDL in the development of osteoporosis given its influence on osteoblastic viability.

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

    PubMed Central

    Burns, John H. R.; Takabayashi, Misaki

    2011-01-01

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

  19. 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.

  20. Effect of Antibiotics against Mycoplasma sp. on Human Embryonic Stem Cells Undifferentiated Status, Pluripotency, Cell Viability and Growth

    PubMed Central

    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 PlasmocinTM 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 PlasmocinTM 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 PlasmocinTM 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 PlasmocinTM 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. PMID:23936178

  1. Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer.

    PubMed

    Poff, A M; Ari, C; Arnold, P; Seyfried, T N; D'Agostino, D P

    2014-10-01

    Cancer cells express an abnormal metabolism characterized by increased glucose consumption owing to genetic mutations and mitochondrial dysfunction. Previous studies indicate that unlike healthy tissues, cancer cells are unable to effectively use ketone bodies for energy. Furthermore, ketones inhibit the proliferation and viability of cultured tumor cells. As the Warburg effect is especially prominent in metastatic cells, we hypothesized that dietary ketone supplementation would inhibit metastatic cancer progression in vivo. Proliferation and viability were measured in the highly metastatic VM-M3 cells cultured in the presence and absence of β-hydroxybutyrate (βHB). Adult male inbred VM mice were implanted subcutaneously with firefly luciferase-tagged syngeneic VM-M3 cells. Mice were fed a standard diet supplemented with either 1,3-butanediol (BD) or a ketone ester (KE), which are metabolized to the ketone bodies βHB and acetoacetate. Tumor growth was monitored by in vivo bioluminescent imaging. Survival time, tumor growth rate, blood glucose, blood βHB and body weight were measured throughout the survival study. Ketone supplementation decreased proliferation and viability of the VM-M3 cells grown in vitro, even in the presence of high glucose. Dietary ketone supplementation with BD and KE prolonged survival in VM-M3 mice with systemic metastatic cancer by 51 and 69%, respectively (p < 0.05). Ketone administration elicited anticancer effects in vitro and in vivo independent of glucose levels or calorie restriction. The use of supplemental ketone precursors as a cancer treatment should be further investigated in animal models to determine potential for future clinical use.

  2. Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer

    PubMed Central

    Poff, AM; Ari, C; Arnold, P; Seyfried, TN; D’Agostino, DP

    2014-01-01

    Cancer cells express an abnormal metabolism characterized by increased glucose consumption owing to genetic mutations and mitochondrial dysfunction. Previous studies indicate that unlike healthy tissues, cancer cells are unable to effectively use ketone bodies for energy. Furthermore, ketones inhibit the proliferation and viability of cultured tumor cells. As the Warburg effect is especially prominent in metastatic cells, we hypothesized that dietary ketone supplementation would inhibit metastatic cancer progression in vivo. Proliferation and viability were measured in the highly metastatic VM-M3 cells cultured in the presence and absence of β-hydroxybutyrate (βHB). Adult male inbred VM mice were implanted subcutaneously with firefly luciferase-tagged syngeneic VM-M3 cells. Mice were fed a standard diet supplemented with either 1,3-butanediol (BD) or a ketone ester (KE), which are metabolized to the ketone bodies βHB and acetoacetate. Tumor growth was monitored by in vivo bioluminescent imaging. Survival time, tumor growth rate, blood glucose, blood βHB and body weight were measured throughout the survival study. Ketone supplementation decreased proliferation and viability of the VM-M3 cells grown in vitro, even in the presence of high glucose. Dietary ketone supplementation with BD and KE prolonged survival in VM-M3 mice with systemic metastatic cancer by 51 and 69%, respectively (p < 0.05). Ketone administration elicited anticancer effects in vitro and in vivo independent of glucose levels or calorie restriction. The use of supplemental ketone precursors as a cancer treatment should be further investigated in animal models to determine potential for future clinical use. PMID:24615175

  3. A possible regulatory role of glyoxalase I in cell viability of human prostate cancer.

    PubMed

    Davidson, Scott D; Milanesa, Dan M; Mallouh, Camille; Choudhury, Muhammad S; Tazaki, Hiroshi; Konno, Sensuke

    2002-05-01

    A role of glyoxalase I (Gly-I), a detoxifying enzyme, in cell viability of prostate cancer was investigated. Cell extracts obtained from 66 prostate tissue specimens and prostatic cancer PC-3 cells were assayed for Gly-I activity using the spectrophotometric method. Gly-I activity was consistently more than eightfold higher in prostate cancer (CAP) specimens (n = 37) than in non-cancerous (NCP) specimens (n = 29). To understand the importance of such a high Gly-I activity in CAP specimens, the effects of methylglyoxal (MG) on PC-3 cells were examined in vitro. MG, a putative toxic glycolytic metabolite, was capable of inducing severe (> 99%) cell death in 24 h, along with a significant reduction in activities of Gly-I as well as glyceraldehyde 3-phosphate dehydrogenase (G3PDH), a key glycolytic enzyme. However, such severe cell death was effectively (approximately 85%) prevented with N-acetylcysteine (NAC), a precursor of reduced glutathione (GSH) that is an essential cofactor for Gly-I, accompanied by the intact Gly-I and G3PDH activities. Therefore, Gly-I may play a critical detoxifying role in glycolysis to maintain cellular activity and viability of prostatic cancer cells.

  4. Low temperature co-fired ceramic packaging of CMOS capacitive sensor chip towards cell viability monitoring

    PubMed Central

    Kilpijärvi, Joni; Sobocinski, Maciej; Datta-Chaudhuri, Timir; Hassinen, Antti; Prakash, Someshekar B; Möller, Peter; Abshire, Pamela; Kellokumpu, Sakari; Lloyd Spetz, Anita

    2016-01-01

    Cell viability monitoring is an important part of biosafety evaluation for the detection of toxic effects on cells caused by nanomaterials, preferably by label-free, noninvasive, fast, and cost effective methods. These requirements can be met by monitoring cell viability with a capacitance-sensing integrated circuit (IC) microchip. The capacitance provides a measurement of the surface attachment of adherent cells as an indication of their health status. However, the moist, warm, and corrosive biological environment requires reliable packaging of the sensor chip. In this work, a second generation of low temperature co-fired ceramic (LTCC) technology was combined with flip-chip bonding to provide a durable package compatible with cell culture. The LTCC-packaged sensor chip was integrated with a printed circuit board, data acquisition device, and measurement-controlling software. The packaged sensor chip functioned well in the presence of cell medium and cells, with output voltages depending on the medium above the capacitors. Moreover, the manufacturing of microfluidic channels in the LTCC package was demonstrated. PMID:28144536

  5. Viability of fat cells over time after syringe suction lipectomy: the effects of cryopreservation.

    PubMed

    Son, Daegu; Oh, Jaehoon; Choi, Taehyun; Kim, Junhyung; Han, Kihwan; Ha, Seongyun; Lee, Kyungho

    2010-09-01

    The purpose of this study was to determine the late decline in viability of fat cells over time for fat tissue stored at -15 degrees C and -70 degrees C after harvest from abdominal liposuction. A total of 16 females were recruited for this study. The viability of fat cell specimens was measured after freezing for 1, 3, 7, 14, 28, and 56 days. A number of viable mature adipocytes were evaluated by fluorescence microscopy after staining with fluorescein diacetate and propidium iodide. The glycerol-3-phosphate dehydrogenase activity was measured in lipoaspirates before digestion and the XTT reduction assay was performed. In addition, the XTT reduction assay was also performed on isolated lipocytes and preadipocytes.The viability of mature adipocytes was very low for both the -15 degrees C and -70 degrees C samples after 1 day of freezing (13.3% +/- 7.4% and 12.6% +/- 6.3%, respectively). There was no statistically significant difference between the samples stored at the 2 temperatures. The GPDH activity of the lipoaspirates frozen, for 1 day, at -15 degrees C and -70 degrees C was 25.1% +/- 10% and 28.7% +/- 11%, respectively. For the XTT test, the fractional enzyme activity of the lipoaspirates frozen, for 1 day, at -15 degrees C and -70 degrees C was 30.0% +/- 10.9% and 36.1% +/- 12.3%, respectively. In addition, the adipocytes had low activity from day one: 15.4% +/- 7.2% at -15 degrees C and 11.5% +/- 5.6% at -70 degrees C. Furthermore, the preadipocytes had a low activity of 8.0% +/- 6.0% at -15 degrees C and 8.6% +/- 3.8% at -70 degrees C. At 8 weeks, there were few viable mature adipocytes and the activity of the cells was very low by XTT and GPDH testing.The results of this study showed that the viability of adipocytes declined rapidly after frozen storage for 1 day at both -15 degrees C and -70 degrees C, and decreased gradually in storage after 8 weeks; at which time only approximately 5% of the fat cells were alive. These findings suggest that the present

  6. Expression of human eukaryotic initiation factor 3f oscillates with cell cycle in A549 cells and is essential for cell viability

    PubMed Central

    2010-01-01

    Background Transcriptional and postranslational regulation of the cell cycle has been widely studied. However, there is scarce knowledge concerning translational control of this process. Several mammalian eukaryotic initiation factors (eIFs) seem to be implicated in controlling cell proliferation. In this work, we investigated if the human eIF3f expression and function is cell cycle related. Results The human eIF3f expression has been found to be upregulated in growth-stimulated A549 cells and downregulated in G0. Western blot analysis and eIF3f promotor-luciferase fusions revealed that eIF3f expression peaks twice in the cell cycle: in the S and the M phases. Deregulation of eIF3f expression negatively affects cell viability and induces apoptosis. Conclusions The expression pattern of human eIF3f during the cell cycle confirms that this gene is cell division related. The fact that eIF3f expression peaks in two cell cycle phases raises the possibility that this gene may exert a differential function in the S and M phases. Our results strongly suggest that eIF3f is essential for cell proliferation. PMID:20462454

  7. 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

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

    PubMed

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

    2016-05-04

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

  9. Capacitance-based assay for real-time monitoring of endocytosis and cell viability.

    PubMed

    Lee, Rimi; Kim, Jihun; Kim, Sook Young; Jang, Seon Mi; Lee, Sun-Mi; Choi, In-Hong; Park, Seung Woo; Shin, Jeon-Soo; Yoo, Kyung-Hwa

    2012-07-07

    Label-free cell-based assays have emerged as a promising means for high-throughput screening. Most label-free sensors are based on impedance measurements that reflect the passive electrical properties of cells. Here we introduce a capacitance-based assay that measures the dielectric constant (capacitance) of biological cells, and demonstrate the feasibility of analyzing endocytosis and screening chemotherapeutic agents with this assay. Endocytosis induces a change in the zeta potential, leading to a change in the dielectric constant which enables real-time endocytosis monitoring using the capacitance sensor. Additionally, since the dielectric constant is proportional to cell radius and cell volume, cell viability can be estimated from the change in capacitance. Therefore, the capacitance sensor array can also be used for cytotoxicity testing for large-scale chemotherapeutic screening.

  10. Evaluation of the Effect of PEGylated Single-Walled Carbon Nanotubes on Viability and Proliferation of Jurkat Cells

    PubMed Central

    Hadidi, Naghmeh; Hosseini Shirazi, Seyed Farshad; Kobarfard, Farzad; Nafissi-Varchehd, Nastaran; Aboofazeli, Reza

    2012-01-01

    Among the numerous nanosized drug delivery systems currently under investigation, carbon nanotubes (CNTs), regardless of being single or multiple-walled, offer several advantages and are considered as promising candidates for drug targeting. Despite the valuable potentials of CNTs in drug delivery, their toxicity still remains an important issue. After the PEGylation of single-walled CNTs (SWCNTs) with phospholipid-PEG (Pl-PEG) conjugates to prepare water-dispersible nanostructures, the present study was designed to evaluate whether the functionalization with Pl-PEG derivatives could alter the cytotoxic response of cells in culture, affect their viability and proliferation. In-vitro cytotoxicity screens were performed on cultured Jurkat cells. The SWCNTs samples used in this exposure were pristine SWCNTs, Pl-PEG 2000/5000-SWCNTs at various concentrations. Jurkat cells were first incubated for 3 h at 37°C with test materials and seeded in 6-well culture plates at a given concentration. The plates were then incubated for 24, 48 and 72 h at 37°C in a 5% CO2 humidified incubator. Cell Viability and proliferation assay were performed using trypan blue exclusion test and the cell cycle kinetic status of Jurkat cells was analyzed by flow cytometry. Cell morphology was finally studied using double staining technique and a fluorescence microscope. We found that, regardless of the duration of exposure, functionalized SWCNTs were substantially less toxic, compared to pure SWCNTs and that the molecular weight of Pl-PEGs played an important role at higher concentrations. In conclusion, our noncovalent protocol seemed to be effective for increasing SWCNTs biocompatibility. PMID:25317182

  11. Microbubble mediated sonoporation of cells in suspension: clonogenic viability and influence of molecular size on uptake.

    PubMed

    Karshafian, Raffi; Samac, Sanya; Bevan, Peter D; Burns, Peter N

    2010-06-01

    This work investigates whether the application of sonoporation is limited by the size of a macromolecule being delivered and by the ability of cells to proliferate following uptake. KHT-C cells in suspension were exposed to variations in ultrasound pressure (0-570 kPa) and microbubble shell-type (lipid and protein) at fixed settings of 500 kHz centre frequency, 32 micros pulse duration, 3 kHz pulse repetition frequency and 2 min insonation. Reversible permeability (P(R)), defined as the number of cells stained with FITC-dextran and unstained with propidium iodide (i.e., PI-viable), was measured with flow cytometry for marker molecules ranging from 10 kDa to 2 MDa in size. Viable permeability (P(V)) defined as the number of permeabilised cells that maintained their ability to proliferate, was measured by clonogenic assay. Comparable intracellular delivery of all sizes of molecules was achieved, indicating that intracellular delivery of common therapeutic drugs may not be limited by molecular size. Maximum P(R)'s of 80% (at 10 kDa) and 55% (at 10 kDa) were achieved with lipid coated bubbles at 3.3% v/v and protein coated bubbles at 6.7% v/v concentrations. The PI-viability was approximately 80% at 570 kPa in both cases. The maximum P(V) achieved with both agents was 22%, while inducing a lower overall clonogenic viability with the lipid (39%) compared to the protein (56%) shelled bubbles. This study demonstrates that large macromolecules, up to 2 MDa in size, can be delivered with high efficiency to cells which undergo reversible permeabilisation, maintaining long-term viability in approximately half of the cells.

  12. Propofol inhibits lung cancer cell viability and induces cell apoptosis by upregulating microRNA-486 expression

    PubMed Central

    Yang, N.; Liang, Y.; Yang, P.; Yang, T.; Jiang, L.

    2017-01-01

    Propofol is a frequently used intravenous anesthetic agent. Recent studies show that propofol exerts a number of non-anesthetic effects. The present study aimed to investigate the effects of propofol on lung cancer cell lines H1299 and H1792 and functional role of microRNA (miR)-486 in these effects. H1299 and/or H1792 cells were treated with or without propofol and transfected or not with miR-486 inhibitor, and then cell viability and apoptosis were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry. The expression of miR-486 was determined by quantitative real-time polymerase chain reaction (qRT-PCR) with or without propofol treatment. Western blot was performed to analyze the protein expression of Forkhead box, class O (FOXO) 1 and 3, Bcl-2 interacting mediator of cell death (Bim), and pro- and activated caspases-3. Results showed that propofol significantly increased the miR-486 levels in both H1299 and H1792 cells compared to untreated cells in a dose-dependent manner (P<0.05 or P<0.01). Propofol statistically decreased cell viability but increased the percentages of apoptotic cells and protein expressions of FOXO1, FOXO3, Bim, and pro- and activated caspases-3; however, miR-486 inhibitor reversed the effects of propofol on cell viability, apoptosis, and protein expression (P<0.05 or P<0.01). In conclusion, propofol might be an ideal anesthetic for lung cancer surgery by effectively inhibiting lung cancer cell viability and inducing cell apoptosis. Modulation of miR-486 might contribute to the anti-tumor activity of propofol. PMID:28076456

  13. Long-Term IGF-I Exposure Decreases Autophagy and Cell Viability

    PubMed Central

    Bitto, Alessandro; Lerner, Chad; Torres, Claudio; Roell, Michaela; Malaguti, Marco; Perez, Viviana; Lorenzini, Antonello; Hrelia, Silvana; Ikeno, Yuji; Matzko, Michelle Elizabeth; McCarter, Roger; Sell, Christian

    2010-01-01

    A reduction in IGF-I signaling has been found to increase lifespan in multiple organisms despite the fact that IGF-I is a trophic factor for many cell types and has been found to have protective effects against multiple forms of damage in acute settings. The increase in longevity seen in response to reduced IGF-I signaling suggests that there may be differences between the acute and chronic impact of IGF-I signaling. We have examined the possibility that long-term stimulation with IGF-I may have a negative impact at the cellular level using quiescent human fibroblasts. We find that fibroblast cells exposed to IGF-I for 14 days have reduced long-term viability as judged by colony forming assays, which is accompanied by an accumulation of senescent cells. In addition we observe an accumulation of cells with depolarized mitochondria and a reduction in autophagy in the long-term IGF-I treated cultures. An examination of mice with reduced IGF-I levels reveals evidence of enhanced autophagy and fibroblast cells derived from these mice have a larger mitochondrial mass relative to controls indicating that changes in mitochondrial turnover occurs in animals with reduced IGF-I. The results indicate that chronic IGF-I stimulation leads to mitochondrial dysfunction and reduced cell viability. PMID:20830296

  14. The viability and intestinal epithelial cell adhesion of probiotic strain combination--in vitro study.

    PubMed

    Piątek, Jacek; Gibas-Dorna, Magdalena; Olejnik, Anna; Krauss, Hanna; Wierzbicki, Krzysztof; Żukiewicz-Sobczak, Wioletta; Głowacki, Maciej

    2012-01-01

    To be effective, probiotic bacteria must exhibit a number of functional characteristics, including the resistance to gastric acidity and the ability to adhere to the intestinal epithelium. In this study, we examined in vitro the viability of lactic acid bacteria (LAB) combination after exposure to low pH, and the adhesion of LAB to Caco-2 cells during coincubation of 9 bacterial strains. To test bacterial viability, 6 commercially available products were incubated in 0.1 N HCl at pH 1.2 for 60 min. The greatest growth inhibition was noted for the non-capsulated product containing the Lactobacillus rhamnosus strain (log reduction of CFU = 6.4), and the best survival observed for the product containing 9 bacterial strains, equipped with a modern capsule made according to the Multi-Resistant Encapsulation technology (log reduction of CFU = 0.1). In the adhesion experiment, the combination of 9 bacterial strains was added to 17-day-old Caco-2 cell culture for 90 min. The greatest efficiency of adhesion was observed for the inoculum containing 5.5x10(8) CFU/mL/9.6 cm(2) of Caco-2 and the dose of probiotic bacteria of 190 cells per one Caco-2 cell. As a result, approximately 157 bacterial cells adhered to one Caco-2 cell. The results indicate that the combination of 9 bacterial strains in the examined product is characterized as highly adhesive.

  15. Monitoring of microbial cell viability using nanostructured electrodes modified with Graphene/Alumina nanocomposite.

    PubMed

    Hassan, Rabeay Y A; Mekawy, Moataz M; Ramnani, Pankaj; Mulchandani, Ashok

    2017-05-15

    Microbial infections are rapidly increasing; however most of the existing microbiological and molecular detection methods are time consuming and/or cannot differentiate between the viable and dead cells which may overestimate the risk of infections. Therefore, a bioelectrochemical sensing platform with a high potential to the microbial-electrode interactions was designed based on decorated graphene oxide (GO) sheet with alumina (Al2O3) nanocrystals. GO-Al2O3 nanocomposite was synthesized using self-assembly of GO and Al2O3 and characterized using the scanning electron microscopy (SEM), transmission electron microscopy (TEM), x-ray diffraction (XRD), Raman-spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Enhancement of electrocatalytic activity of the composite-modified electrode was demonstrated. Thus, using the GO-Al2O3 nanocomposite modified electrode, the cell viability was determined by monitoring the bioelectrochemical response of the living microbial cells (bacteria and yeast) upon stimulation with carbon source. The bioelectrochemical assay was optimized to obtain high sensitivity and the method was applied to monitor cell viability and screen susceptibility of metabolically active cells (E. coli, B. subtilis, Enterococcus, P. aeruginosa and Salmonella typhi) to antibiotics such as ampicillin and kanamycin. Therefore, the developed assay is suitable for cell proliferation and cytotoxicity testing.

  16. Bcl-2 expression in synovial fibroblasts is essential for maintaining mitochondrial homeostasis and cell viability.

    PubMed

    Perlman, H; Georganas, C; Pagliari, L J; Koch, A E; Haines, K; Pope, R M

    2000-05-15

    The regulation of proliferation and cell death is vital for homeostasis, but the mechanism that coordinately balances these events in rheumatoid arthritis (RA) remains largely unknown. In RA, the synovial lining thickens in part through increased proliferation and/or decreased synovial fibroblast cell death. Here we demonstrate that the anti-apoptotic protein, Bcl-2, is highly expressed in RA compared with osteoarthritis synovial tissues, particularly in the CD68-negative, fibroblast-like synoviocyte population. To determine the importance of endogenous Bcl-2, an adenoviral vector expressing a hammerhead ribozyme to Bcl-2 (Ad-Rbz-Bcl-2) mRNA was employed. Ad-Rbz-Bcl-2 infection resulted in reduced Bcl-2 expression and cell viability in synovial fibroblasts isolated from RA and osteoarthritis synovial tissues. In addition, Ad-Rbz-Bcl-2-induced mitochondrial permeability transition, cytochrome c release, activation of caspases 9 and 3, and DNA fragmentation. The general caspase inhibitor zVAD.fmk blocked caspase activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation, but not loss of transmembrane potential or viability, indicating that cell death was independent of caspase activation. Ectopically expressed Bcl-xL inhibited Ad-Rbz-Bcl-2-induced mitochondrial permeability transition and apoptosis in Ad-Rbz-Bcl-2-transduced cells. Thus, forced down-regulation of Bcl-2 does not induce a compensatory mechanism to prevent loss of mitochondrial integrity and cell death in human fibroblasts.

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

    PubMed

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

    2008-01-15

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

  18. 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.

  19. 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

  20. 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.

  1. Impact of high glucose and AGEs on cultured kidney-derived cells. Effects on cell viability, lysosomal enzymes and effectors of cell signaling pathways.

    PubMed

    Peres, Giovani B; Schor, Nestor; Michelacci, Yara M

    2017-04-01

    We have previously reported decreased expression and activities of lysosomal cathepsins B and L in diabetic kidney. Relevant morphological changes were observed in proximal tubules, suggesting that these cells are implicated in the early stages of the disease. The aim of the present study was to investigate the mechanisms that lead to these changes. The effects of high glucose (HG) and advanced glycation end products (AGEs) on cell viability, lysosomal enzymes and other effectors of cell signaling of cultured kidney cells were studied. HG increased viable mesangial cells (ihMC) in 48 h, while epithelial tubular cells were not affected (LLC-PK1 and MDCK). In contrast, the number of viable cells was markedly decreased, for all cell lines, by AGE-BSA. Concerning lysosomal enzymes, the main cysteine-protease expressed by these cells was cathepsin B, and its concentration was much higher in epithelial than in mesangial cells. Exposure to HG had no effect on the cathepsin B activity, but AGE-BSA caused a marked decrease in LLC-PK1, and increased the enzyme activities in the other cell lines. The levels of nitric oxide (NO) was increased by AGE-BSA in all cell lines, suggesting oxidative stress, and Western blotting has shown that, among the investigated proteins, cathepsin B, mTOR and transcription factor EB (TFEB) were the most significantly affected by exposure to AGE-BSA. As mTOR induces anabolism and inhibits autophagy, and TFEB is a master transcription factor for lysosomal enzymes, it is possible that this pathway plays a role in the inhibition of lysosomal enzymes in proximal tubule cells.

  2. OAZ1 knockdown enhances viability and inhibits ER and LHR transcriptions of granulosa cells in geese

    PubMed Central

    Ma, Rong; He, Hui; Yi, Zhixin; Chen, Ziyu

    2017-01-01

    An increasing number of studies suggest that ornithine decarboxylase antizyme 1 (OAZ1), which is regarded as a tumor suppressor gene, regulates follicular development, ovulation, and steroidogenesis. The granulosa cells in the ovary play a critical role in these ovarian functions. However, the action of OAZ1 mediating physiological functions of granulosa cells is obscure. OAZ1 knockdown in granulosa cells of geese was carried out in the current study. The effect of OAZ1 knockdown on polyamine metabolism, cell proliferation, apoptosis, and hormone receptor transcription of primary granulosa cells in geese was measured. The viability of granulosa cells transfected with the shRNA OAZ1 at 48 h was significantly higher than the control (p<0.05). The level of putrescine and spermidine in granulosa cells down-regulating OAZ1 was 7.04- and 2.11- fold higher compared with the control, respectively (p<0.05). The CCND1, SMAD1, and BCL-2 mRNA expression levels in granulosa cells down-regulating OAZ1 were each significantly higher than the control, respectively (p<0.05), whereas the PCNA and CASPASE 3 expression levels were significantly lower than the control (p<0.05). The estradiol concentration, ER and LHR mRNA expression levels were significantly lower in granulosa cells down-regulating OAZ1 compared with the control (p<0.05). Taken together, our results indicated that OAZ1 knockdown elevated the putrescine and spermidine contents and enhanced granulosa cell viability and inhibited ER and LHR transcriptions of granulosa cells in geese. PMID:28362829

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

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

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

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

    PubMed Central

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

    2012-01-01

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

  6. How plasma induced oxidation, oxygenation, and de-oxygenation influences viability of skin cells

    NASA Astrophysics Data System (ADS)

    Oh, Jun-Seok; Strudwick, Xanthe; Short, Robert D.; Ogawa, Kotaro; Hatta, Akimitsu; Furuta, Hiroshi; Gaur, Nishtha; Hong, Sung-Ha; Cowin, Allison J.; Fukuhara, Hideo; Inoue, Keiji; Ito, Masafumi; Charles, Christine; Boswell, Roderick W.; Bradley, James W.; Graves, David B.; Szili, Endre J.

    2016-11-01

    The effect of oxidation, oxygenation, and de-oxygenation arising from He gas jet and He plasma jet treatments on the viability of skin cells cultured in vitro has been investigated. He gas jet treatment de-oxygenated cell culture medium in a process referred to as "sparging." He plasma jet treatments oxidized, as well as oxygenated or de-oxygenated cell culture medium depending on the dissolved oxygen concentration at the time of treatment. He gas and plasma jets were shown to have beneficial or deleterious effects on skin cells depending on the concentration of dissolved oxygen and other oxidative molecules at the time of treatment. Different combinations of treatments with He gas and plasma jets can be used to modulate the concentrations of dissolved oxygen and other oxidative molecules to influence cell viability. This study highlights the importance of a priori knowledge of the concentration of dissolved oxygen at the time of plasma jet treatment, given the potential for significant impact on the biological or medical outcome. Monitoring and controlling the dynamic changes in dissolved oxygen is essential in order to develop effective strategies for the use of cold atmospheric plasma jets in biology and medicine.

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

    PubMed

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

    2012-06-01

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

  8. Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

    PubMed

    de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A

    2013-12-31

    Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the

  9. Comparative assessment of the cytotoxicity of six anti-inflammatory eyedrops in four cultured ocular surface cell lines, as determined by cell viability scores

    PubMed Central

    Ayaki, Masahiko; Iwasawa, Atsuo; Niwano, Yoshimi

    2012-01-01

    Purpose Anti-inflammatory eyedrops are often used in the treatment of corneal epithelial disorders. In the present study, we evaluated the cytotoxicity of six anti-inflammatory eyedrops in four ocular surface cell lines. Methods The cytotoxicity of six commercially available anti-inflammatory ophthalmic solutions (ie, diclofenac, bromfenac, pranoprofen, betamethasone, and fluoromethorone) was assessed in three corneal cell lines and one conjunctival cell line. Cell viability was determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide and neutral red assays after exposing the cells to 10, 30, and 60 minutes of onefold, twofold, and tenfold dilutions of the drugs. Cytotoxicity was compared using the cell viability score (CVS), an integrated cytotoxic parameter that takes various factors into account, such as dilution by tear fluid or concentration by evaporation, drug exposure time, and ocular surface cell type. Results Based on the CVS scores, the order of the anti-inflammatory eyedrops tested from least to most cytotoxic, with the active ingredient %CVS50, and %CVS40/80 for each solution given in parentheses, was as follows: Rinderon® (betamethasone, 100%, 100%) >0.02% Flumethoron® (fluoromethorone, 68%, 22%) = 0.1% Flumethoron® (fluoromethorone, 76%, 22%) >Bronuck® (0.1% bromfenac, 53%, −8%) = Diclod® (0.1% diclofenac, 44%, −15%) = Niflan® (pranoprofen, 50%, −19%). Rinderon® exhibited the least toxicity of all the anti-inflammatory eyedrops tested. Eyedrops containing non-steroidal anti-inflammatory drugs exhibited greater cytotoxicity than those containing steroids with benzalkonium at comparable concentrations. Concentration was the most significant factor affecting cell viability. Conclusion The cytotoxicity of the anti-inflammatory eyedrops evaluated in the present study depended on both the pharmaceutical components and preservatives. The CVS is a concise indicator of drug cytotoxicity. PMID:23185116

  10. The Association of VDAC with Cell Viability of PC12 Model of Huntington’s Disease

    PubMed Central

    Karachitos, Andonis; Grobys, Daria; Kulczyńska, Klaudia; Sobusiak, Adrian; Kmita, Hanna

    2016-01-01

    It is becoming increasingly apparent that mitochondria dysfunction plays an important role in the pathogenesis of Huntington’s disease (HD), but the underlying mechanism is still elusive. Thus, there is a still need for further studies concerning the upstream events in the mitochondria dysfunction that could contribute to cell death observed in HD. Taking into account the fundamental role of the voltage-dependent anion-selective channel (VDAC) in mitochondria functioning, it is reasonable to consider the channel as a crucial element in HD etiology. Therefore, we applied inducible PC12 cell model of HD to determine the relationship between the effect of expression of wild type and mutant huntingtin (Htt and mHtt, respectively) on cell survival and mitochondria functioning in intact cells under conditions of undergoing cell divisions. Because after 48 h of Htt and mHtt expression differences in mitochondria functioning co-occurred with differences in the cell viability, we decided to estimate the effect of Htt and mHtt expression lasted for 48 h on VDAC functioning. Therefore, we isolated VDAC from the cells and tested the preparations by black lipid membrane system. We observed that the expression of mHtt, but not Htt, resulted in changes of the open state conductance and voltage-dependence when compared to control cells cultured in the absence of the expression. Importantly, for all the VDAC preparations, we observed a dominant quantitative content of VDAC1, and the quantitative relationships between VDAC isoforms were not changed by Htt and mHtt expression. Thus, Htt and mHtt-mediated functional changes of VDAC, being predominantly VDAC1, which occur shortly after these protein appearances in cells, may result in differences concerning mitochondria functioning and viability of cells expressing Htt and mHtt. The assumption is important for better understanding of cytotoxicity as well as cytoprotection mechanisms of potential clinical application. PMID

  11. 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

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

    PubMed Central

    Ebrahimi, Aida; Alam, Muhammad Ashraful

    2016-01-01

    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. PMID:27303045

  13. 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

  14. Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality

    PubMed Central

    Angel, Stephanie; von Briesen, Hagen; Oh, Young-Joo; Baller, Marko K.; Zimmermann, Heiko

    2016-01-01

    Cryopreservation of biological materials such as cells, tissues, and organs is a prevailing topic of high importance. It is employed not only in many research fields but also in the clinical area. Cryopreservation is of great importance for reproductive medicine and clinical studies, as well as for the development of vaccines. Peripheral blood mononuclear cells (PBMCs) are commonly used in vaccine research where comparable and reliable results between different research institutions and laboratories are of high importance. Whereas freezing and thawing processes are well studied, controlled, and standardized, storage conditions are often disregarded. To close this gap, we investigated the influence of suboptimal storage conditions during low-temperature storage on PBMC viability, recovery, and T cell functionality. For this purpose, PBMCs were isolated and exposed with help of a robotic system in a low-temperature environment from 0 up to 350 temperature fluctuation cycles in steps of 50 cycles to simulate storage conditions in large biorepositories with sample storage, removal, and sorting functions. After the simulation, the viability, recovery, and T cell functionality were analyzed to determine the number of temperature rises, which ultimately lead to significant cell damage. All studied parameters decreased with increasing number of temperature cycles. Sometimes after as little as only 50 temperature cycles, a significant effect was observed. These results are very important for all fields in which cell cryopreservation is employed, particularly for clinical and multicenter studies wherein the comparability and reproducibility of results play a crucial role. To obtain reliable results and to maintain the quality of the cells, not only the freezing and thawing processes but also the storage conditions should be controlled and standardized, and any deviations should be documented. PMID:27792414

  15. 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...

  16. Optimization of temperature, sugar concentration, and inoculum size to maximize ethanol production without significant decrease in yeast cell viability.

    PubMed

    Laluce, Cecilia; Tognolli, João Olimpio; de Oliveira, Karen Fernanda; Souza, Crisla Serra; Morais, Meline Rezende

    2009-06-01

    Aiming to obtain rapid fermentations with high ethanol yields and a retention of high final viabilities (responses), a 2(3) full-factorial central composite design combined with response surface methodology was employed using inoculum size, sucrose concentration, and temperature as independent variables. From this statistical treatment, two well-fitted regression equations having coefficients significant at the 5% level were obtained to predict the viability and ethanol production responses. Three-dimensional response surfaces showed that increasing temperatures had greater negative effects on viability than on ethanol production. Increasing sucrose concentrations improved both ethanol production and viability. The interactions between the inoculum size and the sucrose concentrations had no significant effect on viability. Thus, the lowering of the process temperature is recommended in order to minimize cell mortality and maintain high levels of ethanol production when the temperature is on the increase in the industrial reactor. Optimized conditions (200 g/l initial sucrose, 40 g/l of dry cell mass, 30 degrees C) were experimentally confirmed and the optimal responses are 80.8 +/- 2.0 g/l of maximal ethanol plus a viability retention of 99.0 +/- 3.0% for a 4-h fermentation period. During consecutive fermentations with cell reuse, the yeast cell viability has to be kept at a high level in order to prevent the collapse of the process.

  17. Enumeration and viability of rare cells in a microfluidic disk via positive selection approach.

    PubMed

    Chen, Ken-Chao; Pan, Yu-Cheng; Chen, Chen-Lin; Lin, Ching-Hung; Huang, Chiun-Sheng; Wo, Andrew M

    2012-10-15

    Recent studies have shown that specific rare cells in the blood can serve as an indicator of cancer prognosis, among other purposes. This article demonstrates the concept of separating and detecting rare cells from peripheral blood mononuclear cells via an economical microfluidic disk with a model system. MCF7, labeled with magnetic beads, was used to simulate circulating tumor cells as a target. Jurkat clone E6-1 was used to simulate leukocytes or other cells abundant in human blood. A tailored multistage magnet maximized the magnetic field to ensure optimal trapping efficiency. Results indicate that the yield of detected MCF7 was consistent at approximately 80% when fewer than hundreds of MCF7 cells were mixed in greater than 1 million Jurkat cells. The 80% yield also held for 10 MCF7 in 100 million Jurkat (rarity of 10(7)). Compared with the results from autoMACS, the performance was at least 20% higher and was more independent of the number of Jurkat. The viability of the enriched cells was approximately 90 ± 20%, showing that this method caused little damage to trapped cells. The microfluidic disk should be applicable for separation and detection of various rare cells, such as circulating tumor cells and circulating endothelial cells in human blood.

  18. 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.

  19. Increase in the nitric oxide release without changes in cell viability of macrophages after laser therapy with 660 and 808 nm lasers.

    PubMed

    Silva, Igor Henrique Morais; de Andrade, Samantha Cardoso; de Faria, Andreza Barkokebas Santos; Fonsêca, Deborah Daniela Diniz; Gueiros, Luiz Alcino Monteiro; Carvalho, Alessandra Albuquerque Tavares; da Silva, Wylla Tatiana Ferreira; de Castro, Raul Manhães; Leão, Jair Carneiro

    2016-12-01

    The aim of this study was to evaluate the influence of low-level laser therapy (LLLT) with different parameters and wavelengths on nitric oxide (NO) release and cell viability. Irradiation was performed with Ga-Al-As laser, continuous mode and wavelengths of 660 and 808 nm at different energy and power densities. For each wavelength, powers of 30, 50, and 100 mW and times of 10, 30, and 60 s were used. NO release was measured using Griess reaction, and cell viability was evaluated by mitochondrial reduction of bromide 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to formazan. LLLT promoted statistically significant changes in NO release and MTT value only at the wavelength of 660 nm (p < 0.05). LLLT also promoted an increase in the NO release and cell viability when the energy densities 64 (p = 0.04) and 214 J/cm(2) (p = 0.012), respectively, were used. LLLT has a significant impact on NO release without affecting cell viability, but the significance of these findings in the inflammatory response needs to be further studied.

  20. Application of cyclic biamperometry to viability and cytotoxicity assessment in human corneal epithelial cells.

    PubMed

    Rahimi, Mehdi; Youn, Hyun-Yi; McCanna, David J; Sivak, Jacob G; Mikkelsen, Susan R

    2013-05-01

    The application of cyclic biamperometry to viability and cytotoxicity assessments of human corneal epithelial cells has been investigated. Electrochemical measurements have been compared in PBS containing 5.0 mM glucose and minimal essential growth medium. Three different lipophilic mediators including dichlorophenol indophenol, 2-methyl-1,4-naphthoquinone (also called menadione or vitamin K3) and N,N,N',N'-tetramethyl-p-phenylenediamine have been evaluated for shuttling electrons across the cell membrane to the external medium. Transfer of these electrons to ferricyanide in the extra cellular medium results in the accumulation of ferrocyanide. The amount of ferrocyanide is then determined using cyclic biamperometry and is related to the extent of cell metabolic activity and therefore cell viability. To illustrate cytotoxicity assessment of chemicals, hydrogen peroxide, benzalkonium chloride and sodium dodecyl sulfate have been chosen as sample toxins, the cytotoxicities of which have been evaluated and compared to values reported in the literature. Similar values have been reported using colorimetric assays; however, the simplicity of this electrochemical assay can, in principle, open the way to miniaturization onto lab-on-chip devices and its incorporation into tiered-testing approaches for cytotoxicity assessment.

  1. 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.

  2. MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets.

    PubMed

    Stockert, Juan C; Blázquez-Castro, Alfonso; Cañete, Magdalena; Horobin, Richard W; Villanueva, Angeles

    2012-12-01

    Although MTT is widely used to assess cytotoxicity and cell viability, the precise localization of its reduced formazan product is still unclear. In the present study the localization of MTT formazan was studied by direct microscopic observation of living HeLa cells and by colocalization analysis with organelle-selective fluorescent probes. MTT formazan granules did not colocalize with mitochondria as revealed by rhodamine 123 labeling or autofluorescence. Likewise, no colocalization was observed between MTT formazan granules and lysosomes labeled by neutral red. Taking into account the lipophilic character and lipid solubility of MTT formazan, an evaluation of the MTT reaction was performed after treatment of cells with sunflower oil emulsions to induce a massive occurrence of lipid droplets. Under this condition, lipid droplets revealed a large amount of MTT formazan deposits. Kinetic studies on the viability of MTT-treated cells showed no harmful effects at short times. Quantitative structure-activity relations (QSAR) models were used to predict and explain the localization of both the MTT tetrazolium salt and its formazan product. These predictions were in agreement with experimental observations on the accumulation of MTT formazan product in lipid droplets.

  3. Effect of Four Different Media on Periodontal Ligament Cells Viability of Dry- Stored Dog Teeth

    PubMed Central

    Moazzami, Fariborz; Asheghi, Bahar; Sahebi, Safoura

    2017-01-01

    Statement of the Problem: The maintenance of viable periodontal ligament cells is the most important issue in the long-term preservation of avulsed teeth. Purpose: The aim of this study was to assess aloe vera as a new storage media in maintaining the cell viability of dry-stored teeth in comparison with soy milk, Hank`s balanced salt solution (HBSS), and milk. Materials and Method: Twenty one extracted dog premolar teeth were dried for 30 minutes and stored in soy milk, HBSS, milk, and aloe vera extract (50%) for 45 minutes (n=6 for each). Furthermore, positive and two negative control groups (n=6), corresponding to 0 min, 30 min, and 2-hour drying times were also prepared respectively. The number of viable cells was counted following storage using Trypan blue exclusion. Data were statistically analyzed using the one-way ANOVA and post hoc Tukey-HSD test. Results: Statistical analysis showed no significant differences in cell viability among aloe vera, soymilk, and HBSS- stored teeth; however, they were all superior to milk. Conclusion: Aloe vera extract can be recommended as a suitable storage media for avulsed teeth. PMID:28280756

  4. Candida albicans RHO1 is required for cell viability in vitro and in vivo.

    PubMed

    Smith, Susan E; Csank, Csilla; Reyes, Guadalupe; Ghannoum, Mahmoud A; Berlin, Vivian

    2002-05-01

    In Saccharomyces cerevisiae, Rho1p plays an important role in cell wall integrity by regulating beta-1,3-glucan synthase, Pkc1p and the actin cytoskeleton. To determine the physiological role of Rho1p in the dimorphic fungus Candida albicans, the major human fungal pathogen, we constructed mutants that conditionally express Rho1p from the glucose-repressible phosphoenolpyruvate carboxykinase promoter (pPCK1). We examined the growth of these cells in a range of conditions. Depletion of Rho1p from yeast cells resulted in cell death, lysis, and aggregation. The Rho1p conditional mutant was inviable on 10% serum indicating that Rho1p was also required for hyphal viability. Furthermore, in a mouse model of systemic candidiasis, strains dependent on pPCK1-driven RHO1 expression failed to colonise the kidneys and establish disease, suggesting that the level of glucose in serum was sufficient to repress the pPCK1 and that Rho1p-depleted strains were inviable within the host. Therefore, Rho1p is essential for the viability of C. albicans in vitro and in vivo.

  5. Impact of graphene oxide on viability of Chinese hamster ovary and mouse hepatoma MH-22A cells.

    PubMed

    Batiuskaite, Danute; Grinceviciute, Nora; Snitka, Valentinas

    2015-08-01

    The evaluation of the cyto- and bio-compatibility is a critical step in the development of graphene oxide (GO) as a new promising material for in vivo biomedical applications. In this study, we report the impact of GO, with and without the addition of bovine serum albumin, on healthy (Chinese hamster ovary) and a cancer (mouse hepatoma MH-22A) cells viability and the estimation of the intracellular distribution of GO inside the cells in vitro. The viability tests were performed using a colony formation assay. The intracellular distribution of GO was estimated using Raman spectroscopy and imaging. The viability of both cell lines decreased with increasing concentration of graphene oxide (12.5-50.0 μg/ml): in the case of Chinese hamster ovary cells viability decreased from 44% to 11%, in the case of mouse hepatoma MH-22A cells--from 22% to 3%. These cell lines significantly differed in their response to GO and GO-BSA formulations. The results of viability tests correlate with results of atomic force microscopy and Raman spectroscopy and imaging findings. The GO influence on cell morphology changes, cell structure, cells colony growth dynamics and GO accumulation inside the cells was higher in the case of mouse hepatoma MH-22A cells.

  6. 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.

  7. Riboflavin deprivation inhibits macrophage viability and activity - a study on the RAW 264.7 cell line.

    PubMed

    Mazur-Bialy, Agnieszka Irena; Buchala, Beata; Plytycz, Barbara

    2013-08-28

    Riboflavin, or vitamin B2, as a precursor of the coenzymes FAD and FMN, has an indirect influence on many metabolic processes and determines the proper functioning of several systems, including the immune system. In the human population, plasma riboflavin concentration varies from 3·1 nM (in a moderate deficiency, e.g. in pregnant women) to 10·4 nM (in healthy adults) and 300 nM (in cases of riboflavin supplementation). The purpose of the present study was to investigate the effects of riboflavin concentration on the activity and viability of macrophages, i.e. on one of the immunocompetent cell populations. The study was performed on the murine monocyte/macrophage RAW 264.7 cell line cultured in medium with various riboflavin concentrations (3·1, 10·4, 300 and 531 nM). The results show that riboflavin deprivation has negative effects on both the activity and viability of macrophages and reduces their ability to generate an immune response. Signs of riboflavin deficiency developed in RAW 264.7 cells within 4 d of culture in the medium with a low riboflavin concentration (3·1 nM). In particular, the low riboflavin content reduced the proliferation rate and enhanced apoptotic cell death connected with the release of lactate dehydrogenase. The riboflavin deprivation impaired cell adhesion, completely inhibited the respiratory burst and slightly impaired phagocytosis of the zymosan particles. In conclusion, macrophages are sensitive to riboflavin deficiency; thus, a low riboflavin intake in the diet may affect the immune system and may consequently decrease proper host immune defence.

  8. Cell viability studies of PEG-thiol treated gold nanorods as optoacoustic contrast agents

    NASA Astrophysics Data System (ADS)

    Manohar, Srirang; Rayavarapu, Rajagopal; Petersen, Wilma; van Leeuwen, Ton G.

    2009-02-01

    Rod shaped gold nanoparticles are synthesized using cetyltriammonium bromide (CTAB) as a major component of growth solutions. This surfactant is toxic to cells, but is at the moment unavoidable when monodisperse and high yield nanorods are to be synthesized. CTAB is found coating side walls of the nanoparticles and plays a role in maintaining colloidal stability. It may be displaced using thiolated PEG which is non-toxic to cells. Here we report on systematic studies of cell viability of such PEGylated nanorods on an SKBR3 cell-line using the MTS assay. These PEGylated particles are characterized using electron microscopy, optical spectroscopy and zeta potential measurements. It is expected that such treatment will be crucial in making nanorods compatible for in vivo biomedical applications.

  9. 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

    2010-08-06

    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.

  10. Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 2. Viability of encapsulated marrow stromal osteoblasts cultured on crosslinking poly(propylene fumarate).

    PubMed

    Payne, Richard G; McGonigle, Joseph S; Yaszemski, Michael J; Yasko, Alan W; Mikos, Antonios G

    2002-11-01

    The effect of temporary encapsulation of rat marrow stromal osteoblasts in crosslinked gelatin microparticles on cell viability and proliferation was investigated in this study for microparticles placed on a crosslinking poly(propylene fumarate) (PPF) composite over a 7 day time period. Encapsulated cells were seeded on crosslinking PPF composites at times up to 10 min following initiation of the crosslinking reaction, and also on fully crosslinked PPF composites and tissue culture polystyrene controls, with a cell seeding density of 5.3 x 10(4) cells/cm2. The crosslinked PPF composite exhibited an average gel point of 10.3 min and an average maximum crosslinking temperature of 47.5 degrees C. Cell viability and proliferation were assessed by DNA and 3H-thymidine assays and the results were compared with those for nonencapsulated cells. The results showed that the addition time of cells to a crosslinking PPF composite had a large effect on cell viability and proliferation for both encapsulated and nonencapsulated cells with more surviving cells added at later time points. Most importantly, the temporary encapsulation of cells significantly enhanced cell viability at earlier time points. The data indicate that the presence of gelatin microparticles does not affect the crosslinking of a PPF composite. They further suggest that the temporary encapsulation of cells in crosslinked gelatin microparticles may preserve the viability of cells contained in an actively crosslinking PPF composite used as an injectable polymeric scaffold serving also as a carrier for osteogenic cell populations.

  11. Multinucleated Giant Cancer Cells Produced in Response to Ionizing Radiation Retain Viability and Replicate Their Genome

    PubMed Central

    Mirzayans, Razmik; Andrais, Bonnie; Scott, April; Wang, Ying W.; Kumar, Piyush; Murray, David

    2017-01-01

    Loss of wild-type p53 function is widely accepted to be permissive for the development of multinucleated giant cells. However, whether therapy-induced multinucleation is associated with cancer cell death or survival remains controversial. Herein, we demonstrate that exposure of p53-deficient or p21WAF1 (p21)-deficient solid tumor-derived cell lines to ionizing radiation (between 2 and 8 Gy) results in the development of multinucleated giant cells that remain adherent to the culture dish for long times post-irradiation. Somewhat surprisingly, single-cell observations revealed that virtually all multinucleated giant cells that remain adherent for the duration of the experiments (up to three weeks post-irradiation) retain viability and metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and the majority (>60%) exhibit DNA synthesis. We further report that treatment of multinucleated giant cells with pharmacological activators of apoptosis (e.g., sodium salicylate) triggers their demise. Our observations reinforce the notion that radiation-induced multinucleation may reflect a survival mechanism for p53/p21-deficient cancer cells. With respect to evaluating radiosensitivity, our observations underscore the importance of single-cell experimental approaches (e.g., single-cell MTT) as the creation of viable multinucleated giant cells complicates the interpretation of the experimental data obtained by commonly-used multi-well plate colorimetric assays. PMID:28208747

  12. Intercellular cytosolic transfer correlates with mesenchymal stromal cell rescue of umbilical cord blood cell viability during ex vivo expansion

    PubMed Central

    Chu, Pat P. Y.; Bari, Sudipto; Fan, Xiubo; Gay, Florence P. H.; Ang, Justina M. L.; Chiu, Gigi N. C.; Lim, Sai K.; Hwang, William Y. K.

    2012-01-01

    Background aims. Mesenchymal stromal cells (MSC) have been observed to participate in tissue repair and to have growth-promoting effects on ex vivo co-culture with other stem cells. Methods. In order to evaluate the mechanism of MSC support on ex vivo cultures, we performed co-culture of MSC with umbilical cord blood (UCB) mononuclear cells (MNC) (UCB-MNC). Results. Significant enhancement in cell growth correlating with cell viability was noted with MSC co-culture (defined by double-negative staining for Annexin-V and 7-AAD; P<0.01). This was associated with significant enhancement of mitochondrial membrane potential (P<0.01). We postulated that intercellular transfer of cytosolic substances between MSC and UCB-MNC could be one mechanism mediating the support. Using MSC endogenously expressing green fluorescent protein (GFP) or labeled with quantum dots (QD), we performed co-culture of UCB-MNC with these MSC. Transfer of these GFP and QD was observed from MSC to UCB-MNC as early as 24 h post co-culture. Transwell experiments revealed that direct contact between MSC and UCB-MNC was necessary for both transfer and viability support. UCB-MNC tightly adherent to the MSC layer exhibited the most optimal transfer and rescue of cell viability. DNA analysis of the viable, GFP transfer-positive UCB-MNC ruled out MSC transdifferentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB-MNC viability (P< 0.0001). Conclusions. Collectively, the data suggest that intercellular transfer of cytosolic materials could be one novel mechanism for preventing UCB cell death in MSC co-culture. PMID:22775077

  13. 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.

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

    PubMed

    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%.

  15. 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.

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

    PubMed

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

    2013-09-13

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

  17. 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

  18. 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

  19. Silencing of Wnt10B reduces viability of heptocellular carcinoma HepG2 cells

    PubMed Central

    Wu, Guohui; Fan, Xiaoli; Sun, Li

    2015-01-01

    Dysregulation of Wnt-mediated β-catenin signaling is associated with carcinogenesis and progression of hepatocellular carcinoma (HCC). Our previous studies showed that the Wnt10B gene, a member of Wnt gene family, over-activated in HCC tissues and cells. Here we demonstrate that stable silencing of Wnt10B reduces the viability of HCC cells in culture. HepG2, a human HCC cell line, was cultured in vitro and Wnt10B gene in the cells stably silenced, as showed in Western blotting analysis, by the shRNA interference with lentivirus plasmid transfection. Compared to the control (HepG2 cells without Wnt10B silencing), the Wnt10B-silencing cells showed significant reductions in proliferation, colony formation, migration and invasion. Furthermore, serum deprivation-induced apoptotic death, assessed by Hoechst 33342 staining and fluorescent microscopy, increased significantly in the Wnt10B-silencing cells. FACScan analysis indicated an arrest of the cell cycle in the Wnt10B-silencing HCC cells, with significant increases in the number of cells in G0-G1 and S phases. Thus, we hypothesize that Wnt10B plays an oncogenic role in HCC and is a potential therapeutic target. PMID:26269753

  20. A Facile Method for Simultaneously Measuring Neuronal Cell Viability and Neurite Outgrowth

    PubMed Central

    K. Hancock, Michael; Kopp, Leisha; Kaur, Navjot; Hanson, Bonnie J.

    2015-01-01

    Neurite outgrowth is an important morphological phenotype of neuronal cells that correlates with their function and cell health, yet there are limited methods available for measuring this phenomenon. Current approaches to measuring neurite outgrowth are laborious and time-consuming, relying largely upon immunocytochemical staining of neuronal markers (e.g., beta-III tubulin or MAP2) followed by manual or automated microscopy for image acquisition and analysis. Here we report the development of a quick and simple dual-color fluorescent dye-based staining method that allows for the simultaneous measurement of neuronal cell health and relative neurite outgrowth from the same sample. An orangered fluorescent dye that stains cell membrane surfaces is used as an indirect reporter of changes in relative neurite outgrowth due to alterations in the number or length of membrane projections emanating from neuronal cell bodies. Cell viability is assessed simultaneously via the use of a cell-permeant dye that is converted by intracellular esterase activity from a non-fluorescent substrate to a green-fluorescent product. Using Neuroscreen-1 cells (a PC-12 subclone), primary rat cortex neurons, and human induced pluripotent stem cell (iPSC)-derived neurons, we demonstrate that this multiplex assay allows for rapid visualization and unbiased, quantitative plate reader analysis of neuronal cell health and neurite outgrowth. PMID:25853055

  1. Fe-Mn alloys for metallic biodegradable stents: degradation and cell viability studies.

    PubMed

    Hermawan, Hendra; Purnama, Agung; Dube, Dominique; Couet, Jacques; Mantovani, Diego

    2010-05-01

    Biodegradable stents have shown their potential to be a valid alternative for the treatment of coronary artery occlusion. This new class of stents requires materials having excellent mechanical properties and controllable degradation behaviour without inducing toxicological problems. The properties of the currently considered gold standard material for stents, stainless steel 316L, were approached by new Fe-Mn alloys. The degradation characteristics of these Fe-Mn alloys were investigated including in vitro cell viability. A specific test bench was used to investigate the degradation in flow conditions simulating those of coronary artery. A water-soluble tetrazolium test method was used to study the effect of the alloy's degradation product to the viability of fibroblast cells. These tests have revealed the corrosion mechanism of the alloys. The degradation products consist of metal hydroxides and calcium/phosphorus layers. The alloys have shown low inhibition to fibroblast cells' metabolic activities. It is concluded that they demonstrate their potential to be developed as degradable metallic biomaterials.

  2. 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

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

    PubMed

    Winkelströter, Lizziane Kretli; De Martinis, Elaine C P

    2015-03-01

    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.

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

    PubMed

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

    2005-07-01

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

  5. Modulation of Autophagy by a Thioxanthone Decreases the Viability of Melanoma Cells.

    PubMed

    Lima, Raquel T; Sousa, Diana; Paiva, Ana M; Palmeira, Andreia; Barbosa, João; Pedro, Madalena; Pinto, Madalena M; Sousa, Emília; Vasconcelos, M Helena

    2016-10-10

    (1) Background: Our previous studies unveiled the hit thioxanthone TXA1 as an inhibitor of P-glycoprotein (drug efflux pump) and of human tumor cells growth, namely of melanoma cells. Since TXA1 is structurally similar to lucanthone (an autophagy inhibitor and apoptosis inducer) and to N(10)-substituted phenoxazines (isosteres of thioxanthones, and autophagy inducers), this study aimed at further assessing its cytotoxic mechanism and evaluating its potential as an autophagy modulator in A375-C5 melanoma cells; (2) Methods: Flow cytometry with propidium iodide (PI) for cell cycle profile analysis; Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry with Annexin V/PI labeling and Western blot for apoptosis analysis were conducted. A pharmacophore approach was used for mapping TXA1 onto pharmacophores for autophagy induction. Autophagy analyses included transmission electron microscopy for visualization of autophagic structures, fluorescence microscopy for observation of monodansylcadaverine (MDC) staining, pattern of LC3 expression in the cells and acridine orange staining, and Western blot for autophagic proteins expression; (3) Results: TXA1 induced autophagy of melanoma cells at the GI50 concentration (3.6 μM) and apoptosis at twice that concentration. Following treatment with TXA1, autophagic structures were observed, together with the accumulation of autophagosomes and the formation of autophagolysosomes. An increase in LC3-II levels was also observed, which was reverted by 3-methyladenine (3-MA) (an early stage autophagy-inhibitor) but further increased by E-64d/pepstatin (late-stage autophagy inhibitors). Finally, 3-MA also reverted the effect of TXA1 in cellular viability; (4) Conclusion: TXA1 decreases the viability of melanoma cells by modulation of autophagy and may, therefore, serve as a lead compound for the development of autophagy modulators with antitumor activity.

  6. Ursolic acid and resveratrol synergize with chloroquine to reduce melanoma cell viability.

    PubMed

    Junco, Jacob J; Mancha-Ramirez, Anna; Malik, Gunjan; Wei, Sung-Jen; Kim, Dae Joon; Liang, Huiyun; Slaga, Thomas J

    2015-04-01

    Malignant melanoma is associated with a 5-year survival rate of less than 20% once metastasized. Malignant melanoma cells exhibit increased levels of autophagy, a process of intracellular digestion that allows cells to survive various stresses including chemotherapies, resulting in reduced patient survival. Autophagy can be inhibited by chemicals like chloroquine (CQ), which prevents fusion of autophagosomes to lysosomes, resulting in autophagosome accumulation in most systems. Here, we describe how tested CQ to see whether it could sensitize B16F10 metastatic mouse melanoma cells to the anticancer activities of the natural compounds ursolic acid (UA) and resveratrol (RES). CQ with UA or RES strongly and synergistically reduced the viability of B16F10 mouse melanoma and A375 human melanoma cells. Surprisingly, flow cytometry of acridine orange-stained cells showed that UA or RES in combination with CQ significantly reduced autophagosome levels. Western blotting analysis revealed that CQ plus UA or RES paradoxically increased LC3II, indicative of autophagosome accumulation. In addition, CQ plus RES synergistically decreased the levels of both autophagy initiator beclin-1 and autophagy supporter p62. These results indicate that CQ with UA or RES strongly and synergistically reduces the viability of B16F10 and A375 melanoma cells. However, studies on B16F10 cells have shown that the synergistic effect was not mediated by inhibition of autophagy induced by UA or RES. These compounds are well-tolerated in humans, and CQ has shown promise as an adjuvant therapy. These combinations may be valuable treatment strategies for melanoma.

  7. Cell viability and chondrogenic differentiation capability of human mesenchymal stem cells after iron labeling with iron sucrose.

    PubMed

    Papadimitriou, Nikolaos; Thorfve, Anna; Brantsing, Camilla; Junevik, Katarina; Baranto, Adad; Barreto Henriksson, Helena

    2014-11-01

    For evaluation of cell therapy strategies using human mesenchymal stem cells (hMSCs), it is important to be able to trace transplanted cells and their distribution in tissues, for example, cartilage, over time. The aim of the study was to determine effects on cell viability, traceability, and chondrogenic differentiation of hMSCs after iron labeling with iron sucrose. hMSCs were collected (seven donors, 13-57 years) from patients undergoing spinal surgery. Two subsets of experiments were performed. (1) Iron labeling of hMSCs: 1 mg/mL of Venofer(®) (iron sucrose) was added (16 h) to cultures. hMSCs were examined for uptake of iron sucrose (Prussian blue staining) and cell viability (flow cytometry). (2) Iron-labeled hMSCs (passage 4) (n=4, pellet mass), 200,000 cells/tube, were cultured (DMEM-HG) with 10 ng/mL TGFβ and compared with controls (from each donor). The pellets were harvested at days 7, 14, and 28. Real-time PCR, IHC, and histology were used to evaluate SOX9, ACAN, C6S, and COL2A1 expression. Mean number of cells containing iron deposits was 98.1% and mean cell viability was 92.7% (no significant difference compared with unlabeled control cells). Pellets containing iron-labeled cells expressed COL2A1 on protein level (all time points), in similar levels as controls, and glycosaminoglycan accumulation was observed in iron-labeled pellets (day 14 or day 28). Results were supported by the expression of chondrogenic genes SOX9, ACAN, and COL2A1. The results in vitro indicate that iron sucrose can be used as a cell tracer for evaluation of cellular distribution in vivo after transplantation of MSCs and thus contribute with important knowledge when exploring new treatment strategies for degenerated cartilaginous tissues.

  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.

  9. 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.

  10. 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.

  11. ATM participates in the regulation of viability and cell cycle via ellipticine in bladder cancer

    PubMed Central

    Tao, Shuixiang; Meng, Shuai; Zheng, Xiangyi; Xie, Liping

    2017-01-01

    Ellipticine, an alkaloid isolated from Apocyanaceae plants, has been demonstrated to exhibit antitumor activity in several cancers. However, the effect and the mechanisms underlying its action have not been investigated in human bladder cancer cells. The aim of the present study was to investigate the effect and mechanism of ellipticine on the behavior of T-24 bladder cancer cells. T-24 cells were treated with varying concentrations and durations of ellipticine. Cell viability was evaluated by Cell Counting Kit-8 assay. Cell motility was analyzed by Transwell migration assay. Flow cytometry, reverse transcription-quantitative polymerase chain reaction and western blot analyses were performed to detect the cell cycle and signaling pathways involved. The results demonstrated that ellipticine suppressed proliferation and inhibited the migration ability of T-24 bladder cancer cells in a dose- and time-dependent manner, and resulted in G2/M cell cycle arrest. The mechanism of this action was demonstrated to be due to ellipticine-triggered activation of the ATM serine/threonine kinase pathway. These data therefore suggest that ellipticine may be effective towards treating human bladder cancer. PMID:28138703

  12. Exploring the dark side of MTT viability assay of cells cultured onto electrospun PLGA-based composite nanofibrous scaffolding materials.

    PubMed

    Qi, Ruiling; Shen, Mingwu; Cao, Xueyan; Guo, Rui; Tian, Xuejiao; Yu, Jianyong; Shi, Xiangyang

    2011-07-21

    One major method used to evaluate the biocompatibility of porous tissue engineering scaffolding materials is MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT cell viability assay is based on the absorbance of the dissolved MTT formazan crystals formed in living cells, which is proportional to the number of viable cells. Due to the strong dye sorption capability of porous scaffolding materials, we propose that the cell viability determined from the MTT assay is likely to give a false negative result. In this study, we aim to explore the effect of the adsorption of MTT formazan on the accuracy of the viability assay of cells cultured onto porous electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers, HNTs (halloysite nanotubes)/PLGA, and CNTs (multiwalled carbon nanotubes)/PLGA composite nanofibrous mats. The morphology of electrospun nanofibers and L929 mouse fibroblasts cultured onto the nanofibrous scaffolds were observed using scanning electron microscopy. The viability of cells proliferated for 3 days was evaluated through the MTT assay. In the meantime, the adsorption of MTT formazan onto the same electrospun nanofibers was evaluated and the standard concentration-absorbance curve was obtained in order to quantify the contribution of the adsorbed MTT formazan during the MTT cell viability assay. We show that the PLGA, and the HNTs- or CNTs-doped PLGA nanofibers display appreciable MTT formazan dye sorption, corresponding to 35.6-50.2% deviation from the real cell viability assay data. The better dye sorption capability of the nanofibers leads to further deviation from the real cell viability. Our study gives a general insight into accurate MTT cytotoxicity assessment of various porous tissue engineering scaffolding materials, and may be applicable to other colorimetric assays for analyzing the biological properties of porous scaffolding materials.

  13. 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.

  14. The viability of mouse spermatogonial germ cells on a novel scaffold, containing human serum albumin and calcium phosphate nanoparticles

    PubMed Central

    Yadegar, Mona; Hekmatimoghaddam, Seyed Hossein; Nezami Saridar, Saeide; Jebali, Ali

    2015-01-01

    Background: In spermatogenesis, spermatogonial cells differentiate to the haploid gametes. It has been shown that spermatogenesis can be done at in vitro condition. In vitro spermatogenesis may provide an open window to treat male infertility. Objective: The aim of this study was to evaluate the effects of a novel scaffold containing human serum albumin (HSA)/tri calcium phosphate nanoparticles (TCP NPs) on the mouse spermatogonial cell line (SCL). Materials and Methods: First, TCP NPs were synthesized by reaction of calcium nitrate and diammonium phosphate at pH 13. Then, serial concentrations of TCP NPs were separately added to 500 mg/mL HSA, and incubated in the 100oC water for 30 min. In the next step, each scaffold was cut (2×2mm), placed into sterile well of microplate, and then incubated for 1, 2, and 3 days at 37oC with mouse SCL. After incubation, the cytotoxicity of the scaffolds was evaluated by different tests including 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) assay, vital staining, and cell counting. On the other hand, the release of TCP NPs and HSA from the scaffolds was measured. Results: Based on microscopic observation, the size of cavities for all scaffolds was near 200-500 µm, and the size of TCP NPs was near 50-100 nm. All toxicity tests showed that the increase of TCP concentration in the scaffold did not affect mouse SCL. It means that the percentage of cell viability, LDH release, vital cells, and cell quantity was 85%, 105%, 90%, and 110%, respectively. But, the increase of incubation time led to increase of LDH release (up to 115%) and cell count (up to 115%). Also, little decrease of cell viability and vital cells was seen when incubation time was increased. Here, no release of TCP NPs and HSA was seen after increase of TCP concentration and incubation time. Conclusion: It can be concluded that the increase of TCP concentration in HSA/ TCP NPs scaffold does not lead to

  15. 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.

  16. 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

  17. Post-irradiation viability and cytotoxicity of natural killer cells isolated from human peripheral blood using different methods.

    PubMed

    Hietanen, Tenho; Pitkänen, Maunu; Kapanen, Mika; Kellokumpu-Lehtinen, Pirkko-Liisa

    2016-01-01

    Purpose We compared the pre- and post-irradiation viability and cytotoxicity of human peripheral natural killer cell (NK) populations obtained using different isolation methods. Material and methods Three methods were used to enrich total NK cells from buffy coats: (I) a Ficoll-Paque gradient, plastic adherence and a nylon wool column; (II) a discontinuous Percoll gradient; or (III) the Dynal NK cell isolation kit. Subsequently, CD16(+) and CD56(+) NK cell subsets were collected using (IV) flow cytometry or (V) magnetic-activated cell sorting (MACS) NK cell isolation kits. The yield, viability, purity and cytotoxicity of the NK cell populations were measured using trypan blue exclusion, flow cytometry using propidium iodide and (51)Cr release assays after enrichments as well as viability and cytotoxicity after a single radiation dose. Results The purity of the preparations, as measured by the CD16(+) and CD56(+) cell content, was equally good between methods I-III (p = 0.323), but the content of CD16(+) and CD56(+) cells using these methods was significantly lower than that using methods IV and V (p = 0.005). The viability of the cell population enriched via flow cytometry (85.5%) was significantly lower than that enriched via other methods (99.4-98.0%, p = 0.003). The cytotoxicity of NK cells enriched using methods I-III was significantly higher than that of NK cells enriched using methods IV and V (p = 0.000). In vitro the NK cells did not recover cytotoxic activity following irradiation. In addition, we detected considerable inter-individual variation in yield, cytotoxicity and radiation sensitivity between the NK cells collected from different human donors. Conclusions The selection of the appropriate NK cell enrichment method is very important for NK cell irradiation studies. According to our results, the Dynal and MACS NK isolation kits best retained the killing capacity and the viability of irradiated NK cells.

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

    PubMed

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

    2017-01-16

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

  19. 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

  20. 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.

  1. 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.

  2. A cell cycle-regulated bacterial DNA methyltransferase is essential for viability.

    PubMed Central

    Stephens, C; Reisenauer, A; Wright, R; Shapiro, L

    1996-01-01

    The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the alpha subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division. Images Fig. 2 Fig. 3 PMID:8577742

  3. 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.

  4. Ultrasound-microbubble mediated cavitation of plant cells: effects on morphology and viability.

    PubMed

    Qin, Peng; Xu, Lin; Zhong, Wenjing; Yu, Alfred C H

    2012-06-01

    The interaction between ultrasound pulses and microbubbles is known to generate acoustic cavitation that may puncture biological cells. This work presents new experimental findings on the bioeffects of ultrasound-microbubble mediated cavitation in plant cells with emphasis on direct observations of morphological impact and analysis of viability trends in tobacco BY-2 cells that are widely studied in higher plant physiology. The tobacco cell suspensions were exposed to 1 MHz ultrasound pulses in the presence of 1% v/v microbubbles (10% duty cycle; 1 kHz pulse repetition frequency; 70 mm between probe and cells; 1-min exposure time). Few bioeffects were observed at low peak negative pressures (<0.4 MPa) where stable cavitation presumably occurred. In contrast, at 0.9 MPa peak negative pressure (with more inertial cavitation activities according to our passive cavitation detection results), random pores were found on tobacco cell wall (observed via scanning electron microscopy) and enhanced exogenous uptake into the cytoplasm was evident (noted in our fluorescein isothiocyanate dextran uptake analysis). Also, instant lysis was observed in 23.4% of cells (found using trypan blue staining) and programmed cell death was seen in 23.3% of population after 12 h (determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling [TUNEL]). These bioeffects generally correspond in trend with those for mammalian cells. This raises the possibility of developing ultrasound-microbubble mediated cavitation into a targeted gene transfection paradigm for plant cells and, conversely, adopting plant cells as experimental test-beds for sonoporation-based gene therapy in mammalian cells.

  5. The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability

    PubMed Central

    Lange, Sabine S.; Tomida, Junya; Boulware, Karen S.; Bhetawal, Sarita; Wood, Richard D.

    2016-01-01

    DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/- Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/- Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents. PMID:26727495

  6. Nanofibrous chitosan-polyethylene oxide engineered scaffolds: a comparative study between simulated structural characteristics and cells viability.

    PubMed

    Kazemi Pilehrood, Mohammad; Dilamian, Mandana; Mirian, Mina; Sadeghi-Aliabadi, Hojjat; Maleknia, Laleh; Nousiainen, Pertti; Harlin, Ali

    2014-01-01

    3D nanofibrous chitosan-polyethylene oxide (PEO) scaffolds were fabricated by electrospinning at different processing parameters. The structural characteristics, such as pore size, overall porosity, pore interconnectivity, and scaffold percolative efficiency (SPE), were simulated by a robust image analysis. Mouse fibroblast cells (L929) were cultured in RPMI for 2 days in the presence of various samples of nanofibrous chitosan/PEO scaffolds. Cell attachments and corresponding mean viability were enhanced from 50% to 110% compared to that belonging to a control even at packed morphologies of scaffolds constituted from pores with nanoscale diameter. To elucidate the correlation between structural characteristics within the depth of the scaffolds' profile and cell viability, a comparative analysis was proposed. This analysis revealed that larger fiber diameters and pore sizes can enhance cell viability. On the contrary, increasing the other structural elements such as overall porosity and interconnectivity due to a simultaneous reduction in fiber diameter and pore size through the electrospinning process can reduce the viability of cells. In addition, it was found that manipulation of the processing parameters in electrospinning can compensate for the effects of packed morphologies of nanofibrous scaffolds and can thus potentially improve the infiltration and viability of cells.

  7. Effects of combination of melatonin and laser irradiation on ovarian cancer cells and endothelial lineage viability.

    PubMed

    Akbarzadeh, Maryam; Nouri, Mohammad; Banekohal, Maryam Vahidi; Cheraghi, Omid; Tajalli, Habib; Movassaghpour, Aliakbar; Soltani, Sina; Cheraghi, Hadi; Feizy, Navid; Montazersaheb, Soheila; Rahbarghazi, Reza; Samadi, Nasser

    2016-11-01

    The main goal of anti-cancer therapeutic approaches is to induce apoptosis in tumor masses but not in the normal tissues. Nevertheless, the combination of photodynamic irradiation with complementary oncostatic agents contributes to better therapeutic performance. Here, we applied two different cell lines; SKOV3 ovarian carcinoma cells and HUVECs umbilical cord cells as in vitro models to pinpoint whether pharmacological concentration of melatonin in combination with photodynamic therapy induces cell cytotoxicity. The cells were separately treated with various concentrations of melatonin (0 to 10 mM) and photodynamic irradiation alone or in combination. Cells were preliminary exposed to increasing concentrations of melatonin for 24 h and subsequently underwent laser irradiation for 60 s with an output power of 80 mW in continuous mode at 675 nm wavelength and a total light dose of 13.22 J/cm(2). Cell viability, apoptosis/necrosis rates, and reactive oxygen species levels as well as heat shock protein 70 expression were monitored after single and combined treatments. A statistical analysis was performed by applying one-way analysis of variance (ANOVA) and post hoc Tukey's test. Combination treatment of both cell lines caused a marked increase in apoptosis/necrosis rate, reactive oxygen species generation, and heat shock protein 70 expression compared to incubation of the cells with each agent alone (p < 0.05). SKOV3 cancer cells expressed higher level of heat shock protein 70 under experimental procedure as compared to HUVECs (p < 0.05). Our results introduce melatonin as a potent stimulus for enhancing the efficacy of laser on induction of apoptosis in tumor cells.

  8. 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.

  9. Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles

    PubMed Central

    Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel

    2016-01-01

    Summary Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle–cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN. PMID:27826507

  10. 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

  11. [Simultaneous staining with fluorescein diacetate-propidium iodide to determine isolated cochlear outer hair cell viability of guinea pig].

    PubMed

    Yu, Q; Shi, H; Wang, J

    1995-01-01

    A simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is discribed for use in the determination of isolated cochlear outer hair cell viability. With exciter light, viable cells fluoresce bright green, while nonviable cells are bright red. In cell culture and cytotoxicity studies, double-staining with FDA-PI is a accurate method to discriminate between live and nonviable cells.

  12. Ferulic acid decreases cell viability and colony formation while inhibiting migration of MIA PaCa-2 human pancreatic cancer cells in vitro.

    PubMed

    Fahrioğlu, Umut; Dodurga, Yavuz; Elmas, Levent; Seçme, Mücahit

    2016-01-15

    Novel and combinatorial treatment methods are becoming sought after entities in cancer treatment and these treatments are even more valuable for pancreatic cancer. The scientists are always on the lookout for new chemicals to help them in their fight against cancer. In this study, we examine the effects of ferulic acid (FA), a phenolic compound, on gene expression, viability, colony formation and migration/invasion in the cultured MIA PaCa-2 human pancreatic cancer cell. Cytotoxic effects of FA were determined by using trypan blue dye exclusion test and Cell TiterGlo (CTG) assay. IC50 dose in MIA PaCa-2 cells was detected as 500μM/ml at the 72nd hour. Expression profiles of certain cell cycle and apoptosis genes such as CCND1 (cyclin D1),CDK4, CDK6, RB, p21, p16, p53, caspase-3, caspase-9, caspase-8, caspase-10, Bcl-2, BCL-XL,BID, DR4,DR5,FADD,TRADD,PARP, APAF, Bax, Akt, PTEN, PUMA, NOXA, MMP2, MMP9, TIMP1 and TIMP2 were determined by real-time PCR. The effect of FA on cell viability was determined by CellTiter-Glo® Luminescent Cell Viability Assay. Additionally, effects of FA on colony formation and invasion were also investigated. It was observed that FA caused a significant decrease in the expression of CCND1, CDK 4/6, Bcl2 and caspase 8 and 10 in the MIA PaCa-2 cells while causing an increase in the expression of p53, Bax, PTEN caspase 3 and 9. FA was observed to decrease colony formation while inhibiting cell invasion and migration as observed by the BioCoat Matrigel Invasion Chamber guide and colony formation assays. In conclusion, FA is thought to behave as an anti-cancer agent by affecting cell cycle, apoptotic, invasion and colony formation behavior of MIA PaCa-2 cells. Therefore, FA is placed as a strong candidate for further studies aimed at finding a better, more effective treatment approach for pancreatic cancer.

  13. 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.

  14. 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

  15. The effect of saturated fatty acids on methanogenesis and cell viability of Methanobrevibacter ruminantium.

    PubMed

    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.

  16. TIA-1 or TIAR is required for DT40 cell viability.

    PubMed

    Le Guiner, Caroline; Gesnel, Marie-Claude; Breathnach, Richard

    2003-03-21

    TIA-1 and TIAR are a pair of related RNA-binding proteins which have been implicated in apoptosis. We show that chicken DT40 cells with both tia-1 alleles and one tiar allele disrupted (tia-1(-/-)tiar(-/+) cells) are viable. However, their growth and survival in medium containing low serum levels is significantly reduced compared with DT40 cells. The remaining intact tiar allele in tia-1(-/-)tiar(-/+) cells can only be disrupted if TIA-1 expression is first restored to the cells by transfection of a TIA-1 expression vector. We conclude that DT40 cells require either TIA-1 or TIAR for viability. TIA-1 overexpression in tia-1(-/-)tiar(-/+) cells leads to a radical drop in TIAR levels, by inducing efficient splicing of two tiar alternative exons carrying in-frame stop codons. In wild-type DT40 cells, tiar transcripts including these exons can also be detected. These transcripts increase significantly in abundance in cycloheximide-treated cells, suggesting that splicing of the exons exposes mRNAs to nonsense-mediated mRNA decay. TIA-1 or TIAR depletion leads to a marked drop in splicing of the exons. The human tiar gene contains a corresponding pair of TIA-1-inducible alternative exons, and we show that there is very high sequence conservation between chickens and humans of the exon pair and parts of the flanking introns. The TIA-1/TIAR responsiveness of these alternative tiar exons is likely to be of physiological importance for controlling TIAR levels.

  17. Modulation of viability and apoptosis of UVB-exposed human keratinocyte HaCaT cells by aqueous methanol extract of laver (Porphyra yezoensis).

    PubMed

    Kim, Saerong; You, Dong Hun; Han, Taejun; Choi, Eun-Mi

    2014-12-01

    We investigated the effect of 80% methanol extract of laver (Porphyra yezoensis) on the UVB-exposed HaCaT cells, human keratinocytes. The laver extract showed absorbance spectrum characteristic of porphyra-334 or shinorine, major mycosporine-like amino acids (MAAs) in red algae, and contained phenolic compounds. UVB exposure decreased cell viability and increased apoptotic cell fractions, and it also decreased the ratio of reduced (GSH) to oxidized glutathione (GSSG) and the total glutathione content. Post-treatment with the laver extract significantly increased the net viability and also the apoptotic cell fractions of UVB-exposed cells. The extract caused increase in GSH/GSSG ratio, yet it exacerbated the decrease in glutathione content in the UVB-exposed cells. These effects of the laver extract were also manifested in the sham-exposed cells, suggesting that those effects might be general phenomena caused by the laver extract. The extract treatment enhanced the UVB-induced phosphorylation of JNK and ERK, affecting more the latter. Our results suggest that the post-treatment with laver extract may protect UVB-exposed skin cells not only by increasing overall cell proliferation but also by enhancing apoptosis of damaged cells, via activating JNK and ERK signaling pathways, in which modulation of the content and redox status of glutathione may take significant parts.

  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. Withagulatin A inhibits hepatic stellate cell viability and procollagen I production through Akt and Smad signaling pathways

    PubMed Central

    Liu, Qiong; Chen, Jing; Wang, Xu; Yu, Liang; Hu, Li-hong; Shen, Xu

    2010-01-01

    Aim: To investigate the effects of the natural product Withagulatin A on hepatic stellate cell (HSC) viability and type I procollagen production. The potential mechanism underlying the pharmacological actions was also explored. Methods: The effect of Withagulatin A on cell viability was evaluated in HSC and LX-2 cells using a sulforhodamine B (SRB) assay. Cell cycle distribution was analyzed using flow cytometry. Type I procollagen gene expression was determined using real-time PCR. Regulation of signaling molecules by Withagulatin A was detected using Western blotting. Results: Primary rat HSCs and the human hepatic stellate cell line LX-2 treated with Withagulatin A (0.625-20 μmol/L) underwent a dose-dependent decrease in cell viability, which was associated with S phase arrest and the induction of cell apoptosis. In addition, the natural product decreased phosphorylation of the Akt/mTOR/p70S6K pathway that controls cell proliferation and survival. Furthermore, Withagulatin A (1, 2 μmol/L) inhibited transforming growth factor-β (TGF-β) stimulated type I procollagen gene expression, which was attributable to the suppression of TGF-β stimulated Smad2 and Smad3 phosphorylation. Conclusion: Our results demonstrated that Withagulatin A potently inhibited HSC viability and type I procollagen production, thereby implying that this natural product has potential use in the development of anti-fibrogenic reagents for the treatment of hepatic fibrosis. PMID:20644552

  20. Cell viability and adhesion on diamond-like carbon films containing titanium dioxide nanoparticles

    NASA Astrophysics Data System (ADS)

    Wachesk, C. C.; Pires, C. A. F.; Ramos, B. C.; Trava-Airoldi, V. J.; Lobo, A. O.; Pacheco-Soares, C.; Marciano, F. R.; Da-Silva, N. S.

    2013-02-01

    The combination of low friction, wear resistance, high hardness, biocompatibility and chemical inertness makes diamond-like carbon (DLC) films suitable in a numerous applications in biomedical engineering. The cell viability and adhesion of L929 mouse fibroblasts was investigated using two different colorimetric assays: (i) 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT), and (ii) lactate dehydrogenase (LDH). The films were growth on 316L stainless steel substrates using plasma enhanced chemical vapor deposition technique from a dispersion of TiO2 nanopowder in hexane. The increasing concentration of TiO2 nanoparticles in DLC films enhanced the mitochondrial activity and decreases the LDH activity on these samples. Fluorescence and scanning electron microscopy corroborate the results. These experiments show the potential use of DLC and TiO2-DLC films in biomedical applications.

  1. miR-433 reduces cell viability and promotes cell apoptosis by regulating MACC1 in colorectal cancer

    PubMed Central

    Li, Jiaxin; Mao, Xuping; Wang, Xing; Miao, Ganggang; Li, Jiaxin

    2017-01-01

    MicroRNAs (miRNAs) are reported to have important roles in regulating the progression of numerous human cancers, although little is known regarding the role of miRNAs in colorectal cancer. The present study aimed to investigate the role of microRNA-433 (miR-433) in colorectal cancer. The expression levels of miR-433 and its target gene metastasis associated in colon cancer-1 (MACC1) in colorectal cancer tissues were evaluated using reverse transcription-quantitative polymerase chain reaction and western blotting. Furthermore, flow cytometry and MTT assays were used to examine the apoptosis, cell cycle distribution and viability of human colorectal cancer cells, and luciferase reporter and western blot assays were performed to verify the regulatory mechanism of miR-433 on MACC1. In addition, caspase-3 and caspase-9 expression were examined using western blotting. It was demonstrated that miR-433 expression was downregulated in colorectal cancer tissues and cell lines. Artificial upregulation of miR-433 in colorectal cancer cell lines using miR-433 mimics revealed that upregulation of miR-433 was able to reduce the viability and promote the apoptosis of colorectal cancer cells by downregulating MACC1. Taken together, these results suggested that miR-433 may have an important role in the pathogenesis of colorectal cancer. PMID:28123526

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

    PubMed

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

    2014-11-01

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

  3. Cannabinoids synergize with carfilzomib, reducing multiple myeloma cells viability and migration

    PubMed Central

    Offidani, Massimo; Amantini, Consuelo; Gentili, Silvia; Soriani, Alessandra; Cardinali, Claudio; Leoni, Pietro; Santoni, Giorgio

    2016-01-01

    Several studies showed a potential anti-tumor role for cannabinoids, by modulating cell signaling pathways involved in cancer cell proliferation, chemo-resistance and migration. Cannabidiol (CBD) was previously noted in multiple myeloma (MM), both alone and in synergy with the proteasome inhibitor bortezomib, to induce cell death. In other type of human cancers, the combination of CBD with Δ9-tetrahydrocannabinol (THC) was found to act synergistically with other chemotherapeutic drugs suggesting their use in combination therapy. In the current study, we evaluated the effects of THC alone and in combination with CBD in MM cell lines. We found that CBD and THC, mainly in combination, were able to reduce cell viability by inducing autophagic-dependent necrosis. Moreover, we showed that the CBD-THC combination was able to reduce MM cells migration by down-regulating expression of the chemokine receptor CXCR4 and of the CD147 plasma membrane glycoprotein. Furthermore, since the immuno-proteasome is considered a new target in MM and also since carfilzomib (CFZ) is a new promising immuno-proteasome inhibitor that creates irreversible adducts with the β5i subunit of immuno-proteasome, we evaluated the effect of CBD and THC in regulating the expression of the β5i subunit and their effect in combination with CFZ. Herein, we also found that the CBD and THC combination is able to reduce expression of the β5i subunit as well as to act in synergy with CFZ to increase MM cell death and inhibits cell migration. In summary, these results proved that this combination exerts strong anti-myeloma activities. PMID:27769052

  4. 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.

  5. 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.

  6. Depletion of hCINAP by RNA interference causes defects in Cajal body formation, histone transcription, and cell viability.

    PubMed

    Zhang, Jinfang; Zhang, Feiyun; Zheng, Xiaofeng

    2010-06-01

    hCINAP is a highly conserved and ubiquitously expressed protein in eukaryotic organisms and its overexpression decreases the average number of Cajal bodies (CBs) with diverse nuclear functions. Here, we report that hCINAP is associated with important components of CBs. Depletion of hCINAP by RNA interference causes defects in CB formation and disrupts subcellular localizations of its components including coilin, survival motor neurons protein, spliceosomal small nuclear ribonucleoproteins, and nuclear protein ataxia-telangiectasia. Moreover, knockdown of hCINAP expression results in marked reduction of histone transcription, lower levels of U small nuclear RNAs (U1, U2, U4, and U5), and a loss of cell viability. Detection of increased caspase-3 activities in hCINAP-depleted cells indicate that apoptosis is one of the reasons for the loss of viability. Altogether, these data suggest that hCINAP is essential for the formation of canonical CBs, histone transcription, and cell viability.

  7. Effects of rice straw on the cell viability, photosynthesis, and growth of Microcystis aeruginosa

    NASA Astrophysics Data System (ADS)

    Su, Wen; Hagström, Johannes A.; Jia, Yuhong; Lu, Yaping; Kong, Fanxiang

    2014-01-01

    Rice straw is supposed to be an environment-friendly biomaterial for inhibiting the growth of harmful blooms of the cyanobacterium Microcystis aeruginosa. However, its potential mechanism is not well known. To explore this mechanism, the growth, cell viability (esterase activity, membrane potential, and membrane integrity), photosynthesis, and cell size of M. aeruginosa were determined using flow cytometry and Phyto-PAM after exposure to rice straw extracts (RSE). The results show that doses from 2.0 to 10.0 g/L of RSE efficiently inhibited the alga for 15 days, while the physiologic and morphologic responses of the cyanobacteria were time-dependent. RSE interfered with the cell membrane potential, cell size, and in vivo chlorophyll- a fluorescence on the first day. After 7 days of exposure, RSE was transported into the cytosol, which disrupted enzyme activity and photosynthesis. The cyanobacteria then started to repair its physiology (enzyme activity, photosynthesis) and remained viable, suggesting that rice straw act as an algistatic agent.

  8. The proprotein convertase furin is required to maintain viability of alveolar rhabdomyosarcoma cells

    PubMed Central

    Jaaks, Patricia; Meier, Gianmarco; Alijaj, Nagjie; Brack, Eva; Bode, Peter; Koscielniak, Ewa; Wachtel, Marco; Schäfer, Beat W.; Bernasconi, Michele

    2016-01-01

    Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Success of current therapies is still limited and outcome is particularly poor for metastatic alveolar rhabdomyosarcoma (aRMS). We previously identified the proprotein convertase furin as potential target for specific drug delivery with RMS-homing peptides. Furin is a protease that converts inactive precursor proteins into bioactive proteins and peptides. In this study, we investigate the biological role of furin in aRMS progression in vitro and in vivo. Furin expression was confirmed in over 86% RMS biopsies in a tissue microarray (n=89). Inducible furin silencing in vitro led to significant impairment of cell viability and proliferation in all investigated aRMS cell lines, but not in MRC5 fibroblasts. Furthermore, the aRMS cell lines Rh3 and Rh4 revealed to be very sensitive to furin silencing, undergoing caspase-dependent cell death. Notably, furin silencing in vivo led to complete remission of established Rh4 tumors and to delayed growth in Rh30 tumors. Taken together, these findings identify furin as an important factor for aRMS progression and survival. Thus, we propose furin as a novel therapeutic target for treatment of aRMS. PMID:27572312

  9. 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

  10. Viability, differentiation capacity, and detectability of super-paramagnetic iron oxide-labeled muscle precursor cells for magnetic-resonance imaging.

    PubMed

    Azzabi, Fahd; Rottmar, Markus; Jovaisaite, Virginija; Rudin, Markus; Sulser, Tullio; Boss, Andreas; Eberli, Daniel

    2015-02-01

    Cell therapies are a promising approach for the treatment of a variety of human conditions including stress urinary incontinence, but their success greatly depends on the biodistribution, migration, survival, and differentiation of the transplanted cells. Noninvasive in vivo cell tracking therefore presents an important aspect for translation of such a procedure into the clinics. Upon labeling with superparamagnetic iron oxide (SPIO) nanoparticles, cells can be tracked by magnetic resonance imaging (MRI), but possible adverse effect of the labeling have to be considered when labeling stem cells with SPIOs. In this study, human muscle precursor cells (hMPC) were labeled with increasing concentrations of SPIO nanoparticles (100-1600 μg/mL) and cell viability and differentiation capacity upon labeling was assessed in vitro. While a linear dependence between cell viability and nanoparticle concentration could be observed, differentiation capacity was not affected by the presence of SPIOs. Using a nude mouse model, a concentration (400 μg/mL) could be defined that allows reliable detection of hMPCs by MRI but does not influence myogenic in vivo differentiation to mature and functional muscle tissue. This suggests that such an approach can be safely used in a clinical setting to track muscle regeneration in patients undergoing cell therapy without negative effects on the functionality of the bioengineered muscle.

  11. Viability, Differentiation Capacity, and Detectability of Super-Paramagnetic Iron Oxide-Labeled Muscle Precursor Cells for Magnetic-Resonance Imaging

    PubMed Central

    Azzabi, Fahd; Rottmar, Markus; Jovaisaite, Virginija; Rudin, Markus; Sulser, Tullio; Boss, Andreas

    2015-01-01

    Cell therapies are a promising approach for the treatment of a variety of human conditions including stress urinary incontinence, but their success greatly depends on the biodistribution, migration, survival, and differentiation of the transplanted cells. Noninvasive in vivo cell tracking therefore presents an important aspect for translation of such a procedure into the clinics. Upon labeling with superparamagnetic iron oxide (SPIO) nanoparticles, cells can be tracked by magnetic resonance imaging (MRI), but possible adverse effect of the labeling have to be considered when labeling stem cells with SPIOs. In this study, human muscle precursor cells (hMPC) were labeled with increasing concentrations of SPIO nanoparticles (100–1600 μg/mL) and cell viability and differentiation capacity upon labeling was assessed in vitro. While a linear dependence between cell viability and nanoparticle concentration could be observed, differentiation capacity was not affected by the presence of SPIOs. Using a nude mouse model, a concentration (400 μg/mL) could be defined that allows reliable detection of hMPCs by MRI but does not influence myogenic in vivo differentiation to mature and functional muscle tissue. This suggests that such an approach can be safely used in a clinical setting to track muscle regeneration in patients undergoing cell therapy without negative effects on the functionality of the bioengineered muscle. PMID:24988198

  12. Fibroblasts maintain the phenotype and viability of the rat heparin-containing mast cell in vitro

    SciTech Connect

    Levi-Schaffer, F.; Austen, K.F.; Caulfield, J.P.; Hein, A.; Bloes, W.F.; Stevens, R.L.

    1985-11-01

    Rat serosal heparin-containing mast cells (HP-MC) were maintained in vitro for as long as 30 days when co-cultured with mouse skin-derived 3T3 fibroblasts. In contrast, when the mast cells were cultured alone, on fibronectin-, gelatin-, or dermal-collagen-coated dishes, on acid and heat-killed fibroblasts in the presence or absence of 24 hr fibroblast-conditioned medium, or on a monolayer of mouse serosal macrophages, they failed to adhere to the dishes, released significant amounts of their histamine and lactate dehydrogenase, and stained with trypan blue, indicating a loss of viability. The rat serosal HP-MC cultured with the 3T3 fibroblasts became so adherent to the fibroblasts that the two cell types could be separated from one another only by trypsinization. The cultured HP-MC stained with both alcian blue and safranin and continued to synthesize proteoglycan at a rate comparable to that of freshly isolated cells. The /sup 35/S-labeled proteoglycan synthesized by these cultured cells, like that produced by freshly isolated rat serosal HP-MC, was a 750,000 to 1,000,000 m.w. proteoglycan containing only heparin glycosaminoglycans of 50,000 to 100,000 m.w. As assessed by electron microscopy, many of the cultured HP-MC resembled freshly isolated cells except that some secretory granules had fused with one another in some cells. These results demonstrate that the in vivo differentiated rat HP-MC maintain their histology, morphology, immunologic responsiveness, histamine content, and ability to synthesize heparin proteoglycan when co-cultured with living fibroblasts.

  13. 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

  14. Allyl-isatin suppresses cell viability, induces cell cycle arrest, and promotes cell apoptosis in hepatocellular carcinoma HepG2 cells.

    PubMed

    Bian, Weihua; An, Yukuan; Qu, Huiqing; Yang, Yue; Yang, Junhou; Xu, Yanyan

    2016-06-01

    The anticancer effect of the newly synthesized isatin derivative, N-allyl-isatin (Allyl-I), was evaluated in vitro with human hepatocellular carcinoma HepG2 cells. Cell viability was detected by cell counting kit-8 (CCK8) assay. Acridine orange (AO)/ethidium bromide (EB) double staining was used to observe the cell morphology. Flow cytometry was used to assess the effects of Allyl-I on the cell cycle, apoptosis rate, and mitochondrial membrane potential (MMP). Western blot analysis was performed to detect the influence of Ally1-I on the expression of cytochrome c (cyt c), Bax, Bcl-2, and cleaved caspase-3. Allyl-I significantly inhibited HepG2 cell viability in a time- and dose-dependent manner. Allyl-I can induce cell cycle arrest in HepG2 cells at the G2/M phase. Apoptotic nuclear morphological changes were observed after AO/EB double staining. Fluorescein isothiocyanate-conjugated Annexin V (Annexin V-FITC) and propidium iodide (PI) double staining showed that the apoptotic rates significantly increased in the presence of Allyl-I. Rhodamine 123 staining indicated that Allyl-I can decrease the MMP. Allyl-I also altered the expression of mitochondrial apoptosis-related proteins. Protein levels of cyt c and cleaved caspase-3 were upregulated following Allyl-I treatment. By contrast, the Bcl-2/Bax ratio decreased. Results suggest that Allyl-I suppresses cell viability, induces cell cycle arrest, and promotes cell apoptosis in HepG2 cells. Furthermore, the induction of apoptosis might be correlated with the mitochondrial pathway.

  15. 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.

  16. 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

  17. Potassium uptake system Trk2 is crucial for yeast cell viability during anhydrobiosis.

    PubMed

    Borovikova, Diana; Herynkova, Pavla; Rapoport, Alexander; Sychrova, Hana

    2014-01-01

    Yeasts grow at very different potassium concentrations, adapting their intracellular cation levels to changes in the external environment. Potassium homeostasis is maintained with the help of several transporters mediating the uptake and efflux of potassium with various affinities and mechanisms. In the model yeast Saccharomyces cerevisiae, two uptake systems, Trk1 and Trk2, are responsible for the accumulation of a relatively high intracellular potassium content (200-300 mM) and the efflux of surplus potassium is mediated by the Tok1 channel and active exporters Ena ATPase and Nha1 cation/proton antiporter. Using a series of deletion mutants, we studied the role of individual potassium transporters in yeast cell resistance to dehydration. The Trk2 transporter (whose role in S. cerevisiae physiology was not clear) is important for cell viability in the stationary phase of growth and, moreover, it plays a crucial role in the yeast survival of dehydration/rehydration treatments. Mutants lacking the TRK2 gene accumulated significantly lower amounts of potassium ions in the stationary culture growth phase, and these lower amounts correlated with decreased resistance to dehydration/rehydration stress. Our results showed Trk2 to be the major potassium uptake system in stationary cells, and potassium content to be a crucial parameter for desiccation survival.

  18. Stn1 is critical for telomere maintenance and long-term viability of somatic human cells

    PubMed Central

    Boccardi, Virginia; Razdan, Neetu; Kaplunov, Jessica; Mundra, Jyoti J; Kimura, Masayuki; Aviv, Abraham; Herbig, Utz

    2015-01-01

    Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging-associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA-mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long-term viability of normal somatic mammalian cells. PMID:25684230

  19. In vitro degradation and cell viability assessment of Zn-3Mg alloy for biodegradable bone implants.

    PubMed

    Dambatta, M S; Murni, N S; Izman, S; Kurniawan, D; Froemming, G R A; Hermawan, H

    2015-05-01

    This article reports the in vitro degradation and cytotoxicity assessment of Zn-3Mg alloy developed for biodegradable bone implants. The alloy was prepared using casting, and its microstructure was composed of Mg2Zn11 intermetallic phase distributed within a Zn-rich matrix. The degradation assessment was done using potentiodynamic polarization and electrochemical impedance spectrometry. The cell viability and the function of normal human osteoblast cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and alkaline phosphatase extracellular enzyme activity assays. The results showed that the degradation rate of the alloy was slower than those of pure Zn and pure Mg due to the formation of a high polarization resistance oxide film. The alloy was cytocompatible with the normal human osteoblast cells at low concentrations (<0.5 mg/mL), and its alkaline phosphatase activity was superior to pure Mg. This assessment suggests that Zn-3Mg alloy has the potential to be developed as a material for biodegradable bone implants, but the toxicity limit must be carefully observed.

  20. 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.

  1. Dragon's Blood Sap (Croton Lechleri) As Storage Medium For Avulsed Teeth: In Vitro Study Of Cell Viability.

    PubMed

    Martins, Christine Men; Hamanaka, Elizane Ferreira; Hoshida, Thayse Yumi; Sell, Ana Maria; Hidalgo, Mirian Marubayashi; Silveira, Catarina Soares; Poi, Wilson Roberto

    2016-01-01

    Tooth replantation success depends on the condition of cementum periodontal ligament after tooth avulsion; which is influenced by storage medium. The dragon's blood (Croton lechleri) sap has been suggested as a promising medium because it supports collagen formation and exhibits healing, anti-inflammatory and antimicrobial properties. Thus, the aim of this study was to evaluate the efficacy of dragon's blood sap as a storage medium for avulsed teeth through evaluation of functional and metabolic cell viability. This in vitro study compared the efficacy of different storage media to maintain the viability of human peripheral blood mononuclear and periodontal ligament cells. A 10% dragon's blood sap was tested while PBS was selected as its control. Ultra pasteurized whole milk was used for comparison as a commonly used storage medium. DMEM and distilled water were the positive and negative controls, respectively. The viability was assessed through trypan blue exclusion test and colorimetric MTT assay after 1, 3, 6, 10 and 24 h of incubation. The dragon's blood sap showed promising results due to its considerable maintenance of cell viability. For trypan blue test, the dragon's blood sap was similar to milk (p<0.05) and both presented the highest viability values. For MTT, the dragon's blood sap showed better results than all storage media, even better than milk (p<0.05). It was concluded that the dragon's blood sap was as effective as milk, the gold standard for storage medium. The experimental sap preserved the membrane of all cells and the functional viability of periodontal ligament cells.

  2. 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

  3. Collagen-based silver nanoparticles: Study on cell viability, skin permeation, and swelling inhibition.

    PubMed

    Cardoso, Vinicius Saura; de Carvalho Filgueiras, Marcelo; Dutra, Yago Medeiros; Teles, Ramon Handerson Gomes; de Araújo, Alyne Rodrigues; Primo, Fernando Lucas; Mafud, Ana Carolina; Batista, Larissa Fernandes; Mascarenhas, Yvonne Primerano; Paino, Iêda Maria Martinez; Zucolotto, Valtencir; Tedesco, Antonio Claudio; Silva, Durcilene Alves; Leite, José Roberto S A; Dos Santos, José Ribeiro

    2017-05-01

    Collagen is considered the most abundant protein in the animal kingdom, comprising 30% of the total amount of proteins and 6% of the human body by weight. Studies that examine the interaction between silver nanoparticles and proteins have been highlighted in the literature in order to understand the stability of the nanoparticle system, the effects observed in biological systems, and the appearance of new chemical pharmaceutical products. The objective of this study was to analyze the behavior of silver nanoparticles stabilized with collagen (AgNPcol) and to check the skin permeation capacity and action in paw edema induced by carrageenan. AgNPcol synthesis was carried out using solutions of reducing agent sodium borohydride (NaBH4), silver nitrate (AgNO3) and collagen. Characterization was done by using dynamic light scattering (DLS) and X-ray diffraction (XRD) and AFM. Cellular viability testing was performed by using flow cytometry in human melanoma cancer (MV3) and murine fibroblast (L929) cells. The skin permeation study was conducted using a Franz diffusion cell, and the efficiency of AgNPcol against the formation of paw edema in mice was evaluated. The hydrodynamic diameter and zeta potential of AgNPcol were 140.7±7.8nm and 20.1±0.7mV, respectively. AgNPcol failed to induce early apoptosis, late apoptosis, and necrosis in L929 cells; however, it exhibited enhanced toxicity in cancer cells (MV3) compared to normal cells (L929). AgNPcol demonstrated increased toxicological effects in cancer MV3 cells, promoting skin permeation, and preventing paw edema.

  4. 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.

  5. 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

  6. AMP-activated protein kinase supports the NGF-induced viability of human HeLa cells to glucose starvation.

    PubMed

    Ting, Luo; Bo, Wan; Li, Ruwei; Chen, Xinya; Wang, Yingli; Jun, Zhou; Yu, Long

    2010-07-01

    As an important cellular energy regulation kinase, AMP-activated protein kinase (AMPK) has been demonstrated as a key molecule in the development of tolerance to nutrient starvation. Activation of AMPK includes the phosphorylation of Thr172 of the alpha-subunit. Nerve growth factor (NGF) was originally isolated for its ability to stimulate both survival and differentiation in peripheral neurons, but many investigations have shown that the NGF also plays an important role in survival, growth and invasion of many human cancers. In this study, we used CCK-8 cell viability assay to find that NGF could facilitate the viability of HeLa cells following glucose deprivation while not in glucose-normal control groups. This effect of NGF-induced viability promotion to glucose starvation can be suppressed by Compound C, a specific inhibitor of AMPK. Meanwhile, western blot analysis showed that AMPKalpha1/alpha2 Thr172 phosphorylation level in HeLa cells was up-regulated after NGF treatment under glucose starvation, and Compound C was able to reduce the AMPKalpha1/alpha2 Thr172 phosphorylation level which was up-regulated by NGF in HeLa cells. Taken together, these results indicate that AMP-activated protein kinase supports the NGF-induced viability of human HeLa cells to glucose starvation.

  7. 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

  8. Oxidized phospholipids derived from ozone-treated lung surfactant extract reduce macrophage and epithelial cell viability.

    PubMed

    Uhlson, Charis; Harrison, Kathleen; Allen, Corrie B; Ahmad, Shama; White, Carl W; Murphy, Robert C

    2002-07-01

    Ozone is known to be a highly toxic gas present in the urban air which exerts its effect on pulmonary tissue through its facile chemical reactions with target molecules in the airway. One of the first barriers encountered by ozone is epithelial lining fluid which contains pulmonary surfactant rich in glycerophosphocholine lipids. The reaction of ozone with calf lung surfactant extract was found to result in the production of 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (16:0a/9-al-GPCho) as an expected product of the ozonolysis of abundant unsaturated phospholipids containing unsaturated fatty acyl groups with a double bond at carbon-9. This oxidized phospholipid was identified as a biologically active product in that it reduced elicited macrophage viability by necrosis with an ED(50) of 6 microM. Further studies of the biological activity of 16:0a/9-al-GPCho revealed that concentrations from 100 to 200 nM initiated apoptosis in pulmonary epithelial-like A549 cells as assessed by TUNEL staining, nuclear size, and caspase-3 activation with loss of viability indicated by reduction of mitochondrial dehydrogenase activity. The release of IL-8, a neutrophil chemokine, from A549 cells was also stimulated by 50-100 nM 16:0a/9-al-GPCho. Exposure of calf lung surfactant to low levels of ozone (62.5, 125, and 250 ppb) for various time periods from 2 to 48 h in a feedback-regulated ozone exposure chamber resulted in a dose- and time-dependent increase in the formation of 16:0a/9-al-GPCho as measured by a specific and sensitive LC/MS/MS assay. The quantity of this biologically active chain-shortened glycerophosphocholine lipid generated even at 125 ppb ozone for 2-4 h (50-100 nM) was consistent with this product mediating the toxic effects of ozone on cells in contact with surfactant.

  9. Mouse granulated metrial gland cells require contact with stromal cells to maintain viability

    PubMed Central

    STEWART, I. J.

    2000-01-01

    Granulated metrial gland (GMG) cells differentiate in the uterine wall in pregnancy in mice but the mechanisms which control their differentiation and maintenance are unknown. In vivo, GMG cells share an intimate association with fibroblast-like stromal cells. The importance of this association has been assessed by examining the effects of withdrawal of stromal cell contact on GMG cell maintenance in vitro. When single cell suspensions of cells were prepared from mouse metrial glands there was a steady decline in numbers with days of culture but usually some remained at 7 d of culture. The ability of metrial gland cells to kill Wehi 164 target cells in 51Cr-release cytotoxicity assays was retained by cells cultured for at least 3 d. When explants of metrial gland were maintained in culture to allow GMG cell migration onto the culture flask, the attached GMG cells were lost by 1 d later. Overall, these results suggest that a juxtacrine regulatory mechanism maintains GMG cells. The rapid loss of unsupported GMG cells in culture has major implications in the design of assays to examine GMG cell function. PMID:11117633

  10. Primate-specific oestrogen-responsive long non-coding RNAs regulate proliferation and viability of human breast cancer cells

    PubMed Central

    Lin, Chin-Yo; Dachet, Fabien; Cai, Juan; Ju, Donghong; Goldstone, Amanda; Wood, Emily J.; Liu, Ka; Jia, Hui; Kosir, Mary A.; Thepsuwan, Pattaraporn

    2016-01-01

    Long non-coding RNAs (lncRNAs) are transcripts of a recently discovered class of genes which do not code for proteins. LncRNA genes are approximately as numerous as protein-coding genes in the human genome. However, comparatively little remains known about lncRNA functions. We globally interrogated changes in the lncRNA transcriptome of oestrogen receptor positive human breast cancer cells following treatment with oestrogen, and identified 127 oestrogen-responsive lncRNAs. Consistent with the emerging evidence that most human lncRNA genes lack homologues outside of primates, our evolutionary analysis revealed primate-specific lncRNAs downstream of oestrogen signalling. We demonstrate, using multiple functional assays to probe gain- and loss-of-function phenotypes in two oestrogen receptor positive human breast cancer cell lines, that two primate-specific oestrogen-responsive lncRNAs identified in this study (the oestrogen-repressed lncRNA BC041455, which reduces cell viability, and the oestrogen-induced lncRNA CR593775, which increases cell viability) exert previously unrecognized functions in cell proliferation and growth factor signalling pathways. The results suggest that oestrogen-responsive lncRNAs are capable of altering the proliferation and viability of human breast cancer cells. No effects on cellular phenotypes were associated with control transfections. As heretofore unappreciated components of key signalling pathways in cancers, including the MAP kinase pathway, lncRNAs hence represent a novel mechanism of action for oestrogen effects on cellular proliferation and viability phenotypes. This finding warrants further investigation in basic and translational studies of breast and potentially other types of cancers, has broad relevance to lncRNAs in other nuclear hormone receptor pathways, and should facilitate exploiting and targeting these cell viability modulating lncRNAs in post-genomic therapeutics. PMID:28003470

  11. 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

  12. Iron regulatory protein-2 knockout increases perihematomal ferritin expression and cell viability after intracerebral hemorrhage.

    PubMed

    Chen, Mai; Awe, Olatilewa O; Chen-Roetling, Jing; Regan, Raymond F

    2010-06-14

    Iron is deposited in perihematomal tissue after an intracerebral hemorrhage (ICH), and may contribute to oxidative injury. Cell culture studies have demonstrated that enhancing ferritin expression by targeting iron regulatory protein (IRP) binding activity reduces cellular vulnerability to iron and hemoglobin. In order to assess the therapeutic potential of this approach after striatal ICH, the effect of IRP1 or IRP2 gene knockout on ferritin expression and injury was quantified. Striatal ferritin in IRP1 knockout mice was similar to that in wild-type controls 3 days after stereotactic injection of artificial CSF or autologous blood. Corresponding levels in IRP2 knockouts were increased by 11-fold and 8.4-fold, respectively, compared with wild-type. Protein carbonylation, a sensitive marker of hemoglobin neurotoxicity, was increased by 2.4-fold in blood-injected wild-type striata, was not altered by IRP1 knockout, but was reduced by approximately 60% by IRP2 knockout. Perihematomal cell viability in wild-type mice, assessed by MTT assay, was approximately half of that in contralateral striata at 3 days, and was significantly increased in IRP2 knockouts but not in IRP1 knockouts. Protection was also observed when hemorrhage was induced by collagenase injection. These results suggest that IRP2 binding activity reduces ferritin expression in the striatum after ICH, preventing an optimal response to elevated local iron concentrations. IRP2 binding activity may be a novel therapeutic target after hemorrhagic CNS injuries.

  13. Iron Regulatory Protein-2 Knockout Increases Perihematomal Ferritin Expression and Cell Viability after Intracerebral Hemorrhage

    PubMed Central

    Chen, Mai; Awe, Olatilewa O.; Chen-Roetling, Jing; Regan, Raymond F.

    2010-01-01

    Iron is deposited in perihematomal tissue after an intracerebral hemorrhage (ICH), and may contribute to oxidative injury. Cell culture studies have demonstrated that enhancing ferritin expression by targeting iron regulatory protein (IRP) binding activity reduces cellular vulnerability to iron and hemoglobin. In order to assess the therapeutic potential of this approach after striatal ICH, the effect of IRP1 or IRP2 gene knockout on ferritin expression and injury was quantified. Striatal ferritin in IRP1 knockout mice was similar to that in wild-type controls three days after stereotactic injection of artificial CSF or autologous blood. Corresponding levels in IRP2 knockouts were increased by 11-fold and 8.4-fold, respectively, compared with wild-type. Protein carbonylation, a sensitive marker of hemoglobin neurotoxicity, was increased by 2.4-fold in blood-injected wild-type striata, was not altered by IRP1 knockout, but was reduced by approximately 60% by IRP2 knockout. Perihematomal cell viability in wild-type mice, assessed by MTT assay, was approximately half of that in contralateral striata at three days, and was significantly increased in IRP2 knockouts but not in IRP1 knockouts. Protection was also observed when hemorrhage was induced by collagenase injection. These results suggest that IRP2 binding activity reduces ferritin expression in the striatum after ICH, preventing an optimal response to elevated local iron concentrations. IRP2 binding activity may be a novel therapeutic target after hemorrhagic CNS injuries. PMID:20399759

  14. YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability.

    PubMed

    Idziorek, T; Estaquier, J; De Bels, F; Ameisen, J C

    1995-09-25

    In the absence of cell permeabilization, the impermeant nuclear dye YOPRO-1 permits accurate analysis of apoptosis using cytofluorometry or fluorescent microscopy. Several immune cell populations were studied including dexamethasone-treated thymocytes, irradiated peripheral blood mononuclear cells and a growth factor-depleted tumor B cell line. Excellent correlation values were found with acridine orange using cytofluorometry and with eosin-hematoxylin using optical microscopy. Under fluorescent microscopy, YOPRO-1-fluorescent cells demonstrate the morphological features of cells undergoing apoptosis such as nuclear shrinkage and fragmentation. An important characteristic of the dye that differs from all other nuclear dyes previously used for the detection of apoptosis is that it does not label living cells. Cell sorting after flow cytofluorometry analysis confirmed that only the apoptotic cell population was labelled with YOPRO-1. Further studies showed that while incubation of living cells with Hoechst 33342 almost completely abrogated the capacity of T cells to proliferate in response to several stimuli, YOPRO-1 had no inhibitory effect. This new simple, rapid and reproducible use of the YOPRO-1 dye should prove useful in the analysis of apoptotic cells as well as for investigations of the functional properties of living cells in a culture containing apoptotic cells.

  15. 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

  16. Controlling laser-induced jet formation for bioprinting mesenchymal stem cells with high viability and high resolution.

    PubMed

    Ali, Muhammad; Pages, Emeline; Ducom, Alexandre; Fontaine, Aurelien; Guillemot, Fabien

    2014-09-12

    Laser-assisted bioprinting is a versatile, non-contact, nozzle-free printing technique which has demonstrated high potential for cell printing with high resolution. Improving cell viability requires determining printing conditions which minimize shear stress for cells within the jet and cell impact at droplet landing. In this context, this study deals with laser-induced jet dynamics to determine conditions from which jets arise with minimum kinetic energies. The transition from a sub-threshold regime to jetting regime has been associated with a geometrical parameter (vertex angle) which can be harnessed to print mesenchymal stem cells with high viability using slow jet conditions. Finally, hydrodynamic jet stability is also studied for higher laser pulse energies which give rise to supersonic but turbulent jets.

  17. 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.

  18. 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.

  19. 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

    2016-01-12

    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.

  20. 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

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

    PubMed Central

    Braun Miyara, Sigal; Ezra, David

    2016-01-01

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

  2. 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

  3. 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.

  4. Effect of different substrates on the performance, bacterial diversity, and bacterial viability in microbial fuel cells.

    PubMed

    Chae, Kyu-Jung; Choi, Mi-Jin; Lee, Jin-Wook; Kim, Kyoung-Yeol; Kim, In S

    2009-07-01

    Four microbial fuel cells (MFCs) were inoculated with anaerobic sludge and fed four different substrates for over one year. The Coulombic efficiency (CE) and power output varied with different substrates, while the bacterial viability was similar. Acetate fed-MFC showed the highest CE (72.3%), followed by butyrate (43.0%), propionate (36.0%) and glucose (15.0%). Glucose resulted in the lowest CE because of its fermentable nature implying its consumption by diverse non-electricity-generating bacteria. 16S rDNA sequencing results indicated phylogenetic diversity in the communities of all anode biofilms, and there was no single dominant bacterial species. A relative abundance of beta-Proteobacteria but an absence of gamma-Proteobacteria was observed in all MFCs except for propionate-fed system in which Firmicutes dominating. The glucose-fed-MFC showed the widest community diversity, resulting in the rapid generation of current without lag time when different substrates were suddenly fed. Geobacter-like species with the most representative Geobactersulfurreducens PCA(T) were integral members of the bacterial community in all MFCs except for the propionate-fed system.

  5. 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.

  6. Cell viability and MRI performance of highly efficient polyol-coated magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Arteaga-Cardona, Fernando; Gutiérrez-García, Eric; Hidalgo-Tobón, Silvia; López-Vasquez, Ciro; Brito-Barrera, Yazmín A.; Flores-Tochihuitl, Julia; Angulo-Molina, Aracely; Reyes-Leyva, Julio R.; González-Rodríguez, Roberto; Coffer, Jeffery L.; Pal, Umapada; Diaz-Conti, Mario Pérez-Peña; Platas-Neri, Diana; Dies-Suarez, Pilar; Fonseca, Rebeca Sosa; Arias-Carrión, Oscar; Méndez-Rojas, Miguel A.

    2016-11-01

    This work aimed at determining conditions that would allow us to control the size of the NPs and create a system with characteristics apt for biomedical applications. We describe a comprehensive study on the synthesis and physical characterization of two highly sensitive sets of triethylene glycol (TREG) and polyethylene glycol (PEG)-coated superparamagnetic iron oxide nanoparticles (SPIONs) to be evaluated for use as magnetic resonance (MR) contrast agents. The ferrofluids demonstrated excellent colloidal stability in deionized water at pH 7.0 as indicated by dynamic light scattering (DLS) data. The magnetic relaxivities, r 2, were measured on a 1.5 T clinical MRI instrument. Values in the range from 205 to 257 mM-1 s-1 were obtained, varying proportionally to the SPIONs' sizes and coating nature. Further in vitro cell viability tests and in vivo biodistribution analyses of the intravenously administered nanoparticles showed that the prepared systems have good biocompatibility and migrate to several organs, mainly the meninges, spleen, and liver. Based on these results, our findings demonstrated the potential utility of these nanosystems as clinical contrast agents for MR imaging.

  7. ZnO nanoparticles induced effects on nanomechanical behavior and cell viability of chitosan films

    PubMed Central

    Jayasuriya, Ambalangodage C.; Aryaei, Ashkan; Jayatissa, Ahalapitiya H.

    2014-01-01

    The aim of this paper is to develop novel chitosan-Zinc oxide nanocomposite films for biomedical applications. The films were fabricated with 1, 5, 10 and 15% w/w of Zinc Oxide (ZnO) nanoparticles (NPs) incorporated with chitosan (CS) using a simple method. The prepared nanocomposite films were characterized using atomic force microscopy, Raman and X-Ray diffraction studies. In addition, nano and micro mechanical properties were measured. It was found that the microhardness, nanohardness and its corresponding elastic modulus increased with the increasing of ZnO NPs percentage in the CS films. However, the ductility of films decreased as the percentage of ZnO NPs increased. Cell attachment and cytotoxicity of the prepared films at day two and five were evaluated in vitro using osteoblasts (OBs). It was observed that OB viability decreased in films with higher than 5% ZnO NPs. This result suggests that although ZnO NPs can improve the mechanical properties of pure CS films, only a low percentage of ZnO NPs can be applied for biomedical and bioengineering applications because of the cytotoxicity effects of these particles. PMID:23910265

  8. Effect of deoxycholic acid on Ca2+ movement, cell viability and apoptosis in human gastric cancer cells.

    PubMed

    Chien, Jau-Min; Chou, Chiang-Ting; Liang, Wei-Zhe; Cheng, Jin-Shiung; Chang, Hong-Tai; Tseng, Hui-Wen; Kuo, Soong-Yu; Kuo, Chun-Chi; Chen, Fu-An; Shieh, Pochuen; Ho, Chin-Man; Lin, Jia-Rong; Kuo, Daih-Huang; Jan, Chung-Ren

    2015-02-01

    Deoxycholic acid (DOA) is one of the secondary bile acids used as a mild detergent for the isolation of membrane associated proteins. This study examined whether the secondary bile acid, DOA, altered Ca(2+) movement, cell viability and apoptosis in SCM1 human gastric cancer cells. The Ca(2+)-sensitive fluorescent dye fura-2 was used to measure [Ca(2+)]i. DOA-evoked [Ca(2+)]i rises concentration dependently. The response was reduced by removing extracellular Ca(2+). DOA-evoked Ca(2+) entry was inhibited by store-operated Ca(2+) channel inhibitors (nifedipine, econazole and SKF96365), the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA) and the PKC inhibitor GF109203X. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) abolished DOA-evoked [Ca(2+)]i rises. Conversely, treatment with DOA abolished TG-evoked [Ca(2+)]i rises. Inhibition of phospholipase C with U73122 abolished DOA-evoked [Ca(2+)]i rises. At 100-500 μM, DOA decreased cell viability, which was not changed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). DOA between 100 and 300 μM also induced apoptosis. Collectively, in SCM1 cells, DOA-induced [Ca(2+)]i rises by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via store-operated Ca(2+) channels. DOA also caused Ca(2+)-independent apoptosis.

  9. 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

  10. Effects of lead uptake on microtubule cytoskeleton organization and cell viability in the seagrass Cymodocea nodosa.

    PubMed

    Malea, Paraskevi; Adamakis, Ioannis-Dimosthenis S; Kevrekidis, Theodoros

    2014-06-01

    The effects of lead uptake on microtubule integrity and cell viability in intermediate-juvenile leaf blades of the seagrass Cymodocea nodosa were investigated under laboratory conditions in increasing exposure concentrations (0.1, 0.25, 0.5, 5, 10, 20 and 40mg/L). Uptake kinetics was generally fitted well to the Michaelis-Menten equation. The equilibrium concentration and the velocity of lead uptake tended to increase as the exposure concentration increased up to 5-10mg/L; equilibrium concentration values at most of the treatments were comparable to reported lead concentrations in seagrass leaves. Lead caused a drastic change in the microtubule organization; microtubule depolymerization was observed after 3-7 days of exposure, depending on metal dosage. This observation indicates that microtubule integrity could be utilized as an early biomarker of emerging lead contamination. Cell death starting to occur at later time than microtubule disturbance was also observed at all of the treatments. Microtubule depolymerization expressed as percentage of fluorescence intensity reduction and cell mortality expressed as percentage of dead cells (blue stained) increased with time. Toxic effects were first detected during or at the beginning of the steady state-phase of lead uptake. The lowest experimental tissue lead concentrations associated with the onset of toxic effects (18.33-20.24μg/g dry wt, 0.1-0.25mg/L treatments, 7th day) were comparable to or lower than lead concentrations measured in leaves of C. nodosa and other seagrass species from various geographical areas, probably implying that lead may play a important role in the worldwide decline of seagrass meadows. These lowest tissue concentrations were exceeded up to the 3rd day of incubation at higher exposure concentrations, but microtubule depolymerization at 0.5mg/L and cell death at 0.5-20mg/L were first detected at later time. The time period required for the onset of microtubule depolymerization tended to

  11. 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.

  12. Mitochondrial DNA ligase is dispensable for the viability of cultured cells but essential for mtDNA maintenance.

    PubMed

    Shokolenko, Inna N; Fayzulin, Rafik Z; Katyal, Sachin; McKinnon, Peter J; Wilson, Glenn L; Alexeyev, Mikhail F

    2013-09-13

    Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ(0) phenotype.

  13. 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.

  14. Chemical composition and effect on intestinal Caco-2 cell viability and lipid profile of fixed oil from Cynomorium coccineum L.

    PubMed

    Rosa, Antonella; Rescigno, Antonio; Piras, Alessandra; Atzeri, Angela; Scano, Paola; Porcedda, Silvia; Zucca, Paolo; Assunta Dessì, M

    2012-10-01

    Cynomorium coccineum L. is a non-photosynthetic plant, spread over Mediterranean countries, amply used in traditional medicine. We investigated the composition and effect on intestinal Caco-2 cell viability and lipid profile of fixed oil obtained from dried stems of the plant. Oil isolation has been performed by supercritical fractioned extraction with CO2. 13C NMR spectroscopy has been used to study the molecular composition of oil lipids; fatty acid composition was identified using GC and HPLC techniques. The fixed oil was composed mainly by triacylglycerols and derivates. The main fatty acids were 18:1 n-9 (38%), 18:2 n-6 (20%), 16:0 (15%), and 18:3 n-3 (10.8%). The oil showed a significant in vitro inhibitory effect on the growth of colon cancer undifferentiated Caco-2 cells. Moreover, cell viability, lipid composition, and lipid peroxidation were measured in intestinal epithelial cells (differentiated Caco-2 cells) after 24 h incubation with fixed oil. The oil did not show a toxic effect on colon epithelial cell viability but induced a significant change in fatty acid composition, with a significant accumulation of the essential fatty acids 18:3 n-3 and 18:2 n-6. The results showed remarkable biological activity of Maltese mushroom oil, and qualify it as a potential resource for food/pharmaceutical applications.

  15. Antagonism of serotonin receptor 1B decreases viability and promotes apoptosis in the COS canine osteosarcoma cell line.

    PubMed

    Viall, A K; Goodall, C P; Stang, B; Marley, K; Chappell, P E; Bracha, S

    2016-06-01

    Serotonin receptor 1B (5HTR1B) traditionally exhibits anti-proliferative activity in osteoblasts. We examined the expression and function of 5HTR1B in the COS canine osteosarcoma cell line and normal canine osteoblasts. Equal levels of 5HTR1B gene and protein expression were found between normal and malignant osteoblasts. Treatment with serotonin enhanced viability of osteosarcoma cells but not normal osteoblasts. Challenge with the 5HTR1B agonist anpirtoline caused no change in cell viability. Rather incubation with the specific receptor antagonist SB224289 caused reduction in osteoblast viability, with this effect more substantial in osteosarcoma cells. Investigation of this inhibitory activity showed 5HTR1B antagonism induces apoptosis in malignant cells. Evaluation of phosphorylated levels of CREB and ERK, transcriptional regulators associated with serotonin receptor signalling in osteoblasts, revealed aberrant 5HTR1B signalling in COS. Our results confirm the presence of 5HTR1B in a canine osteosarcoma cell line and highlight this receptor as a possible novel therapeutic target.

  16. Mitochondrial DNA Ligase Is Dispensable for the Viability of Cultured Cells but Essential for mtDNA Maintenance*

    PubMed Central

    Shokolenko, Inna N.; Fayzulin, Rafik Z.; Katyal, Sachin; McKinnon, Peter J.; Wilson, Glenn L.; Alexeyev, Mikhail F.

    2013-01-01

    Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ0 phenotype. PMID:23884459

  17. TRIB3 enhances cell viability during glucose deprivation in HEK293-derived cells by upregulating IGFBP2, a novel nutrient deficiency survival factor.

    PubMed

    Örd, Tiit; Örd, Daima; Adler, Priit; Vilo, Jaak; Örd, Tõnis

    2015-10-01

    Glucose deprivation occurs in several human diseases, including infarctions and solid tumors, and leads to cell death. In this article, we investigate the role of the pseudokinase Tribbles homolog 3 (TRIB3) in the cellular stress response to glucose starvation using cell lines derived from HEK293, which is highly glycolytic under standard conditions. Our results show that TRIB3 mRNA and protein levels are strongly upregulated in glucose-deprived cells via the induction of activating transcription factor 4 (ATF4) by the endoplasmic reticulum (ER) stress sensor kinase PERK. Cell survival in glucose-deficient conditions is enhanced by TRIB3 overexpression and reduced by TRIB3 knockdown. Genome-wide gene expression profiling uncovered approximately 40 glucose deprivation-responsive genes that are affected by TRIB3, including several genes involved in signaling processes and metabolism. Based on transcription factor motif analysis, the majority of TRIB3-downregulated genes are target genes of ATF4, which TRIB3 is known to inhibit. The gene most substantially upregulated by TRIB3 is insulin-like growth factor binding protein 2 (IGFBP2). IGFBP2 mRNA and protein levels are downregulated in cells subjected to glucose deprivation, and reduced IGFBP2 expression aggravates cell death during glucose deficiency, while overexpression of IGFBP2 prolongs cell survival. Moreover, IGFBP2 silencing abrogates the pro-survival effect of TRIB3. Since TRIB3 augments IGFBP2 expression in glucose-starved cells, the data indicate that IGFBP2 contributes to the attenuation of cell death by TRIB3. These results implicate TRIB3 and IGFBP2, both of which are known to be overexpressed in several types of cancers, as pro-survival modulators of cell viability in nutrient-deficient microenvironments.

  18. Effects of Antrodia camphorata extracts on the viability, apoptosis, [Ca2+]i, and MAPKs phosphorylation of OC2 human oral cancer cells.

    PubMed

    Huang, Chorng-Chih; Cheng, He-Hsiung; Wang, Jue-Long; Cheng, Jin-Shiung; Chai, Kuo-Liang; Fang, Yi-Chien; Kuo, Chun-Chi; Chu, Sau-Tung; Ho, Chin-Man; Lin, Ko-Long; Tsai, Jeng-Yu; Jan, Chung-Ren

    2009-06-30

    The effect of Antrodia camphorata (AC) on human oral cancer cells has not been explored. This study examined the effect of AC on the viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca2+ regulation of OC2 human oral cancer cells. AC at a concentration of 25 microM induced an increase in cell viability, but AC at concentrations > or = 50 microg/ml decreased viability in a concentration-dependent manner. AC at concentrations of 100-200 microg/ml induced apoptosis in a concentration-dependent manner as demonstrated by propidium iodide staining. AC (25 microg/ml) did not alter basal [Ca2+]i, but decreased the [Ca2+]i increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin, and histamine increased cell viability whereas thapsigargin decreased it. AC (25 microg/ml) pretreatment failed to alter ATP-induced increase in viability, potentiated bradykinin-induced increase in viability, decreased histamine-induced increase in viability and reversed thapsigargin-induced decrease in viability. Immunoblotting suggested that AC induced phosphorylation of ERK and JNK MAPKs, but not p38 MAPK. Collectively, for OC2 cells, AC exerted multiple effects on their viability and [Ca2+]i, induced their ERK and JNK MAPK phosphorylation, and probably evoked their apoptosis.

  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. Comparison of Periodontal Ligament Stem Cells Isolated from the Periodontium of Healthy Teeth and Periodontitis-Affected Teeth

    PubMed Central

    Soheilifar, Sara; Amiri, Iraj; Bidgoli, Mohsen; Hedayatipanah, Morad

    2016-01-01

    Objectives: Stem cell (SC) therapy is a promising technique for tissue regeneration. This study aimed to compare the viability and proliferation ability of periodontal ligament stem cells (PDLSCs) isolated from the periodontium of healthy and periodontitis-affected teeth to obtain an autologous, easily accessible source of SCs for tissue regeneration in periodontitis patients. Materials and Methods: The PDLSCs were isolated from the roots of clinically healthy premolars extracted for orthodontic purposes and periodontally involved teeth with hopeless prognosis (with and without phase I periodontal treatment). Cells were cultured and viability and proliferation ability of third passage cells in each group were evaluated using the methyl thiazol tetrazolium assay. The results were statistically analyzed using t-test. Results: No SCs could be obtained from periodontitis-affected teeth without phase I periodontal treatment. The viability of cells was 0.86±0.13 OD/540 in healthy group and 0.4±0.25 OD/540 in periodontitis-affected group (P=0.035). The proliferation ability (population doubling time) of cells obtained from healthy teeth was 4.22±1.23 hours. This value was 2.3±0.35 hours for those obtained from periodontitis-affected teeth (P=0.02). Conclusions: Viability and proliferation ability of cells isolated from the periodontium of healthy teeth were significantly greater than those of cells isolated from the periodontitis-affected teeth. PMID:28127319

  1. Equol, an Isoflavone Metabolite, Regulates Cancer Cell Viability and Protein Synthesis Initiation via c-Myc and eIF4G*

    PubMed Central

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

    2015-01-01

    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

  2. Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

    NASA Astrophysics Data System (ADS)

    Zhao, Guoping; Chen, Shaopeng; Zhao, Ye; Zhu, Lingyan; Huang, Pei; Bao, Lingzhi; Wang, Jun; Wang, Lei; Wu, Lijun; Wu, Yuejin; Xu, An

    2010-02-01

    Magnetic resonance image (MRI) systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields (SMF). With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid (AL) cells, Chinese hamster ovary (CHO) cells, DNA double-strand break repair deficient mutant (XRS-5) cells, and human primary skin fibroblasts (AG1522) cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster cells after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

  3. Sodium Nitroprusside Changed The Metabolism of Mesenchymal Stem Cells to An Anaerobic State while Viability and Proliferation Remained Intact

    PubMed Central

    Pari, Sadiyeh; Abnosi, Mohammad Hussein; Pakyari, Reza

    2017-01-01

    Objective We used sodium nitroprusside (SNP), a nitric oxide (NO) releasing molecule, to understand its effect on viability and proliferation of rat bone marrow mesenchymal stem cells (BM-MSCs). Materials and Methods This experimental study evaluated the viability and morphology of MSCs in the presence of SNP (100 to 2000 µM) at 1, 5, and 15 hours. We chose the 100, 1000, and 2000 µM concentrations of SNP for one hour exposure for further analyses. Cell proliferation was investigated by the colony forming assay and population doubling number (PDN). Na+, K+, and Ca2+ levels as well as activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT) were measured. Results The viability of MSCs dose-dependently reduced from 750 µM at one hour and 250 µM at 5 and 15 hours. The 100 µM caused no change in viability, however we observed a reduction in the cytoplasmic area at 5 and 15 hours. This change was not observed at one hour. The one hour treatment with 100 µM of SNP reduced the mean colony numbers but not the diameter when the cells were incubated for 7 and 14 days. In addition, one hour treatment with 100 µM of SNP significantly reduced ALT, AST, and ALP activities whereas the activity of LDH increased when incubated for 24 hours. The same treatment caused an increase in Ca2+ and reduction in Na+ content. The 1000 and 2000 µM concentrations reduced all the factors except Ca2+ and LDH which increased. Conclusion The high dose of SNP, even for a short time, was toxic. The low dose was safe with respect to viability and proliferation, especially over a short time. However elevated LDH activity might increase anaerobic metabolism. PMID:28367425

  4. Enantiomeric CopA3 dimer peptide suppresses cell viability and tumor xenograft growth of human gastric cancer cells.

    PubMed

    Lee, Joon Ha; Kim, In-Woo; Shin, Yong Pyo; Park, Ho Jin; Lee, Young Shin; Lee, In Hee; Kim, Mi-Ae; Yun, Eun-Young; Nam, Sung-Hee; Ahn, Mi-Young; Kang, Dongchul; Hwang, Jae Sam

    2016-03-01

    The CopA3 dimer peptide is a coprisin analog that has an anticancer effect against human cancer cells in vitro. In this study, we investigated the anticancer activity of the enantiomeric CopA3 dimer peptide in human gastric cancer cell lines as well as in an in vivo tumor xenograft model. Enantiomeric CopA3 reduced gastric cancer cell viability and exhibited cytotoxicity against cancer cells. Enantiomeric CopA3-induced cell death was mediated by specific interactions with phosphatidylserine and phosphatidylcholine, membrane components that are enriched in cancer cells, in a calcein leakage assay. Moreover, acridine orange/ethidium bromide staining, flow cytometric analysis, and Western blot analysis showed that enantiomeric CopA3 induced apoptotic and necrotic gastric cancer cell death. The antitumor effect was also observed in a mouse tumor xenograft model in which intratumoral inoculation of the peptide resulted in a significant decrease in the SNU-668 gastric cancer tumor volume. In addition, periodic acid-Schiff and hematoxylin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed apoptotic and necrotic cell death in tumor masses treated with greater than 150 μg CopA3. Collectively, these results indicate that the enantiomeric CopA3 dimer peptide induces apoptosis and necrosis of gastric cancer cells in vitro and in vivo, indicating that the peptide is a potential candidate for the treatment of gastric cancer, which is a common cause of cancer and cancer deaths worldwide.

  5. Effects of Ciprofloxacin-Containing Antimicrobial Scaffolds on Dental Pulp Stem Cell Viability — In Vitro Studies

    PubMed Central

    Kamocki, Krzysztof; Nör, Jacques E.; Bottino, Marco C.

    2017-01-01

    Objective A combination of antibiotics, including but not limited to metronidazole (MET) and ciprofloxacin (CIP), has been indicated to eradicate bacteria in necrotic immature permanent teeth prior to regenerative procedures. It has been shown clinically that antibiotic pastes may lead to substantial stem cell death. The aim of this study was to synthesize scaffolds containing various concentrations of CIP to enhance cell viability while preserving antimicrobial properties. Design Polydioxanone (PDS)-based electrospun scaffolds were processed with decreasing CIP concentrations (25 – 1 wt.%) and morphologically evaluated using scanning electron microscopy (SEM). Cytotoxicity assays were performed to determine whether the amount of CIP released from the scaffolds would lead to human dental pulp stem cell (hDPSC) toxicity. Similarly, WST-1 assays were performed to evaluate the impact of CIP release on hDPSC proliferation. Pure PDS scaffolds and saturated double antibiotic solution MET/CIP (DAP) served as both positive and negative controls, respectively. Antibacterial efficacy against E. faecalis (Ef) was tested. Results A significant decrease in hDPSC’ viability at concentrations 5–25 wt.% was observed. However, concentrations below 5 wt.% did not impair cell viability. Data from the WST-1 assays indicated no detrimental impact on cell proliferation for scaffolds containing 2.5 wt.% CIP or less. Significant antimicrobial properties were seen for CIP-scaffolds at lower concentrations (i.e., 1 and 2.5 wt.%). Conclusion The obtained data demonstrated that a reduced concentration of CIP incorporated into PDS-based scaffolds maintains its antimicrobial properties while enhancing viability and proliferation of dental pulp stem cells. PMID:26042622

  6. 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.

  7. Impedimetric and optical interrogation of single cells in a microfluidic device for real-time viability and chemical response assessment.

    PubMed

    James, Conrad D; Reuel, Nigel; Lee, Eunice S; Davalos, Rafael V; Mani, Seethambal S; Carroll-Portillo, Amanda; Rebeil, Roberto; Martino, Anthony; Apblett, Christopher A

    2008-01-18

    We report here a non-invasive, reversible method for interrogating single cells in a microfluidic flow-through system. Impedance spectroscopy of cells held at a micron-sized pore under negative pressure is demonstrated and used to determine the presence and viability of the captured cell. The cell capture pore is optimized for electrical response and mechanical interfacing to a cell using a deposited layer of parylene. Changes in the mechanical interface between the cell and the chip due to chemical exposure or environmental changes can also be assessed. Here, we monitored the change in adhesion/spreading of RAW264.7 macrophages in response to the immune stimulant lipopolysaccharide (LPS). This method enables selective, reversible, and quantitative long-term impedance measurements on single cells. The fully sealed electrofluidic assembly is compatible with long-term cell culturing, and could be modified to incorporate single cell lysis and subsequent intracellular separation and analysis.

  8. Xanthohumol, a Prenylated Chalcone from Hops, Inhibits the Viability and Stemness of Doxorubicin-Resistant MCF-7/ADR Cells.

    PubMed

    Liu, Ming; Yin, Hua; Qian, Xiaokun; Dong, Jianjun; Qian, Zhonghua; Miao, Jinlai

    2016-12-28

    Xanthohumol is a unique prenylated flavonoid in hops (Humulus lupulus L.) and beer. Xanthohumol has been shown to possess a variety of pharmacological activities. There is little research on its effect on doxorubicin-resistant breast cancer cells (MCF-7/ADR) and the cancer stem-like cells exiting in this cell line. In the present study, we investigate the effect of xanthohumol on the viability and stemness of MCF-7/ADR cells. Xanthohumol inhibits viability, induces apoptosis, and arrests the cell cycle of MCF-7/ADR cells in a dose-dependent manner; in addition, xanthohumol sensitizes the inhibition effect of doxorubicin on MCF-7/ADR cells. Interestingly, we also find that xanthohumol can reduce the stemness of MCF-7/ADR cells evidenced by the xanthohumol-induced decrease in the colony formation, the migration, the percentage of side population cells, the sphere formation, and the down-regulation of stemness-related biomarkers. These results demonstrate that xanthohumol is a promising compound targeting the doxorubicin resistant breast cancer cells and regulating their stemness, which, therefore, will be applied as a potential candidate for the development of a doxorubicin-resistant breast cancer agent and combination therapy of breast cancer.

  9. The viability of cells obtained using the Reamer-Irrigator-Aspirator system and in bone graft from the iliac crest.

    PubMed

    Uppal, H S; Peterson, B E; Misfeldt, M L; Della Rocca, G J; Volgas, D A; Murtha, Y M; Stannard, J P; Choma, T J; Crist, B D

    2013-09-01

    We hypothesised that cells obtained via a Reamer-Irrigator-Aspirator (RIA) system retain substantial osteogenic potential and are at least equivalent to graft harvested from the iliac crest. Graft was harvested using the RIA in 25 patients (mean age 37.6 years (18 to 68)) and from the iliac crest in 21 patients (mean age 44.6 years (24 to 78)), after which ≥ 1 g of bony particulate graft material was processed from each. Initial cell viability was assessed using Trypan blue exclusion, and initial fluorescence-activated cell sorting (FACS) analysis for cell lineage was performed. After culturing the cells, repeat FACS analysis for cell lineage was performed and enzyme-linked immunosorbent assay (ELISA) for osteocalcin, and Alizarin red staining to determine osteogenic potential. Cells obtained via RIA or from the iliac crest were viable and matured into mesenchymal stem cells, as shown by staining for the specific mesenchymal antigens CD90 and CD105. For samples from both RIA and the iliac crest there was a statistically significant increase in bone production (both p < 0.001), as demonstrated by osteocalcin production after induction. Medullary autograft cells harvested using RIA are viable and osteogenic. Cell viability and osteogenic potential were similar between bone grafts obtained from both the RIA system and the iliac crest.

  10. 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

    2014-08-07

    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.

  11. 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.

  12. 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.

  13. 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

  14. Influence of Silica Matrix Composition and Functional Component Additives on the Bioactivity and Viability of Encapsulated Living Cells

    DOE PAGES

    Savage, Travis J.; Dunphy, Darren R.; Harbaugh, Svetlana; ...

    2015-11-06

    We report the remarkable impact encapsulation matrix chemistry can have on the bioactivity and viability of integrated living cells. Two silica chemistries (aqueous silicate and alkoxysilane), and a functional component additive (glycerol), are employed to generate three distinct silica matrices. Moreover, these matrices are used to encapsulate living E. coli cells engineered with a synthetic riboswitch for cell-based biosensing. Following encapsulation, membrane integrity, reproductive capability, and riboswitch-based protein expression levels and rates are measured over a 5 week period. Striking differences in E. coli bioactivity, viability, and biosensing performance are observed for cells encapsulated within the different matrices. E. colimore » cells encapsulated for 35 days in aqueous silicate-based (AqS) matrices showed relatively low membrane integrity, but high reproductive capability in comparison to cells encapsulated in glycerol containing sodium silicate-based (AqS + g) and alkoxysilane-based (PGS) gels. Further, cells in sodium silicate-based matrices showed increasing fluorescence output over time, resulting in a 1.8-fold higher fluorescence level, and a faster expression rate, over cells free in solution. Finally, this unusual and unique combination of biological properties demonstrates that careful design of the encapsulation matrix chemistry can improve functionality of the biocomposite material, and result in new and unexpected physiological states.« less

  15. Influence of Silica Matrix Composition and Functional Component Additives on the Bioactivity and Viability of Encapsulated Living Cells

    SciTech Connect

    Savage, Travis J.; Dunphy, Darren R.; Harbaugh, Svetlana; Kelley-Loughnane, Nancy; Harper, Jason C.; Brinker, C. Jeffrey

    2015-11-06

    We report the remarkable impact encapsulation matrix chemistry can have on the bioactivity and viability of integrated living cells. Two silica chemistries (aqueous silicate and alkoxysilane), and a functional component additive (glycerol), are employed to generate three distinct silica matrices. Moreover, these matrices are used to encapsulate living E. coli cells engineered with a synthetic riboswitch for cell-based biosensing. Following encapsulation, membrane integrity, reproductive capability, and riboswitch-based protein expression levels and rates are measured over a 5 week period. Striking differences in E. coli bioactivity, viability, and biosensing performance are observed for cells encapsulated within the different matrices. E. coli cells encapsulated for 35 days in aqueous silicate-based (AqS) matrices showed relatively low membrane integrity, but high reproductive capability in comparison to cells encapsulated in glycerol containing sodium silicate-based (AqS + g) and alkoxysilane-based (PGS) gels. Further, cells in sodium silicate-based matrices showed increasing fluorescence output over time, resulting in a 1.8-fold higher fluorescence level, and a faster expression rate, over cells free in solution. Finally, this unusual and unique combination of biological properties demonstrates that careful design of the encapsulation matrix chemistry can improve functionality of the biocomposite material, and result in new and unexpected physiological states.

  16. Influence of silica matrix composition and functional component additives on the bioactivity and viability of encapsulated living cells

    SciTech Connect

    Savage, Travis J.; Dunphy, Darren R.; Harbaugh, Svetlana; Kelley-Loughnane, Nancy; Harper, Jason C.; Brinker, C. Jeffrey

    2015-11-06

    The remarkable impact encapsulation matrix chemistry can have on the bioactivity and viability of integrated living cells is reported. Two silica chemistries (aqueous silicate and alkoxysilane), and a functional component additive (glycerol), are employed to generate three distinct silica matrices. These matrices are used to encapsulate living E. coli cells engineered with a synthetic riboswitch for cell-based biosensing. Following encapsulation, membrane integrity, reproductive capability, and riboswitch-based protein expression levels and rates are measured over a 5 week period. Striking differences in E. coli bioactivity, viability, and biosensing performance are observed for cells encapsulated within the different matrices. E. coli cells encapsulated for 35 days in aqueous silicate-based (AqS) matrices showed relatively low membrane integrity, but high reproductive capability in comparison to cells encapsulated in glycerol containing sodium silicate-based (AqS + g) and alkoxysilane-based (PGS) gels. Further, cells in sodium silicate-based matrices showed increasing fluorescence output over time, resulting in a 1.8-fold higher fluorescence level, and a faster expression rate, over cells free in solution. Furthermore, this unusual and unique combination of biological properties demonstrates that careful design of the encapsulation matrix chemistry can improve functionality of the biocomposite material, and result in new and unexpected physiological states.

  17. 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.

  18. Viability and stress protection of chronic lymphoid leukemia cells involves overactivation of mitochondrial phosphoSTAT3Ser727

    PubMed Central

    Capron, C; Jondeau, K; Casetti, L; Jalbert, V; Costa, C; Verhoyen, E; Massé, J M; Coppo, P; Béné, M C; Bourdoncle, P; Cramer-Bordé, E; Dusanter-Fourt, I

    2014-01-01

    Chronic lymphoid leukemia (CLL) is characterized by the accumulation of functionally defective CD5-positive B lymphocytes. The clinical course of CLL is highly variable, ranging from a long-lasting indolent disease to an unpredictable and rapidly progressing leukemia requiring treatment. It is thus important to identify novel factors that reflect disease progression or contribute to its assessment. Here, we report on a novel STAT3-mediated pathway that characterizes CLL B cells-extended viability and oxidative stress control. We observed that leukemic but not normal B cells from CLL patients exhibit constitutive activation of an atypical form of the STAT3 signaling factor, phosphorylated on serine 727 (Ser727) in the absence of detectable canonical tyrosine 705 (Tyr705)-dependent activation in vivo. The Ser727-phosphorylated STAT3 molecule (pSTAT3Ser727) is localized to the mitochondria and associates with complex I of the respiratory chain. This pSer727 modification is further controlled by glutathione-dependent antioxidant pathway(s) that mediate stromal protection of the leukemic B cells and regulate their viability. Importantly, pSTAT3Ser727, but neither Tyr705-phosphorylated STAT3 nor total STAT3, levels correlate with prolonged in vivo CLL B cells survival. Furthermore, STAT3 activity contributes to the resistance to apoptosis of CLL, but not normal B cells, in vitro. These data reveal that mitochondrial (Mt) pSTAT3Ser727 overactivity is part of the antioxidant defense pathway of CLL B cells that regulates their viability. Mt pSTAT3Ser727 appears to be a newly identified cell-protective signal involved in CLL cells survival. Targeting pSTAT3Ser727 could be a promising new therapeutic approach. PMID:25299776

  19. Enrichment and Viability Inhibition of Circulating Tumor Cells on a Dual Acid-Responsive Composite Nanofiber Film.

    PubMed

    Wang, Wenqian; Cheng, Yaya; Li, Yansheng; Zhou, Hao; Xu, Li-Ping; Wen, Yongqiang; Zhao, Liang; Zhang, Xueji

    2017-04-06

    The formation and metastatic colonization of circulating tumor cells (CTCs) are responsible for the vast majority of cancer-related deaths. Over the last decade, drug-delivery systems (DDSs) have rapidly developed with the emergence of nanotechnology; however, most reported tumor-targeting DDSs are able to deliver drugs only to solid tumor cells and not CTCs. Herein, a novel DDS comprising a composite nanofiber film was constructed to inhibit the viability of CTCs. In this system, gold nanoparticles (Au NPs) were functionalized with doxorubicin (DOX) through an acid-responsive cleavable linker to obtain Au-DOX NPs. Then, the Au-DOX NPs were mixed in a solution of an acid-responsive polymer {i.e., poly[2-(dimethylamino)ethyl methacrylate]} to synthesize the nanofiber film through electrospinning technology. After that, the nanofiber film was modified with a specific antibody (i.e., anti-EpCAM) to enrich the concentration of CTCs on the film. Finally, the Au-DOX NPs were released from the nanofiber film, and they consequently inhibited the viability of CTCs by delivering DOX to the enriched CTCs. This composite nanofiber film was able to decrease the viability of CTCs significantly in the suspended and fluid states, and it is expected to limit the migration and proliferation of tumor cells.

  20. Selective inhibitors of aurora kinases inhibit proliferation, reduce cell viability and impair cell cycle progression in papillary thyroid carcinoma cells.

    PubMed

    Baldini, E; Tuccilli, C; Prinzi, N; Sorrenti, S; Antonelli, A; Fallahi, P; Mian, C; Barollo, S; Catania, A; Morrone, S; Tartaglia, F; Mascagni, D; Coccaro, C; Pepe, M; Filippini, A; D'Armiento, M; Ulisse, S

    2015-01-01

    The three members of the Aurora kinase family, Aurora-A, -B and -C, regulate several aspects of the mitotic process, and their aberrant expression and/or function causes mitotic abnormalities leading either to cell death or aneuploidy. They are found overexpressed in several human malignancies, including the papillary thyroid carcinoma (PTC). In the present study, we sought to establish whether Aurora kinase inhibition could be of any therapeutic value in the treatment of aggressive forms of PTC, enduring to radioactive iodide (RAI) ablation. To this end, the effects of selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) were analyzed on 3 human PTC cell lines expressing either wild-type (K1 and TPC1) or mutant p53 (BCPAP). The two inhibitors were capable of reducing cell proliferation in a time- and dose-dependent manner, with IC₅₀ comprised between 65.4 and 114.9 nM for MLN8237, and between 26.6 and 484.6 nM for AZD1152. Immunofluorescence experiments confirmed that AZD1152 inhibited Aurora-B phosphorylation of histone H3 on Ser10, however, it did not affect Aurora-A autophosphorylation. MLN8237 inhibited Aurora-A autophosphorylation as expected, but at concentrations required to achieve the maximum antiproliferative effects it also abolished H3 (Ser10) phosphorylation. Time-lapse videomicroscopy evidenced that both inhibitors prevented the completion of cytokinesis, and cytofluorimetric analysis showed accumulation of cells in G2/M phase and/or polyploidy. Apoptosis was induced in all the cells by both inhibitors independently from the p53 status. In conclusion, in the present preclinical study MLN8237 and AZD1152 have emerged as promising drug candidates for RAI-insensitive PTC.

  1. 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

  2. Effects of Asiasari radix on the morphology and viability of mesenchymal stem cells derived from the gingiva.

    PubMed

    Jeong, Su-Hyeon; Lee, Ji-Eun; Jin, Seong-Ho; Ko, Youngkyung; Park, Jun-Beom

    2014-12-01

    Medicinal herbs used in traditional Oriental medicine, which have been in use clinically for thousands of years, are attractive sources of novel therapeutics or preventatives. Asiasari radix (A. radix) has been suggested for use in the treatment of dental diseases, including toothache and aphthous stomatitis. The aim of this study was to evaluate the effects of A. radix extracts on the morphology and viability of human stem cells derived from the gingiva. An Asiasarum heterotropoides extract was centrifuged and freeze-dried in a lyophilizer. Stem cells derived from the gingiva were grown in the presence of A. radix at concentrations ranging between 0.1 µg/ml and 1 mg/ml (0, 0.1, 1, 10, 100 and 1,000 µg/ml). Cell morphology was evaluated with an optical microscope and the viability of the cells was quantitatively analyzed with a cell counting kit-8 (CCK-8) assay for up to seven days. The untreated control group exhibited normal fibroblast morphology. The shapes of the cells following 0.1, 1, 10 and 100 µg/ml A. radix treatments were similar to those of the control group. However, a significant change was noted in the 1,000 µg/ml group on day 1, when compared with the untreated group. Furthermore, on day 7, the shapes of the cells following 100 and 1,000 µg/ml A. radix treatments were rounder and fewer cells were present, when compared with those of the control group. The cultures that grew in the presence of A. radix did not exhibit any changes in the CCK‑8 assay on day 2; however, significant reductions in cell viability were noticed following 100 and 1,000 µg/ml A. radix treatment on days 5 and 7. Within the limits of this study, A. radix influenced the viability of the stem cells derived from the gingiva. Thus, the direct application of A. radix to oral tissues may produce adverse effects at high doses. Therefore, the concentration and application time of A. radix requires meticulous control to obtain optimal results. These effects require consideration

  3. Caffeic acid reduces the viability and migration rate of oral carcinoma cells (SCC-25) exposed to low concentrations of ethanol.

    PubMed

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

    2014-10-17

    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.

  4. Influence of silica matrix composition and functional component additives on the bioactivity and viability of encapsulated living cells

    DOE PAGES

    Savage, Travis J.; Dunphy, Darren R.; Harbaugh, Svetlana; ...

    2015-11-06

    The remarkable impact encapsulation matrix chemistry can have on the bioactivity and viability of integrated living cells is reported. Two silica chemistries (aqueous silicate and alkoxysilane), and a functional component additive (glycerol), are employed to generate three distinct silica matrices. These matrices are used to encapsulate living E. coli cells engineered with a synthetic riboswitch for cell-based biosensing. Following encapsulation, membrane integrity, reproductive capability, and riboswitch-based protein expression levels and rates are measured over a 5 week period. Striking differences in E. coli bioactivity, viability, and biosensing performance are observed for cells encapsulated within the different matrices. E. coli cellsmore » encapsulated for 35 days in aqueous silicate-based (AqS) matrices showed relatively low membrane integrity, but high reproductive capability in comparison to cells encapsulated in glycerol containing sodium silicate-based (AqS + g) and alkoxysilane-based (PGS) gels. Further, cells in sodium silicate-based matrices showed increasing fluorescence output over time, resulting in a 1.8-fold higher fluorescence level, and a faster expression rate, over cells free in solution. Furthermore, this unusual and unique combination of biological properties demonstrates that careful design of the encapsulation matrix chemistry can improve functionality of the biocomposite material, and result in new and unexpected physiological states.« less

  5. Novel type 1 photosensitizers: viability of leukemia cells exposed to reactive intermediates generated in situ by in vitro photofragmentation

    NASA Astrophysics Data System (ADS)

    Rajagopalan, Raghavan; Karwa, Amol; Lusiak, Przemyslaw M.; Srivastava, Kripa; Poreddy, Amruta R.; Pandurangi, Raghootama S.; Galen, Karen P.; Neumann, William L.; Cantrell, Gary E.; Dorshow, Richard B.

    2009-06-01

    Photodynamic therapy of tumors involving Type 2 photosenstizers has been conspicuously successful, but the Type 1 process, in contrast, has not received much attention despite its considerable potential. Accordingly, several classes of molecules containing fragile bonds such as azido (-N=N=N), azo (-N=N-), sulfenato (-S-O-) and oxaza (-N-O-) functional groups that produce reactive intermediates such as radicals and nitrenes upon photoexcitation were prepared and tested for cell viability using U397 leukemia cell line. The azido photosensitizer was conjugated to leukemia cell binding peptide, SFFWRLS, for targeted cell viability study. The cells were incubated with the photosensitizer at various concentrations, and were illuminated for 5, 10, and 20 minutes. The results show that all the photosensitizers caused cell death compared to the controls when exposed to both the photosensitizers and light. Most importantly, selective cell death was observed with the azido peptide conjugate 6, which clearly demonstrates that these Type 1 sensitizers are useful for phototherapeutic applications.

  6. Investigation of cell viability and morphology in 3D bio-printed alginate constructs with tunable stiffness.

    PubMed

    Shi, Pujiang; Laude, Augustinus; Yeong, Wai Yee

    2017-04-01

    In this article, mouse fibroblast cells (L929) were seeded on 2%, 5%, and 10% alginate hydrogels, and they were also bio-printed with 2%, 5%, and 10% alginate solutions individually to form constructs. The elastic and viscous moduli of alginate solutions, their interior structure and stiffness, interactions of cells and alginate, cell viability, migration and morphology were investigated by rheometer, MTT assay, scanning electron microscope (SEM), and fluorescent microscopy. The three types of bio-printed scaffolds of distinctive stiffness were prepared, and the seeded cells showed robust viability either on the alginate hydrogel surfaces or in the 3D bio-printed constructs. Majority of the proliferated cells in the 3D bio-printed constructs weakly attached to the surrounding alginate matrix. The concentration of alginate solution and hydrogel stiffness influenced cell migration and morphology, moreover the cells formed spheroids in the bio-printed 10% alginate hydrogel construct. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1009-1018, 2017.

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

    PubMed

    Koo, YoungWon; Kim, GeunHyung

    2016-05-20

    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.

  8. Oligomeric TTR V30M aggregates compromise cell viability, erythropoietin gene expression and promoter activity in the human hepatoma cell line Hep3B.

    PubMed

    Moreira, Luciana; Beirão, João Melo; Beirão, Idalina; Pinho e Costa, Paulo

    2015-01-01

    Familial amyloidotic polyneuropathy, ATTRV30M (p. TTRV50M) amyloidosis, is a neurodegenerative disease characterized by systemic extracellular amyloid deposition of a mutant transthyretin, TTR V30M. Anemia, with low erythropoietin (EPO) levels and spared kidney function, affects about 25% of symptomatic patients, suggesting a blockage of EPO-producing cells. Early non-fibrillar TTR aggregates are highly cytotoxic, inducing oxidative stress, the expression of apoptosis-related molecules and secretion of pro-inflammatory cytokines, factors capable of inhibiting EPO production. Low EPO levels in these patients are not related to renal amyloid deposition or the presence of circulating TTR V30M. However, the role of early non-fibrillar TTR aggregates remains unexplored. We used the EPO producing Hep3B human hepatoma cell line to study the effect of TTR oligomeric aggregates on EPO expression. Hep3B cells were incubated with soluble and oligomeric TTR V30M, and cell proliferation as well as caspase 3/7 activation was evaluated. Relative quantification of EPO mRNA transcripts was performed by real-time PCR. Significant reductions in cell viability (13 ± 7.3%) and activation of caspases 3/7 were seen after 24 h in the presence of oligomeric TTR V30M. Also, EPO expression was significantly reduced (50 ± 2.8%), in normoxic conditions. A reporter assay was constructed with a PCR fragment of the EPO promoter linked to the luciferase gene to evaluate the role of transcription factors targeting the promoter. A significant reduction of EPO promoter activity (53 ± 6.5%) was observed in transfected cells exposed to TTR oligomers. Our results show that oligomeric TTR V30M reduces EPO expression, at least in part through inhibition of promoter activity.

  9. Nano-QSAR in cell biology: Model of cell viability as a mathematical function of available eclectic data.

    PubMed

    Toropova, Alla P; Toropov, Andrey A

    2017-03-07

    The prediction of biochemical endpoints is an important task of the modern medicinal chemistry, cell biology, and nanotechnology. Simplified molecular input-line entry system (SMILES) is a tool for representation of the molecular structure. In particular, SMILES can be used to build up the quantitative structure - property/activity relationships (QSPRs/QSARs). The QSPR/QSAR is a tool to predict an endpoint for a new substance, which has not been examined in experiment. Quasi-SMILES are representation of eclectic data related to an endpoint. In contrast to traditional SMILES, which are representation of the molecular structure, the quasi-SMILES are representation of conditions (in principle, the molecular structure also can be taken into account in quasi-SMILES). In this work, the quasi-SMILES were used to build up model for cell viability under impact of the metal-oxides nanoparticles by means of the CORAL software (http://www.insilico.eu/coral). The eclectic data for the quasi-SMILES are (i) molecular structure of metals-oxides; (ii) concentration of the nanoparticles; and (iii) the size of nanoparticles. The significance of different eclectic facts has been estimated. Mechanistic interpretation and the domain of applicability for the model are suggested. The statistical quality of the models is satisfactory for three different random distribution of available data into the training (sub-training and calibration) and the validation sets.

  10. 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.

  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. 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

  13. 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

  14. 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

  15. Strengths and weaknesses in the determination of Saccharomyces cerevisiae cell viability by ATP-based bioluminescence assay.

    PubMed

    Paciello, Lucia; Falco, Francesco Cristino; Landi, Carmine; Parascandola, Palma

    2013-03-05

    Due to its sensitivity and speed of execution, detection of ATP by luciferin-luciferase reaction is a widely spread system to highlight cell viability. The paper describes the methodology followed to successfully run the assay in the presence of yeast cells of two strains of the yeast Saccharomyces cerevisiae, BY4741 and CEN.PK2-1C and emphasizes the importance of correctly determining the contact time between the lysing agent and the yeast cells. Once this was established, luciferin-luciferase reaction was exploited to determine the maximum specific rate of growth, as well as cell viability in a series of routine tests. The results obtained in this preliminary study highlighted that using luciferin-luciferase can imply an over-estimation of maximum specific growth rate with respect to that determined by optical density and/or viable count. On the contrary, the bioluminescence assay gave the possibility to highlight, if employed together with viable count, physiological changes occurring in yeast cells as response to stressful environmental conditions such as those deriving from exposure of yeast cells to high temperature or those depending on the operative conditions applied during fed-batch operations.

  16. PDH45 transgenic rice maintain cell viability through lower accumulation of Na(+), ROS and calcium homeostasis in roots under salinity stress.

    PubMed

    Nath, Manoj; Yadav, Sandep; Kumar Sahoo, Ranjan; Passricha, Nishat; Tuteja, Renu; Tuteja, Narendra

    2016-02-01

    Salinity severely affects the growth/productivity of rice, which is utilized as major staple food crop worldwide. PDH45 (pea DNA helicase 45), a member of the DEAD-box helicase family, actively provides salinity stress tolerance, but the mechanism behind this is not well known. Therefore, in order to understand the mechanism of stress tolerance, sodium ion (Na(+)), reactive oxygen species (ROS), cytosolic calcium [Ca(2+)]cyt and cell viability were analyzed in roots of PDH45 transgenic-IR64 rice lines along with wild-type (WT) IR64 rice under salinity stress (100mM and 200 mM NaCl). In addition, the roots of salinity-tolerant (FL478) and susceptible (Pusa-44) rice varieties were also analyzed under salinity stress for comparative analysis. The results reveal that, under salinity stress (100mM and 200 mM NaCl), roots of PDH45 transgenic lines accumulate lower levels of Na(+), ROS and maintain [Ca(2+)]cyt and exhibit higher cell viability as compared with roots of WT (IR64) plants. Similar results were also obtained in the salinity-tolerant FL478 rice. However, the roots of WT and salinity-susceptible Pusa-44 rice accumulated higher levels of Na(+), ROS and [Ca(2+)]cyt imbalance and lower cell viability during salinity stress, which is in contrast to the overexpressing PDH45 transgenic lines and salinity-tolerant FL478 rice. Further, to understand the mechanism of PDH45 at molecular level, comparative expression profiling of 12 cation transporters/genes was also conducted in roots of WT (IR64) and overexpressing PDH45 transgenic lines (L1 and L2) under salt stress (24h of 200 mM NaCl). The expression analysis results show altered and differential gene expression of cation transporters/genes in salt-stressed roots of WT (IR64) and overexpressing transgenic lines (L1 and L2). These observations collectively suggest that, under salinity stress conditions, PDH45 is involved in the regulation of Na(+) level, ROS production, [Ca(2+)]cyt homeostasis, cell viability and

  17. 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

  18. Effects of four commonly used UV filters on the growth, cell viability and oxidative stress responses of the Tetrahymena thermophila.

    PubMed

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

    2013-11-01

    UV filters are increasingly used in sunscreens and other personal care products. Although their residues have been widely identified in aquatic environment, little is known about the influences of UV filters to protozoan. The growth inhibition effects, cell viability and oxidative stress responses of four commonly used UV filters, 2-ethylhexyl 4-methoxycinnamate (EHMC), benzophenone-3 (BP-3), 4-methyl-benzylidene camphor (4-MBC) and octocrylene (OC), to protozoan Tetrahymena thermophila were investigated in this study. The 24-h EC50 values with 95% confidence intervals for BP-3 and 4-MBC were 7.544 (6.561-8.675) mg L(-1) and 5.125 (4.874-5.388) mg L(-1), respectively. EHMC and OC did not inhibit the growth of T. thermophila after 24h exposure at the tested concentrations. The results of cell viability assays with propidium iodide (PI) staining were consistent with that of the growth inhibition tests. As for BP-3 and 4-MBC, the relatively higher concentrations, i.e. of 10.0 and 15.0 mg L(-1), could lead to the cell membranes impairment after 4h exposure. With the increase of the exposure time to 6h, their adverse effects on cell viability of T. thermophila were observed at the relatively lower concentration groups (1.0 mg L(-1) and 5.0 mg L(-1)). In addition, it is noticeable that at environmentally relevant concentration (1.0 μg L(-1)), BP-3 and 4-MBC could lead to the significant increase of catalase (CAT) activities of the T. thermophila cells. Especially for the BP-3, the oxidative injuries were further confirmed by the reduction of glutathione (GSH) content. It is imperative to further investigate the additive action of UV filters and seek other sensitive endpoint, especially at environmentally relevant concentration.

  19. 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

  20. Punicalagin and catechins contain polyphenolic substructures that influence cell viability and can be monitored by radical chemosensors sensitive to electron transfer.

    PubMed

    Carreras, Anna; Mateos-Martín, María Luisa; Velázquez-Palenzuela, Amado; Brillas, Enric; Sánchez-Tena, Susana; Cascante, Marta; Juliá, Luis; Torres, Josep Lluís

    2012-02-22

    Plant polyphenols may be free radical scavengers or generators, depending on their nature and concentration. This dual effect, mediated by electron transfer reactions, may contribute to their influence on cell viability. This study used two stable radicals (tris(2,3,5,6-tetrachloro-4-nitrophenyl)methyl (TNPTM) and tris(2,4,6-trichloro-3,5-dinitrophenyl)methyl (HNTTM)) sensitive only to electron transfer reduction reactions to monitor the redox properties of polyphenols (punicalagin and catechins) that contain phenolic hydroxyls with different reducing capacities. The use of the two radicals reveals that punicalagin's substructures consisting of gallate esters linked together by carbon-carbon (C-C) bonds are more reactive than simple gallates and less reactive than the pyrogallol moiety of green tea catechins. The most reactive hydroxyls, detected by TNPTM, are present in the compounds that affect HT-29 cell viability the most. TNPTM reacts with C-C-linked gallates and pyrogallol and provides a convenient way to detect potentially beneficial polyphenols from natural sources.

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

    PubMed Central

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

    2016-01-01

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

  2. Short term wear of high Dk soft contact lenses does not alter corneal epithelial cell size or viability

    PubMed Central

    Stapleton, F.; Kasses, S.; Bolis, S.; Keay, L.

    2001-01-01

    BACKGROUND/AIMS—Current contact lenses (CLs) when worn on an extended wear basis cause corneal epithelial alterations. The aim of this study was to evaluate changes in corneal epithelial cell morphology and physiology following short term (3 months) wear of highly oxygen permeable CLs and to compare this with disposable CLs.
METHODS—Subjects were wearers of highly oxygen permeable CLs (n=11, wearing CLs on a 30 night schedule), disposable CL users (n=6, wearing CLs on a 6 night schedule), and non-CL wearers (n=20). Mean CL wear experience was 3 months. Epithelial cells were harvested using corneal cytology and were stained using acridine orange and ethidium bromide. Epithelial cell size and viability were determined.
RESULTS—The majority of epithelial cells recovered were non-viable (71%), and the mean longest cell diameter was 38 (SD 8) µm. Disposable CLs caused an increase in cell size (42 (7) µm) compared with both non-wear (39 (7) µm, p=0.01) and wear of highly oxygen permeable CLs (37 (10) µm, p=0.0049). There was no difference in cell viability between groups.
CONCLUSIONS—Extended wear of disposable CLs caused an 8% increase in cell diameter in harvested corneal epithelial cells following 3 months of CL wear. Cells harvested following 3 months' wear of highly oxygen permeable CLs were indistinguishable from those recovered from non-CL wearers.

 PMID:11159475

  3. Pregnancy persistently affects memory T cell populations.

    PubMed

    Kieffer, Tom E C; Faas, Marijke M; Scherjon, Sicco A; Prins, Jelmer R

    2017-02-01

    Pregnancy is an immune challenge to the maternal immune system. The effects of pregnancy on maternal immunity and particularly on memory T cells during and after pregnancy are not fully known. This observational study aims to show the short term and the long term effects of pregnancy on the constitution, size and activation status of peripheral human memory T-lymphocyte populations. Effector memory (EM) and central memory (CM) T-lymphocytes were analyzed using flow cytometry of peripheral blood from 14 nulligravid, 12 primigravid and 15 parous women that were on average 18 months postpartum. The short term effects were shown by the significantly higher CD4+ EM cell and activated CD4+ memory cell proportions in primigravid women compared to nulligravid women. The persistent effects found in this study were the significantly higher proportions of CD4+ EM, CD4+ CM and activated memory T cells in parous women compared to nulligravid women. In contrast to CD4+ cells, activation status of CD8+ memory cells did not differ between the groups. This study shows that pregnancy persistently affects the pre-pregnancy CD4+ memory cell pool in human peripheral blood. During pregnancy, CD4+ T-lymphocytes might differentiate into EM cells followed by persistent higher proportions of CD4+ CM and EM cells postpartum. The persistent effects of pregnancy on memory T cells found in this study support the hypothesis that memory T cells are generated during pregnancy and that these cells could be involved in the lower complication risks in multiparous pregnancies in humans.

  4. The effect of gamma keratose on cell viability in vitro after thermal stress and the regulation of cell death pathway-specific gene expression.

    PubMed

    Poranki, Deepika R; Van Dyke, Mark E

    2014-05-01

    When skin is thermally burned, transfer of heat energy into the skin results in the destruction of cells. Some of these cells are damaged but may be capable of self-repair and survival, thereby contributing to spontaneous healing of the wound. Keratin protein-based biomaterials have been suggested as potential treatments for burn injury. Isolation of cortical proteins from hair fibers results in an acid soluble fraction of keratin proteins referred to as "gamma" keratose. In the present study, treatment with this fraction dissolved in media was able to maintain cell viability after thermal stress in an in vitro model using primary mouse dermal fibroblasts. PCR array analysis demonstrated that gamma keratose treatment may assist in the survival and salvage of thermally stressed cells by maintaining their viability through regulation of cell death pathway-related genes. Gamma keratose may be a promising biomaterial for burn treatment that aids in spontaneous wound healing from viable tissue surrounding the burn.

  5. In vitro and in vivo inhibition of tumor cell viability by combined dihydroartemisinin and doxorubicin treatment, and the underlying mechanism

    PubMed Central

    Tai, Xiang; Cai, Xiao-Bei; Zhang, Zhang; Wei, Rui

    2016-01-01

    The natural extract artemisinin and its derivatives have good anticancer activity. The present study aimed to investigate the in vitro inhibitory effects of combined dihydroartemisinin (DHA) and doxorubicin (DOX) treatment on a variety of tumor cell lines (HeLa, OVCAR-3, MCF-7, PC-3 and A549), as well as the underlying mechanisms. In addition, the in vivo effects of DHA and DOX were evaluated using a mouse HeLa tumor model. The HeLa, OVCAR-3, MCF-7, PC-3 and A549 cells were treated with a combination of DHA and DOX, and the effect on cell viability was detected by Cell Counting kit-8. The cells were observed under a fluorescence microscope after staining with Hoechst 33258 dye to observe morphological changes in the nuclei in order to determine whether the cells in the treatment group exhibited apoptosis. Apoptosis of the cells was further detected by flow cytometry, and statistical analysis was performed. The specific inhibitors of caspase-3, −8 and −9 were used to determine the intrinsic and extrinsic pathways of cell apoptosis. The cervical cancer HeLa cells treated with the combination of DHA and DOX showed up to a 91.5% decrease in viability, which was higher than that of the same cells treated with DHA or DOX alone at the same concentration, respectively (P<0.01). The optimal concentrations of the drugs used in combination were DHA at 10 µg/ml and DOX at 10 µg/ml. DHA + DOX also had a significant inhibitory effect on the ovarian cancer (OVCAR-3), breast cancer (MCF-7), lung cancer (A549) and prostate cancer (PC-3) cells. The images observed under fluorescence microscope after Hoechst 33258 staining showed marked pyknosis in the cells treated with DHA + DOX, similar to that when treated with DHA or DOX alone, which is typical in apoptosis. As determined by flow cytometry, the apoptotic rate of the cells treated with DHA + DOX at optimal concentrations was up to 90%, which was significantly higher than that of the cells treated with DHA or DOX alone at

  6. 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.

  7. Umbilical cord blood for autologous transfusion in the early postnatal ontogeny: analysis of cell composition and viability during long-term culturing.

    PubMed

    Romanov, Yu A; Balashova, E E; Bystrykh, O A; Titkov, K V; Dugina, T N; Kabaeva, N V; Fedorova, T A; Rogachevskii, O V; Degtyarev, D N; Sukhikh, G T

    2015-02-01

    Changes in cell composition and viability as well as the content and functional activity of hemopoietic progenitor cells were analyzed during long-term (up to 1 month at 4°C) storage of human umbilical cord blood cells. No significant quantitative changes in erythrocytes were found during this period. The total content and viability of leukocytes changed, which resulted in the prevalence of mononuclear cells (lymphocytes and monocytes). Analysis of functional activity of hemopoietic stem cells in semisolid culture revealed a decrease in the relative content of CFU during the first week of storage [corrected] and inability of cells to colony formation after 2 weeks.

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

    PubMed

    Rampersad, Sephra N

    2012-01-01

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

  9. An improved method for the detection of cell surface antigens in samples of low viability using flow cytometry.

    PubMed

    Wing, M G; Montgomery, A M; Songsivilai, S; Watson, J V

    1990-01-24

    A high non-specific background fluorescence signal was observed when cell surface antigen analysis was carried out using flow cytometry on a cell sample which contained a high proportion of dead and dying cells. To overcome this problem it was necessary to analyse the cells in three stages. First the intact cells were identified by their forward (FWD) and 90 degree scatter profile. These cells were gated-on, then analysed on the basis of their FWD scatter and propidium iodide (PI) signal, allowing the dead PI positive cells to be gated out. The PI negative cells were then displayed using their 90 degree scatter and fluorescence signals following staining with the irrelevant antibody control. This revealed a population of dead cells, which despite being PI negative, were non-specifically binding antibody molecules. Such multiparameter analysis permitted the successful analysis of cell surface antigens in preparations of low viability by gating out the high background fluorescence associated with dead PI positive and negative cells.

  10. Cryptococcal Cell Morphology Affects Host Cell Interactions and Pathogenicity

    PubMed Central

    Nielsen, Judith N.; Charlier, Caroline; Baltes, Nicholas J.; Chrétien, Fabrice; Heitman, Joseph; Dromer, Françoise; Nielsen, Kirsten

    2010-01-01

    Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 µm. Cell enlargement was observed in vivo, producing cells up to 100 µm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aΔ pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection. PMID:20585559

  11. Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines.

    PubMed

    Buttke, T M; McCubrey, J A; Owen, T C

    1993-01-04

    A new tetrazolium compound, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), has recently been described which in the presence of phenazine methosulfate (PMS) is reduced by living cells to yield a formazan product that can be assayed colorimetrically. An important advantage of MTS/PMS over other tetrazolium dyes (e.g., MTT) is the aqueous solubility of the reduced formazan product which eliminates the need for detergent solubilization or organic solvent extraction steps. Its advantages over XTT/PMS, another tetrazolium which yields a water-soluble formazan product, include the absorbance range of color produced (515-580 nm as opposed to 450 nm), the rapidity of color development, and the storage stability of the MTS/PMS reagent solution. In the present study, MTS/PMS was used to assay viability and proliferation of the IL-2-dependent HT-2 and CTLL-2 cell lines and the IL-3-dependent FDC-P1 and FL5.12 cell lines. With each cell line, the amount of formazan product was time-dependent and proportional to the number of viable cells. Furthermore, with both HT-2 and CTLL-2 cells it was found that cultures could be simultaneously labeled with MTS/PMS and [3H]thymidine, with relatively little effect of the dye on uptake of the latter. This feature was further capitalized upon in studies with FDC-P1 cells, in which the co-addition of MTS/PMS and [3H]thymidine was used to distinguish between cell viability and proliferation.

  12. Effect of exogenous surfactants on viability and DNA synthesis in A549, immortalized mouse type II and isolated rat alveolar type II cells

    PubMed Central

    2011-01-01

    Background In mechanically ventilated preterm infants with respiratory distress syndrome (RDS), exogenous surfactant application has been demonstrated both to decrease DNA-synthesis but also and paradoxically to increase epithelial cell proliferation. However, the effect of exogenous surfactant has not been studied directly on alveolar type II cells (ATII cells), a key cell type responsible for alveolar function and repair. Objective The aim of this study was to investigate the effects of two commercially available surfactant preparations on ATII cell viability and DNA synthesis. Methods Curosurf® and Alveofact® were applied to two ATII cell lines (human A549 and mouse iMATII cells) and to primary rat ATII cells for periods of up to 24 h. Cell viability was measured using the redox indicator resazurin and DNA synthesis was measured using BrdU incorporation. Results Curosurf® resulted in slightly decreased cell viability in all cell culture models. However, DNA synthesis was increased in A549 and rat ATII cells but decreased in iMATII cells. Alveofact® exhibited the opposite effects on A549 cells and had very mild effects on the other two cell models. Conclusion This study showed that commercially available exogenous surfactants used to treat preterm infants with RDS can have profound effects on cell viability and DNA synthesis. PMID:21324208

  13. Cell viability evaluation of transdifferentiated endothelial-like cells by quantitative electron-probe X-ray microanalysis for tissue engineering.

    PubMed

    Vico, Manuel; Rodríguez-Morata, Alejandro; Garzón, Ingrid; Campos, Fernando; Jaimes-Parra, Boris; Pérez-Köhler, Barbara; Buján, Julia; Alaminos, Miguel; Sánchez-Quevedo, M Carmen

    2015-11-01

    Development of an efficient vascular substitute by tissue engineering is strongly dependent on endothelial cell viability. The aim of this study was to evaluate cell viability of transdifferentiated endothelial-like cells (Tr-ELC) by using for the first time electron probe X-ray microanalysis (EPXMA), not only to accurately analyze cell viability by quantifying the intracellular ionic concentrations, but also to establish their possible use in vascular tissue engineering protocols. Human umbilical cord Wharton's jelly stem cells (HWJSC) and endothelial cells from the human umbilical vein (HUVEC) were isolated and cultured. Transdifferentiation from HWJSC to the endothelial phenotype was induced. EPXMA was carried out to analyze HUVEC, HWJSC and Tr-ELC cells by using a scanning electron microscope equipped with an EDAX DX-4 microanalytical system and a solid-state backscattered electron detector. To determine total ion content, the peak-to-local-background (P/B) ratio method was used with reference to standards composed of dextran containing known amounts of inorganic salts. Our results revealed a high K/Na ratio in Tr-ELC (9.41), in association with the maintenance of the intracellular levels of chlorine, phosphorous and magnesium and an increase of calcium (p=0.031) and sulfur (p=0.022) as compared to HWJSC. Calcium levels were similar for HUVEC and Tr-ELC. These results ensure that transdifferentiated cells are highly viable and resemble the phenotypic and microanalytical profile of endothelial cells. Tr-ELC induced from HWJSC may fulfill the requirements for use in tissue engineering protocols applied to the vascular system at the viability and microanalytical levels.

  14. Effects of exogenous zinc on cell cycle, apoptosis and viability of MDAMB231, HepG2 and 293 T cells.

    PubMed

    Wang, Yan-hong; Li, Ke-jin; Mao, Li; Hu, Xin; Zhao, Wen-jie; Hu, An; Lian, Hong-zhen; Zheng, Wei-juan

    2013-09-01

    As a non-toxic metal to humans, zinc is essential for cell proliferation, differentiation, regulation of DNA synthesis, genomic stability and mitosis. Zinc homeostasis in cells, which is crucial for normal cellular functioning, is maintained by various protein families including ZnT (zinc transporter/SLC30A) and ZIP (Zrt-, Irt-like proteins/SLC39A) that decrease and increase cytosolic zinc availability, respectively. In this study, we investigated the influences of a specific concentration range of ZnSO4 on cell cycle and apoptosis by flow cytometry, and cell viability by MTT method in MDAMB231, HepG2 and 293 T cell lines. Fluorescent sensors NBD-TPEA and the counterstain for nuclei Hoechst 33342 were used to stain the treated cells for observing the localisation and amount of Zn(2+) via laser scanning confocal microscope. It was found that the influence manners of ZnSO4 on cell cycle, apoptosis and cell viability in various cell lines were different and corresponding to the changes of Zn(2+) content of the three cell lines, respectively. The significant increase on intracelluar zinc content of MDAMB231 cells resulted in cell death, G1 and G2/M cell cycle arrest and increased apoptotic fraction. Additionally, the mRNA expression levels of ZnT and ZIP families in the three cell lines, when treated with high concentration of ZnSO4, increased and decreased corresponding to their functions, respectively.

  15. 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

  16. 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.

  17. Geranylgeraniol suppresses the viability of human DU145 prostate carcinoma cells and the level of HMG CoA reductase

    PubMed Central

    Fernandes, Nicolle V.; Yeganehjoo, Hoda; Katuru, Rajasekhar; DeBose-Boyd, Russell A.; Morris, Lindsey L.; Michon, Renee; Yu, Zhi-Ling; Mo, Huanbiao

    2014-01-01

    The rate-limiting enzyme of the mevalonate pathway, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, provides essential intermediates for the prenylation of nuclear lamins and Ras and dolichol-mediated glycosylation of growth factor receptors. The diterpene geranylgeraniol downregulates the level of HMG CoA reductase and suppresses the growth of human liver, lung, ovary, pancreas, colon, stomach, and blood tumors. We evaluated the growth-suppressive activity of geranylgeraniol in human prostate carcinoma cells. Geranylgeraniol induced dose-dependent suppression of the viability of human DU145 prostate carcinoma cells (IC50 = 80 ±18 μmol/L, n =5) following 72-h incubations in 96-well plates. Cell cycle was arrested at the G1 phase with a concomitant decrease in cyclin D1 protein. Geranylgeraniol-induced apoptosis was detected by flow cytometric analysis, fluorescence microscopy following acridine orange and ethidium bromide dual staining, and caspase-3 activation. Geranylgeraniol-induced viability suppression was accompanied by concentration-dependent decrease in the level of HMG CoA reductase protein. As a nonsterol molecule that downregulates HMG CoA reductase in the presence of sterols, geranylgeraniol may have potential in the chemoprevention and/or therapy of human prostate cancer. PMID:24006306

  18. 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.

  19. The synthetic retinoid Am80 delays recovery in a model of multiple sclerosis by modulating myeloid-derived suppressor cell fate and viability.

    PubMed

    Moliné-Velázquez, Verónica; Ortega, María Cristina; Vila del Sol, Virginia; Melero-Jerez, Carolina; de Castro, Fernando; Clemente, Diego

    2014-07-01

    Relapsing-remitting multiple sclerosis (RR-MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). It is characterized by relapsing phases with ongoing neurological affectation that are followed by a remitting period in which inflammatory events are controlled and the patients partially recover. Experimental Autoimmune Encephalomyelitis (EAE) is the animal model most often used to study the inflammatory component of MS. Several cell types are involved in controlling the immune response in EAE and immature myeloid-derived suppressor cells (MDSCs) have emerged as important actors in the immunomodulation that occurs in EAE due to their ability to suppress inflammatory responses by inducing T cell apoptosis. In this study, we assessed whether MDSC differentiation may have consequences on the clinical course of EAE by treating mice around the peak of the clinical course EAE with the MDSC-differentiating agent Am80, an analogue of retinoid acid. Am80 administration abrogates the immunomodulation that occurs in EAE mice through different MDSC-related mechanisms: i) induction of MDSC apoptosis; ii) polarization of MDSCs to mature subsets of myeloid cells (dendritic cells/macrophages/neutrophils); and iii) altering their immunosuppressor phenotype. Consequently, T cell density increases and their viability is promoted, delaying the animal's recovery. Therefore, our data point to MDSC behaviour as a crucial factor in facilitating the transition from the relapsing to the remission phase in EAE, which should be considered for future immune-related therapies for MS.

  20. Fluoride promotes viability and differentiation of osteoblast-like Saos-2 cells via BMP/Smads signaling pathway.

    PubMed

    Huo, Liangliang; Liu, Kangkang; Pei, Junrui; Yang, Yanmei; Ye, Yan; Liu, Yang; Sun, Jing; Han, Hepeng; Xu, Weimin; Gao, Yanhui

    2013-10-01

    The BMP/Smad signaling pathway plays an important role in the viability and differentiation of osteoblast; however, it is not clear whether this pathway is involved in the fluoride-induced osteoblast differentiation. In this study, we investigated the role of BMP/Smad signaling pathway in fluoride-induced osteoblast-like Saos-2 cells differentiation. Cells were exposed to fluoride of different concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mM), and cell proliferation was determined using WST assays. The expression of osteoblast marker genes such as osteocalcin (BGP) and bone alkaline phosphatase (BALP) were detected by qRT-PCR. We found that fluoride enhanced the proliferation of Saos-2 cells in a dose-dependent manner and 0.2 mM of fluoride resulted in a higher expression of osteoblast marker genes. In addition, immunofluorescence analysis showed that the promotion effects of 0.2 mM of fluoride on Saos-2 cells differentiation were associated with the activation of the BMP/Smad pathway. Expression of phosphorylated Smad1/5(p-Smad1/5) was higher in cells exposed to 0.2 mM of fluoride. Plasmid expression vectors encoding the short hairpin RNA (shRNA) targeting Smad4 gene were used to block the BMP/Smad pathway, which resulted in a significantly reduced expression of BGP and BALP as well as their corresponding mRNA. The mRNA levels after transfection remained low even in the presence of fluoride. The present results reveal that BMP/Smad signaling pathway was altered during the period of osteogenesis, and that the activities of p-Smad1/5 were required for Saos-2 cells viability and differentiation induced by fluoride.

  1. Effect of tissue shipping on plasma cell isolation, viability, and RNA integrity in the context of a centralized good laboratory practice-certified tissue banking facility.

    PubMed

    Ahmann, Gregory J; Chng, Wee Joo; Henderson, Kimberly J; Price-Troska, Tammy L; DeGoey, Roberta W; Timm, Michael M; Dispenzieri, Angela; Greipp, Philip R; Sable-Hunt, Alicia; Bergsagel, Leif; Fonseca, Rafael

    2008-03-01

    The Multiple Myeloma Research Consortium has established a tissue bank for the deposition of bone marrow samples from patients with multiple myeloma to be mailed and processed under good laboratory practices. To date, over 1,000 samples have been collected. At this time, limited information is available on shipped bone marrow aspirates in regards to cell viability, yield, purity, and subsequent RNA yield and quality. To test these determinants, we did a pilot study on behalf of the Multiple Myeloma Research Consortium where samples were drawn at Mayo Clinic Rochester (MCR) pooled and split into two equal aliquots. One-half of each sample was processed following good laboratory practices compliant standard operating procedures, immediately after sample procurement, at MCR. The CD138+ cells were stored at -80 degrees C as a Trizol lysate. The other half of the aspirate was sent overnight to Mayo Clinic Scottsdale where they were processed using identical standard operating procedures. The RNA was extracted and analyzed in a single batch at MCR. At both locations, samples were assayed for the following quality determinants: Viability was assessed using a three-color flow cytometric method (CD45, CD38, and 7-AAD). Cell counts were done to determine plasma cell recovery and post-sort purity determined by means of a slide-based immunofluorescent assay. RNA recovery and integrity was assessed using the Agilent Bioanalyzer. Lastly, gene expression profiles were compared to determine the signature emanating from the shipment of samples. Despite minor differences, our results suggest that shipment of samples did not significantly affect these quality determinants in aggregate.

  2. Interspecific differences in cryoresistance of lichen symbiotic algae of genus Trebouxia assessed by cell viability and chlorophyll fluorescence.

    PubMed

    Hájek, J; Váczi, P; Barták, M; Jahnová, L

    2012-06-01

    Unicellular algae of genus Trebouxia are the most frequent symbiotic photobionts found in lichen species adapted to extreme environments. When lichenised, they cope well with freezing temperature of polar regions, high-mountains environments and were successfully tested in open-space experiments. Trebouxia sp. is considered potential model species for exobiological experiments. The aim of this paper is to evaluate cryoresistence of Trebouxia sp. when isolated from lichen thalli and cultivated on media. In our study, six algal strains were exposed to repeated freezing/thawing cycles. The strains of Trebouxia sp. (freshly isolated from lichen Lasallia pustulata), Trebouxia erici, Trebouxia asymmetrica, Trebouxia glomerata, Trebouxia irregularis, and Trebouxia jamesii from culture collection were cooled from 25 to -40 °C at two different rates. The strains were also shock frozen in liquid nitrogen. After repeated treatment, the strains were inoculated and cultivated on a BBM agar for 7 days. Then, cell viability was assessed as relative share of living cells. Potential quantum yield of photochemical reactions in PS II (F(V)/F(M)), and effective quantum yield of photochemical reactions in PS II (Φ(PSII)) were measured. While the slow cooling rate (0.5 °C min(-1)) did not cause any change in viability, F(V)/F(M), and Φ(PSII), the fast cooling rate (6.0 °C min(-1)) caused species-specific decrease in all parameters. The most pronounced interspecific differences in cryoresistance were found after shock freezing and consequent cultivation. While T. asymmetrica and T. jamesii exhibited low viability of living cells (18.9% and 34.7%) and full suppression of photosynthetic processes, the other strains had viability over 60%, and unaffected values of F(V)/F(M), and Φ(PSII). This indicated a high degree of cryoresistance of T. glomerata, T. erici, T. irregularis and Trebouxia sp. strains. These strains could be used for detailed investigation of underlying physiological

  3. Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device: effect of cytostatic drugs.

    PubMed

    Komen, Job; Wolbers, Floor; Franke, Henk R; Andersson, Helene; Vermes, Istvan; van den Berg, Albert

    2008-10-01

    Breast cancer is the leading cause of cancer deaths among non-smoking women worldwide. At the moment the treatment regime is such that patients receive different chemotherapeutic and/or hormonal treatments dependent on the hormone receptor status, the menopausal status and age. However, in vitro sensitivity testing of tumor biopsies could rationalize and improve the choice of chemo- and hormone therapy. Lab-on-a-Chip devices, using microfluidic techniques, make detailed cellular analysis possible using fewer cells, enabling working with a patients' own cells and performing chemo- and hormone sensitivity testing in an ex vivo setting. This article describes the development of two microfluidic devices made in poly(dimethylsiloxane) (PDMS) to validate the cell culture properties and analyze the chemosensitivity of MCF-7 cells (estrogen receptor positive human breast cancer cells) in response to the drug staurosporine (SSP). In both cases, cell viability was assessed using the life-stain Calcein-AM (CAAM) and the death dye propidium iodide (PI). MCF-7 cells could be statically cultured for up to 7 days in the microfluidic chip. A 30 min flow with SSP and a subsequent 24 h static incubation in the incubator induced apoptosis in MCF-7 cells, as shown by a disappearance of the aggregate-like morphology, a decrease in CAAM staining and an increase in PI staining. This work provides valuable leads to develop a microfluidic chip to test the chemosensitivity of tumor cells in response to therapeutics and in this way improve cancer treatment towards personalized medicine.

  4. Dietary oleic and palmitic acids modulate the ratio of triacylglycerols to cholesterol in postprandial triacylglycerol-rich lipoproteins in men and cell viability and cycling in human monocytes.

    PubMed

    López, Sergio; Bermúdez, Beatriz; Pacheco, Yolanda M; López-Lluch, Guillermo; Moreda, Wenceslao; Villar, José; Abia, Rocío; Muriana, Francisco J G

    2007-09-01

    The postprandial metabolism of dietary fats produces triacylglycerol (TG)-rich lipoproteins (TRL) that could interact with circulating cells. We investigated whether the ratios of oleic:palmitic acid and monounsaturated fatty acids (MUFA):SFA in the diet affect the ratio of TG:cholesterol (CHOL) in postprandial TRL of healthy men. The ability of postprandial TRL at 3 h (early postprandial period) and 5 h (late postprandial period) to affect cell viability and cycle in the THP-1 human monocytic cell line was also determined. In a randomized, crossover experiment, 14 healthy volunteers (Caucasian men) ate meals enriched (50 g/m(2) body surface area) in refined olive oil, high-palmitic sunflower oil, butter, and a mixture of vegetable and fish oils, which had ratios of oleic:palmitic acid (MUFA:SFA) of 6.83 (5.43), 2.36 (2.42), 0.82 (0.48), and 13.81 (7.08), respectively. The ratio of TG:CHOL in postprandial TRL was inversely correlated (r = -0.89 to -0.99) with the ratio of oleic:palmitic acid and with the MUFA:SFA ratio in the dietary fats (P < 0.05). Postprandial TRL at 3 h preferentially increased the proportion of necrotic cells, whereas postprandial TRL at 5 h increased the proportion of apoptotic cells (P < 0.05). Cell cycle analysis showed that postprandial TRL blocked the human monocytes in S-phase. Our findings suggest that the level of TG and CHOL into postprandial TRL is associated with the ratios of oleic:palmitic acid and MUFA:SFA in dietary fats, which determines the ability of postprandial TRL to induce cytotoxicity and disturb the cell cycle in THP-1 cells.

  5. Exploring the frontier between life and death in Escherichia coli: evaluation of different viability markers in live and heat- or UV-killed cells.

    PubMed

    Villarino, A; Bouvet, O M; Regnault, B; Martin-Delautre, S; Grimont PAD

    2000-11-01

    A number of methods have been proposed to assess the viability of cells without culture. Each method is based on criteria that reflect different levels of cellular integrity or functionality. As a consequence, the interpretation of viability is often ambiguous. The purposes of this work were to evaluate the capacity of current viability markers to distinguish between live and dead Escherichia coli K-12 cells. Methods that assess 'viability' by the demonstration of metabolic activities (esterase activity, active electron transport chain, transport of glucose), cellular integrity (membrane integrity, presence of nucleic acids) or the building up of cellular material (cell elongation) have been evaluated in live and UV- or heat-killed cells. With live cells, viability markers detected cells in counts similar to the colony count. However, these so-called viability markers could stain dead cells for some time after the lethal treatment. For the UV-killed cells, residual activities were detected even after 48 h of storage at 20 degrees C. However, for heat-treated cells, these activities disappeared within hours after heat treatment. Only a combination of fluorescence in situ hybridization with rRNA probes and cell elongation in response to nutrients (in the presence of an inhibitor of cell division) had the ability to differentiate live from dead cells. Problems in the definition of a viable but nonculturable state are in part due to the lack of a clear definition of bacterial death. We consider death as an irreversible state where no growth, cell elongation or protein synthesis may occur.

  6. Sodium phenylbutyrate antagonizes prostate cancer through the induction of apoptosis and attenuation of cell viability and migration

    PubMed Central

    Xu, Yawen; Zheng, Shaobo; Chen, Binshen; Wen, Yong; Zhu, Shanwen

    2016-01-01

    Background Prostate cancer (PCa) is a leading cause of cancer-related death in men. Sodium phenylbutyrate (SPB) has shown its potential as an anticancer therapy in numerous cancer types. In the present study, we attempted to assess the effect of SPB against PCa and whether this treatment was associated with the regulation of survivin. Methods Two human PCa cancer cell lines, DU145 and PC3, were used in the present study. Cell Counting Kit-8 (CCK-8) assay was conducted to measure the proliferation of PCa cells incubated with SPB. The effect of SPB on the cell apoptosis, cell colony formation ability, and cell morphological change was also assessed. Transwell experiment and Western blotting assay were performed to determine the effect of SPB on the migration and invasion ability of both cell types. Moreover, the expression pattern of survivin and MAPK members in both cell types after the treatment of SPB was also detected. Additionally, an in vivo tumor formation assay was performed to evaluate the treatment potential of SPB against PCa. Results We found that the viability of PCa cells was significantly inhibited by SPB treatment. As illustrated by flow cytometry, for DU145 cell line the average apoptotic rate of SPB-treated cells was significantly lower than that of the control group (P<0.05); similar results were also seen for PC3 (P<0.05). SPB administration also attenuated the colony formation and migration abilities in both cell lines. The expression level of survivin in SPB-treated cells was significantly downregulated, while the phosphorylation of p-38 and ERK was enhanced. Furthermore, in vivo tumor formation of both cell lines was suppressed by SPB as well. Conclusion The above results confirmed the potential of SPB as an effective therapeutic agent for the prevention or treatment of PCa. This amelioration might be due to the blockade of the survivin pathway. PMID:27274278

  7. 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.

  8. 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. Hydrat