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Sample records for activity cell viability

  1. Sphingosine Kinase Activity Is Not Required for Tumor Cell Viability

    PubMed Central

    Brown, Matthew L.; Carlson, Timothy; Coxon, Angela; Fajardo, Flordeliza; Frank, Brendon; Gustin, Darin; Kamb, Alexander; Kassner, Paul D.; Li, Shyun; Li, Yihong; Morgenstern, Kurt; Plant, Matthew; Quon, Kim; Ruefli-Brasse, Astrid; Schmidt, Joanna; Swearingen, Elissa; Walker, Nigel; Wang, Zhulun; Watson, J. E. Vivienne; Wickramasinghe, Dineli; Wong, Mariwil; Xu, Guifen; Wesche, Holger

    2013-01-01

    Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology. PMID:23861887

  2. 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

  3. Transcriptional activity around bacterial cell death reveals molecular biomarkers for cell viability

    PubMed Central

    Kort, Remco; Keijser, Bart J; Caspers, Martien PM; Schuren, Frank H; Montijn, Roy

    2008-01-01

    Background In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis. Results We present microbiological, cytological and molecular analyses of the physiological response to lethal heat stress under accurately defined conditions through systematic sampling of bacteria from a single culture exposed to gradually increasing temperatures. We identified a coherent transcriptional program including known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase. Conclusion The observed coordinated gene expression continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce. Transcription of a very limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria – but silent in dead bacteria-include those of essential genes encoding chaperones of the protein folding machinery and can serve as molecular biomarkers for bacterial cell viability. PMID:19061518

  4. The role of adrenergic activation on murine luteal cell viability and progesterone production.

    PubMed

    Wang, Jing; Tang, Min; Jiang, Huaide; Wu, Bing; Cai, Wei; Hu, Chuan; Bao, Riqiang; Dong, Qiming; Xiao, Li; Li, Gang; Zhang, Chunping

    2016-09-15

    Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating β-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective β-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on β-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating β-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3β-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO. PMID:27173955

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

    PubMed

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

    1976-01-01

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

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

    PubMed

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

    2016-05-01

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

  7. 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...

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

    PubMed Central

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

    2016-01-01

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

  9. 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

  10. 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

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

    PubMed

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

    2006-12-01

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

  12. 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. PMID:24486453

  13. Nanocoating of single cells: from maintenance of cell viability to manipulation of cellular activities.

    PubMed

    Park, Ji Hun; Yang, Sung Ho; Lee, Juno; Ko, Eun Hyea; Hong, Daewha; Choi, Insung S

    2014-04-01

    The chronological progresses in single-cell nanocoating are described. The historical developments in the field are divided into biotemplating, cytocompatible nanocoating, and cells in nano-nutshells, depending on the main research focuses. Each subfield is discussed in conjunction with the others, regarding how and why to manipulate living cells by nanocoating at the single-cell level. PMID:24452932

  14. 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.

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

    PubMed

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

    2015-07-01

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

  16. 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. PMID:26549707

  17. Effect of estradiol and bisphenol A on human hepatoblastoma cell viability and telomerase activity.

    PubMed

    Xu, B L; Zhao, Q Z; Gao, X Y; Hou, G J

    2015-11-01

    Sex hormones from environmental and physiological sources might play a major role in the pathogenesis of hepatoblastoma in children. This study investigated the effects of estradiol and bisphenol A on the proliferation and telomerase activity of human hepatoblastoma HepG2 cells. The cells were divided into 6 treatment groups: control, bisphenol A, estradiol, anti-estrogen ICI 182,780 (hereinafter ICI), bisphenol A+ICI, and estradiol+ICI. Cell proliferation was measured based on average absorbance using the Cell Counting-8 assay. The cell cycle distribution and apoptotic index were determined by flow cytometry. Telomerase activity was detected by polymerase chain reaction and a telomeric repeat amplification protocol assay. A higher cell density was observed in bisphenol A (P<0.01) and estradiol (P<0.05) groups compared with the control group. Cell numbers in S and G2/M phases after treatment for 48 h were higher (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h (P<0.05) were higher in these groups than in the control group. The cell density was also higher in bisphenol A+ICI (P<0.01) and estradiol+ICI (P<0.05) groups compared with the ICI group. Furthermore, cell numbers were increased in S and G2/M phases (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h were higher (P<0.05) in these groups than in the ICI group. Therefore, bisphenol A and estradiol promote HepG2 cell proliferation in vitro by inhibition of apoptosis and stimulation of telomerase activity via an estrogen receptor-dependent pathway. PMID:26397976

  18. Overexpression of interleukin-18 protein reduces viability and induces apoptosis of tongue squamous cell carcinoma cells by activation of glycogen synthase kinase-3β signaling

    PubMed Central

    LIU, WEIWEI; HU, MIN; WANG, YUMEI; SUN, BAOZHEN; GUO, YU; XU, ZHIMIN; LI, JIA; HAN, BING

    2015-01-01

    The aim of this study was to investigate the effects of interleukin-18 (IL-18) expression on regulating the viability and apoptosis of tongue squamous cell carcinoma (TSCC) cells in vitro and examine the underlying molecular events. Human IL-18 cDNA was cloned into the vector pcDNA3.1 (+) and transfected into CRL-1623™ cells. Quantitative reverse transcription-PCR (RT-qPCR), western blot analysis, immunofluorescence, cell viability MTT assay, flow cytometric Annexin V/propidium iodide (PI), Giemsa staining, and caspase-3 activity assay were performed. The data showed that overexpression of IL-18 protein reduced TSCC cell viability by inducing apoptosis. Compared with cells transfected with the control vector, IL-18 expression activated caspase-3, -7, and -9 by inducing their cleavage and increased the expression of interferon (IFN)-γ and cytochrome c mRNA, but reduced cyclin D1 and A1 expression in TSCC cells. IL-18 expression upregulated the expression and phosphorylation of glycogen synthase kinase (GSK)-3β protein in CRL1623 cells, whereas the selective GSK-3β inhibitor kenpaullone antagonized the effects of IL-18 protein on TSCC cells in vitro. The results indicated that IL-18 played an important role in the inhibition of TSCC cell growth and may be further investigated as a novel therapeutic target against TSCC. PMID:25591548

  19. Sustained Akt Activity Is Required to Maintain Cell Viability in Seborrheic Keratosis, a Benign Epithelial Tumor.

    PubMed

    Neel, Victor A; Todorova, Kristina; Wang, Jun; Kwon, Eunjeong; Kang, Minjeong; Liu, Qingsong; Gray, Nathanael; Lee, Sam W; Mandinova, Anna

    2016-03-01

    Seborrheic keratoses (SKs) are common benign skin tumors that share many morphological features with their malignant counterpart, squamous cell carcinoma. SKs frequently have acquired oncogenic mutations in the receptor tyrosine kinase/phosphatidylinositol 3-kinase/Akt signaling cascade. We developed a reliable culture system to study SKs in vitro and screened these cells using a library of selective kinase inhibitors to evaluate effects on cell survival. These benign tumors are sensitive to inhibition by ATP-competitive Akt inhibitors, including A-443654 and GSK690693. RNA interference-mediated Akt suppression mimicked the effects of enzyme inhibition in cultured cells. Akt inhibition suppressed phosphorylation of downstream targets of Akt kinase that are critical for cell survival, including MDM2 and FOXO3a, and induced apoptosis. Cell death was also dependent on p53, mutations in which, although common in cutaneous squamous cell carcinoma, have not been identified in SKs. Intact explants of SKs were also sensitive to Akt inhibition. In addition to the obvious therapeutic implications of these findings, identifying the signaling characteristics that differentiate benign and malignant tumors may inform our understanding of the malignant state. PMID:26739095

  20. Impact of Cyanidin-3-Glucoside on Glycated LDL-Induced NADPH Oxidase Activation, Mitochondrial Dysfunction and Cell Viability in Cultured Vascular Endothelial Cells

    PubMed Central

    Xie, Xueping; Zhao, Ruozhi; Shen, Garry X.

    2012-01-01

    Elevated levels of glycated low density lipoprotein (glyLDL) are frequently detected in diabetic patients. Previous studies demonstrated that glyLDL increased the production of reactive oxygen species (ROS), activated NADPH oxidase (NOX) and suppressed mitochondrial electron transport chain (mETC) enzyme activities in vascular endothelial cells (EC). The present study examined the effects of cyanidin-3-glucoside (C3G), a type of anthocyanin abundant in dark-skinned berries, on glyLDL-induced ROS production, NOX activation and mETC enzyme activity in porcine aortic EC (PAEC). Co-treatment of C3G prevented glyLDL-induced upregulation of NOX4 and intracellular superoxide production in EC. C3G normalized glyLDL-induced inhibition on the enzyme activities of mETC Complex I and III, as well as the abundances of NADH dehydrogenase 1 in Complex I and cytochrome b in Complex III in EC. Blocking antibody for the receptor of advanced glycation end products (RAGE) prevented glyLDL-induced changes in NOX and mETC enzymes. Combination of C3G and RAGE antibody did not significantly enhance glyLDL-induced inhibition of NOX or mETC enzymes. C3G reduced glyLDL-induced RAGE expression with the presence of RAGE antibody. C3G prevented prolonged incubation with the glyLDL-induced decrease in cell viability and the imbalance between key regulators for cell viability (cleaved caspase 3 and B cell Lyphoma-2) in EC. The findings suggest that RAGE plays an important role in glyLDL-induced oxidative stress in vascular EC. C3G may prevent glyLDL-induced NOX activation, the impairment of mETC enzymes and cell viability in cultured vascular EC. PMID:23443099

  1. Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability

    PubMed Central

    Yanagisawa, Tatsuo; Takahashi, Hideyuki; Suzuki, Takehiro; Masuda, Akiko; Dohmae, Naoshi; Yokoyama, Shigeyuki

    2016-01-01

    Translation elongation factor P (EF-P), a ubiquitous protein over the entire range of bacterial species, rescues ribosomal stalling at consecutive prolines in proteins. In Escherichia coli and Salmonella enterica, the post-translational β-lysyl modification of Lys34 of EF-P is important for the EF-P activity. The β-lysyl EF-P modification pathway is conserved among only 26–28% of bacteria. Recently, it was found that the Shewanella oneidensis and Pseudomonas aeruginosa EF-P proteins, containing an Arg residue at position 32, are modified with rhamnose, which is a novel post-translational modification. In these bacteria, EF-P and its Arg modification are both dispensable for cell viability, similar to the E. coli and S. enterica EF-P proteins and their Lys34 modification. However, in the present study, we found that EF-P and Arg32 are essential for the viability of the human pathogen, Neisseria meningitidis. We therefore analyzed the modification of Arg32 in the N. meningitidis EF-P protein, and identified the same rhamnosyl modification as in the S. oneidensis and P. aeruginosa EF-P proteins. N. meningitidis also has the orthologue of the rhamnosyl modification enzyme (EarP) from S. oneidensis and P. aeruginosa. Therefore, EarP should be a promising target for antibacterial drug development specifically against N. meningitidis. The pair of genes encoding N. meningitidis EF-P and EarP suppressed the slow-growth phenotype of the EF-P-deficient mutant of E. coli, indicating that the activity of N. meningitidis rhamnosyl–EF-P for rescuing the stalled ribosomes at proline stretches is similar to that of E. coli β-lysyl–EF-P. The possible reasons for the unique requirement of rhamnosyl–EF-P for N. meningitidis cells are that more proline stretch-containing proteins are essential and/or the basal ribosomal activity to synthesize proline stretch-containing proteins in the absence of EF-P is lower in this bacterium than in others. PMID:26840407

  2. Influence of Cu-Ti thin film surface properties on antimicrobial activity and viability of living cells.

    PubMed

    Wojcieszak, Damian; Kaczmarek, Danuta; Antosiak, Aleksandra; Mazur, Michal; Rybak, Zbigniew; Rusak, Agnieszka; Osekowska, Malgorzata; Poniedzialek, Agata; Gamian, Andrzej; Szponar, Bogumila

    2015-11-01

    The paper describes properties of thin-film coatings based on copper and titanium. Thin films were prepared by co-sputtering of Cu and Ti targets in argon plasma. Deposited coatings consist of 90at.% of Cu and 10at.% of Ti. Characterization of the film was made on the basis of investigations of microstructure and physicochemical properties of the surface. Methods such as scanning electron microscopy, x-ray microanalysis, x-ray diffraction, x-ray photoelectron spectroscopy, atomic force microscopy, optical profilometry and wettability measurements were used to assess the properties of deposited thin films. An impact of Cu-Ti coating on the growth of selected bacteria and viability of the living cells (line L929, NCTC clone 929) was described in relation to the structure, surface state and wettability of the film. It was found that as-deposited films were amorphous. However, in such surroundings the nanocrystalline grains of 10-15nm and 25-35nm size were present. High surface active area with a roughness of 8.9nm, had an effect on receiving relatively high water contact angle value (74.1°). Such wettability may promote cell adhesion and result in an increase of the probability of copper ion transfer from the film surface into the cell. Thin films revealed bactericidal and fungicidal effects even in short term-contact. High activity of prepared films was directly related to high amount (ca. 51 %) of copper ions at 1+ state as x-ray photoelectron spectroscopy results have shown. PMID:26249564

  3. Interleukin 1β and tumor necrosis factor α promote hFOB1.19 cell viability via activating AP1

    PubMed Central

    Ying, Hongliang; Li, Qiang; Zhao, Changfu

    2016-01-01

    Bone trauma healing is a complex physiological process, which may involve the function of various inflammatory cytokines. Our study aimed to explore the roles of inflammatory cytokines in bone trauma healing and reveal the potential mechanism. Concentrations of interleukin (IL)-6, IL-1β and tumor necrosis factor alpha (TNF-α) in peripheral blood serum of bone trauma patients after surgery were determined by ELISA. The human osteoblast hFOB1.19 cell line was cultured to determine the effect of these cytokines in cell viability using MTT assay. In addition, luciferase reporter assay was performed to investigate the activator protein 1 (AP1) transcriptional activity, and small interfering RNA was transfected to inhibit FOS, a component of AP1 molecule. IL-6, IL-1β and TNF-α exhibited higher level in patients with more severe bone traumas after surgery. IL-1β and TNF-α, but not IL-6, induced a significant increase of hFOB1.19 viability after three days of treatment (P < 0.05). IL-1β and TNF-α could activate AP1 transcriptional activity in hFOB1.19 cells (P < 0.001), but the activation was inhibited when cells were pretreated with inhibitor of JNKs, SP600125 (P < 0.001). Besides, the effect of IL-1β and TNF-α on promoting viability was significantly inhibited after knockdown of FOS. These findings indicated that IL-1β and TNF-α played an important role in promoting osteoblast viability via the activation of AP1 transcriptional activity, which was likely to involve the JNK/MAPK signaling pathway. Modulating inflammatory cytokines is a potential strategy for improving the outcome of bone trauma healing. PMID:27347349

  4. 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

  5. Viability, infectivity and fatty acid synthetic activity of Perkinsus marinus meront cells incubated in estuarine and artificial seawater.

    PubMed

    Chu, Fu-Lin E; Lund, Eric D

    2006-07-25

    We investigated the viability and fatty acid synthetic activity of in vitro cultured Perkinsus marinus (Dermo) in lipid-free medium and estuarine water, and the infectivity of P. marinus maintained in artificial seawater (ASW). Viability and fatty acid synthetic activity in 7 d old P. marinus meronts maintained in lipid-free medium and estuarine water were tested. The infectivity of meronts incubated in ASW was examined by first incubating P. marinus meronts in ASW for 2, 3 or 7 d, and then inoculating viable ASW-incubated meronts into the shell cavity of individual oysters Crassostrea virginica. P. marinus infection prevalence and intensity in oysters were determined 9 wk post-inoculation. Heavy mortality occurred in meronts maintained in estuarine water, a drop from an initial value of 100% viable to 7.8 and 6.1% after 3 and 14 d incubation, respectively. Viability was 85 and 67% in meronts maintained in lipid-free medium for 3 and 24 d, respectively. Meronts kept in lipid-free medium for 14 d retained their ability to synthesize fatty acids. Viable meronts incubated in ASW remained infective for up to 7 d. The infection prevalences were 85, 48 and 100%, in the treatments inoculated with viable meronts that were incubated in ASW for 2, 3 and 7 d, respectively. Infection prevalence in the group inoculated with viable meronts immediately after they were transferred to ASW ranged from 61 to 85%. Our results suggest that in nature meronts can survive for at least 14 d outside the host. Viable meronts are not only infective, but are also able to replicate and retain their fatty acid synthetic ability for 7 d. PMID:16956060

  6. Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of Lactococcus lactis in batch fermentations.

    PubMed

    Hansen, Gunda; Johansen, Claus Lindvald; Marten, Gunvor; Wilmes, Jacqueline; Jespersen, Lene; Arneborg, Nils

    2016-07-01

    In this study, we investigated the influence of three extracellular pH (pHex) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pHi) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pHi) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pHex was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point. Furthermore, regardless of the pHex, the acidification activity per cell decreased over time which seemed to be linked to cell shrinkage. Flow cytometric pHi determination demonstrated an increase of the averaged pHi level for higher pH set points, while the pH gradient (pHi-pHex) and the extent of pHi heterogeneity decreased. Cells maintained positive pH gradients at a low pHex of 5.5 and even during substrate limitation at the more widely used pHex 6.5. Moreover, the strain proved able to grow despite small negative or even absent pH gradients at a high pHex of 7.5. The larger pHi heterogeneity at pHex 5.5 and 6.5 was associated with more stressful conditions resulting, e.g., from higher concentrations of non-dissociated lactic acid, while the low pHi heterogeneity at pHex 7.5 most probably corresponded to lower concentrations of non-dissociated lactic acid which facilitated the cells to reach the highest maximum active cell counts of the three pH set points. PMID:27020293

  7. A novel synthetic derivative of the natural product berbamine inhibits cell viability and induces apoptosis of human osteosarcoma cells, associated with activation of JNK/AP-1 signaling.

    PubMed

    Yang, Fan; Nam, Sangkil; Zhao, Robin; Tian, Yan; Liu, Lucy; Horne, David A; Jove, Richard

    2013-11-01

    Osteosarcoma is the most common primary bone tumor in children and adolescents. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have antitumor effects in several cancers. Here, we report that a novel synthetic berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of G292, KHOS, and MG-63 human osteosarcoma cells. Induction of apoptosis in these tumor cells depends on activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Since pan-caspase inhibitor (Z-VAD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) could block the cleavage of PARP, the apoptosis induced by BBMD3 is through intrinsic signaling pathway. BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in increase of phosphorylated c-Jun and total c-Fos, the major components of transcriptional factor AP-1. JNK inhibitor could partially suppress antitumor effect of BBMD3 on osteosarcoma cells. BBMD3 increased the production of reactive oxygen species (ROS) and ROS scavenger, N-acetylcysteine (NAC), could block the phosphorylation of JNK and c-Jun induced by BBMD3. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1 and D2, the positive cell cycle regulators, which is correlated with growth inhibition in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of human osteosarcoma. PMID:24025361

  8. A novel synthetic derivative of the natural product berbamine inhibits cell viability and induces apoptosis of human osteosarcoma cells, associated with activation of JNK/AP-1 signaling

    PubMed Central

    Yang, Fan; Nam, Sangkil; Zhao, Robin; Tian, Yan; Liu, Lucy; Horne, David A; Jove, Richard

    2013-01-01

    Osteosarcoma is the most common primary bone tumor in children and adolescents. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have antitumor effects in several cancers. Here, we report that a novel synthetic berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of G292, KHOS, and MG-63 human osteosarcoma cells. Induction of apoptosis in these tumor cells depends on activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Since pan-caspase inhibitor (Z-VAD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) could block the cleavage of PARP, the apoptosis induced by BBMD3 is through intrinsic signaling pathway. BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in increase of phosphorylated c-Jun and total c-Fos, the major components of transcriptional factor AP-1. JNK inhibitor could partially suppress antitumor effect of BBMD3 on osteosarcoma cells. BBMD3 increased the production of reactive oxygen species (ROS) and ROS scavenger, N-acetylcysteine (NAC), could block the phosphorylation of JNK and c-Jun induced by BBMD3. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1 and D2, the positive cell cycle regulators, which is correlated with growth inhibition in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of human osteosarcoma. PMID:24025361

  9. Evaluation of antibacterial activity and osteoblast-like cell viability of TiN, ZrN and (Ti1-xZrx)N coating on titanium

    PubMed Central

    Park, Sang-Won; Lee, Kwangmin; Kang, In-Chol; Kim, Hyun-Seung

    2015-01-01

    PURPOSE The aim of this study was to evaluate antibacterial activity and osteoblast-like cell viability according to the ratio of titanium nitride and zirconium nitride coating on commercially pure titanium using an arc ion plating system. MATERIALS AND METHODS Polished titanium surfaces were used as controls. Surface topography was observed by scanning electron microscopy, and surface roughness was measured using a two-dimensional contact stylus profilometer. Antibacterial activity was evaluated against Streptococcus mutans and Porphyromonas gingivalis with the colony-forming unit assay. Cell compatibility, mRNA expression, and morphology related to human osteoblast-like cells (MG-63) on the coated specimens were determined by the XTT assay and reverse transcriptase-polymerase chain reaction. RESULTS The number of S. mutans colonies on the TiN, ZrN and (Ti1-xZrx)N coated surface decreased significantly compared to those on the non-coated titanium surface (P<0.05). CONCLUSION The number of P. gingivalis colonies on all surfaces showed no significant differences. TiN, ZrN and (Ti1-xZrx)N coated titanium showed antibacterial activity against S. mutans related to initial biofilm formation but not P. gingivalis associated with advanced periimplantitis, and did not influence osteoblast-like cell viability. PMID:25932316

  10. Cell viability in a wet silica gel.

    PubMed

    Nieto, Alejandra; Areva, Sami; Wilson, Timothy; Viitala, Reeta; Vallet-Regi, Maria

    2009-11-01

    A modified two-step sol-gel route using silicon ethoxide (TEOS) has been used to synthesize amorphous sol-gel-derived silica, which has been successfully used as a cell encapsulation matrix for 3T3 mouse fibroblasts and CRL-2595 epithelial cells due to its non-toxicity. The sol-gel procedure comprised a first, low pH hydrolysis step, followed by a neutral condensation-gelation step. A high water-to-TEOS ratio and the addition of d-glucose as a porogen and source of nutrients were chosen to minimize silica dissolution and improve the biocompatibility of the process. Indeed, the cell integrity in the encapsulation process was preserved by alcohol removal from the starting solution. Cells were then added in a buffered medium, causing rapid gelation and entrapment of the cells within a randomly structured siloxane matrix in the shape of a monolith, which was maintained in the wet state. MTT and alamarBlue assays were used to check the cytotoxicity of the silica gels and the viability of entrapped cells at initial times in contact with silica. To improve cell attachment, cell clumping experiments - where groups of cells were formed - were designed, rendering improved viability. The obtained materials are therefore excellent candidates for designing tissue-culture scaffolds and implantable bioreactors for biomedical applications. PMID:19481618

  11. [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. PMID:22642125

  12. Effects of cryopreservation and hypothermic storage on cell viability and enzyme activity in recombinant encapsulated cells overexpressing alpha-L-iduronidase.

    PubMed

    Mayer, Fabiana Quoos; Baldo, Guilherme; de Carvalho, Talita Giacomet; Lagranha, Valeska Lizzi; Giugliani, Roberto; Matte, Ursula

    2010-05-01

    Here, we show the effects of cryopreservation and hypothermic storage upon cell viability and enzyme release in alginate beads containing baby hamster kidney cells overexpressing alpha-L-iduronidase (IDUA), the enzyme deficient in mucopolysaccharidosis type I. In addition, we compared two different concentrations of alginate gel (1% and 1.5%) in respect to enzyme release from the beads and their shape and integrity. Our results indicate that in both alginate concentrations, the enzyme is released in lower amounts compared with nonencapsulated cells. Alginate 1% beads presented increased levels of IDUA release, although this group presented more deformities when compared with alginate 1.5% beads. Importantly, both encapsulated groups presented higher cell viability after long cryopreservation period and hypothermic storage. In addition, alginate 1.5% beads presented higher enzyme release after freezing protocols. Taken together, our findings suggest a benefic effect of alginate upon cell viability and functionality. These results may have important application for treatment of both genetic and nongenetic diseases using microencapsulation-based artificial organs. PMID:20633158

  13. [The Role of ABCG2 Protein in Maintenance of Viability and Proliferative Activity of Bone Marrow Mesenchymal Stem Cells Under Hypoxic Conditions].

    PubMed

    Poleshko, A G; Volotovski, I D

    2016-01-01

    It has been shown that hypoxia (5% 02) and fibroblast growth factor bFGF reduce the doubling time of bone marrow mesenchymal stem cells under their cultivation in vitro that indicates an increase in cell culture proliferation. It has been found out that low concentrations of O2 and factor bFGF added to the cell culture medium increase an expression of abcg2 gene and its gene protein, ABCG2 transport gene, in mesenchymal stem cells. These events potentiate the effects of hypoxia observed in mesenchymal stem cells. We revealed that blocking of ABCG2 protein functional activity led to increased generation of reactive oxygen species in mesenchymal stem cells. The effect of hypoxia and/or bFGF on protein profile of mesenchymal stem cells was studied. The results represented in this work together with previous data proved a link between ABCG2 protein expression, its activity and maintenance of viability and proliferative activity of mesenchymal stem cells cultivated under hypoxia. ABCG2 acts as protector. PMID:27192835

  14. Effects of G6PD activity inhibition on the viability, ROS generation and mechanical properties of cervical cancer cells.

    PubMed

    Fang, Zishui; Jiang, Chengrui; Feng, Yi; Chen, Rixin; Lin, Xiaoying; Zhang, Zhiqiang; Han, Luhao; Chen, Xiaodan; Li, Hongyi; Guo, Yibin; Jiang, Weiying

    2016-09-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency has been revealed to be involved in the efficacy to anti-cancer therapy but the mechanism remains unclear. We aimed to investigate the anti-cancer mechanism of G6PD deficiency. In our study, dehydroepiandrosterone (DHEA) and shRNA technology were used for inhibiting the activity of G6PD of cervical cancer cells. Peak Force QNM Atomic Force Microscopy was used to assess the changes of topography and biomechanical properties of cells and detect the effects on living cells in a natural aqueous environment. Flow cytometry was used to detect the apoptosis and reactive oxygen species (ROS) generation. Scanning electron microscopy was used to observe cell morphology. Moreover, a laser scanning confocal microscope was used to observe the alterations in cytoskeleton to explore the involved mechanism. When G6PD was inhibited by DHEA or RNA interference, the abnormal Young's modulus and increased roughness of cell membrane were observed in HeLa cells, as well as the idioblasts. Simultaneously, G6PD deficiency resulted in decreased HeLa cells migration and proliferation ability but increased ROS generation inducing apoptosis. What's more, the inhibition of G6PD activity caused the disorganization of microfilaments and microtubules of cytoskeletons and cell shrinkage. Our results indicated the anti-cervix cancer mechanism of G6PD deficiency may be involved with the decreased cancer cells migration and proliferation ability as a result of abnormal reorganization of cell cytoskeleton and abnormal biomechanical properties caused by the increased ROS. Suppression of G6PD may be a promising strategy in developing novel therapeutic methods for cervical cancer. PMID:27217331

  15. IL-2, IL-4, IFN-γ or TNF-α enhances BAFF-stimulated cell viability and survival by activating Erk1/2 and S6K1 pathways in neoplastic B-lymphoid cells.

    PubMed

    Gui, Lin; Zeng, Qingyu; Xu, Zhigang; Zhang, Hai; Qin, Shanshan; Liu, Chunxiao; Xu, Chong; Qian, Zhou; Zhang, Shuangquan; Huang, Shile; Chen, Long

    2016-08-01

    B-cell activating factor of the TNF family (BAFF) has been documented to act as a critical factor in the development of aggressive B lymphocytes and autoimmune diseases. However, the effect of various cytokines on BAFF-elicited neoplastic B-lymphoid cells is not known. In this study, we exhibited that administration of human soluble BAFF (hsBAFF), IL-2, IL-4, IFN-γ, or TNF-α alone increased cell viability and survival in Raji cells concentration-dependently, yet a more robust viability/survival was seen in the cells co-treatment of IL-2, IL-4, IFN-γ, or TNF-α with hsBAFF, respectively. Further research revealed that both Erk1/2 and S6K1 signaling pathways were essential for IL-2, IL-4, IFN-γ, or TNF-α enhancement of the viability/survival in the hsBAFF-stimulated cells, as inhibition of Erk1/2 with U0126 or down-regulation of Erk1/2, or blockage of S6K1 with rapamycin or silencing S6K1, or silencing S6K1/Erk1/2, respectively, reduced the cell viability/survival in the cells treated with/without hsBAFF±IL-2, IL-4, IFN-γ, or TNF-α. These findings indicate that IL-2, IL-4, IFN-γ or TNF-α enhances BAFF-stimulated cell viability/survival by activating Erk1/2 and S6K1 signaling in neoplastic B-lymphoid cells. Our data suggest that modulation of IL-2, IL-4, IFN-γ and/or TNF-α levels, or inhibitors of Erk1/2 or S6K1 may be a new approach to prevent BAFF-induced aggressive B-cell malignancies. PMID:27235588

  16. The Spindle Assembly Checkpoint Is Not Essential for Viability of Human Cells with Genetically Lowered APC/C Activity

    PubMed Central

    Wild, Thomas; Larsen, Marie Sofie Yoo; Narita, Takeo; Schou, Julie; Nilsson, Jakob; Choudhary, Chunaram

    2016-01-01

    Summary The anaphase-promoting complex/cyclosome (APC/C) and the spindle assembly checkpoint (SAC), which inhibits the APC/C, are essential determinants of mitotic timing and faithful division of genetic material. Activation of the APC/C is known to depend on two APC/C-interacting E2 ubiquitin-conjugating enzymes—UBE2C and UBE2S. We show that APC/C activity in human cells is tuned by the combinatorial use of three E2s, namely UBE2C, UBE2S, and UBE2D. Genetic deletion of UBE2C and UBE2S, individually or in combination, leads to discriminative reduction in APC/C function and sensitizes cells to UBE2D depletion. Reduction of APC/C activity results in loss of switch-like metaphase-to-anaphase transition and, strikingly, renders cells insensitive to chemical inhibition of MPS1 and genetic ablation of MAD2, both of which are essential for the SAC. These results provide insights into the regulation of APC/C activity and demonstrate that the essentiality of the SAC is imposed by the strength of the APC/C. PMID:26904940

  17. Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

    SciTech Connect

    Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung; Yoo, Eun-Sun; Chan Ra, Jeong; Ryu, Kyung-Ha

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.

  18. Effects of various agents on flagellar activity, flagellar autotomy and cell viability in four species of Chlamydomonas (chlorophyta: volvocales).

    PubMed

    Lewin, R A; Lee, T H; Fang, L S

    1982-01-01

    Over 200 strains of green algal flagellates, representing about 100 species, were examined for their suitability as experimental organisms for studies of flagellar activity. The cells of all species shed their flagella under unfavourable conditions of temperature or pH, or in the presence of alcohols, detergents or toxic agents of various kinds. For further studies of flagellar activity, motility and autotomy (biologically induced shedding) in particular, we selected four species of Chlamydomonas: C. dysosmos Moewus, C. moewusii Gerloff, C. monoica Strehlow and C. reinhardtii Dangeard. Agents found to inhibit motility without inducing death or flagellar autotomy included azide, arsenite, thiosulphate, cyanide, ferricyanide, hydroxylamine, chloral hydrate, malonate, p-chloro-mercury benzoate and cytochalasin-B, each in a limited range of concentrations which differed according to species and strain. Higher concentrations of these agents caused the flagella to be shed. Since flagellar autotomy is a means by which a cell can quickly reduce the area of its permeable surface, it may have a positive survival value for species liable to be subjected to unfavourable physicochemical conditions. PMID:6764045

  19. Monomeric adiponectin increases cell viability in porcine aortic endothelial cells cultured in normal and high glucose conditions: Data on kinases activation.

    PubMed

    Grossini, Elena; Farruggio, Serena; Qoqaiche, Fatima; Raina, Giulia; Camillo, Lara; Sigaudo, Lorenzo; Mary, David; Surico, Nicola; Surico, Daniela

    2016-09-01

    We found that monomeric adiponectin was able to increase cell viability in porcine aortic endothelial cells (PAE) cultured both in normal and high glucose condition. Moreover, in normal glucose condition monomeric adiponectin increased p38MAPK, Akt, ERK1/2 and eNOS phosphorylation in a dose- and time-dependent way. Also in high glucose condition monomeric adiponectin increased eNOS and above kinases phosphorylation with similar patterns but at lower extent. For interpretation of the data presented in this article, please see the research article "Monomeric adiponectin modulates nitric oxide release and calcium movements in porcine aortic endothelial cells in normal/high glucose conditions" (Grossini et al., in press) [1]. PMID:27583345

  20. 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

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

    PubMed

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

    2016-01-01

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

  2. Effect of anaesthetics and dichlorodifluoromethane on the viability of the cells of Escherichia coli and the activities of some of its enzymes

    SciTech Connect

    Laverty, D.M.; Fennema, O.

    1985-01-01

    Three anaesthetics (halothane, CF/sub 3/CHClBr; Ethrane, F/sub 2/ HCOF/sub 2/CCHClF; cyclopropane) and one other halogenated, short-chain hydrocarbon (F-12, Cl/sub 2/F/sub 2/C) were tested under various conditions to determine their effects on the viability of cells of Escherichia coli and the activities of some of its enzymes. When any of the test chemicals were applied for 60 min at concentrations slightly in excess of saturation, the number of surviving cells decreased substantially, with halothane being the most biocidal of the four chemicals and F-12 the least. Three enzymes (malate dehydrogenase, MD; NADH dehydrogenase; glyceraldehyde-3-phosphate dehydrogenase, GPD) were tested for activity after treatment of E. coli with the test chemicals. In all instances, GPD was least resistant to inactivation and MD was most resistant. Halothane was most inhibitory followed in order by Ethrane, cyclopropane and F-12. Treatment of E. coli with halothane for 60 min at 23 degrees C and a concentration slightly in excess of saturation, resulted in nearly complete inhibition of all three enzymes.

  3. Investigation of the effects of 2.1 GHz microwave radiation on mitochondrial membrane potential (ΔΨm), apoptotic activity and cell viability in human breast fibroblast cells.

    PubMed

    Esmekaya, Meric Arda; Seyhan, Nesrin; Kayhan, Handan; Tuysuz, Mehmet Zahid; Kurşun, Ayşe Canseven; Yağcı, Münci

    2013-01-01

    In the present study we aimed to investigate the effects of 2.1 GHz Wideband Code Division Multiple Access (W-CDMA) modulated Microwave (MW) Radiation on cell survival and apoptotic activity of human breast fibroblast cells. The cell cultures were exposed to W-CDMA modulated MW at 2.1 GHz at a SAR level of 0.607 W/kg for 4 and 24 h. The cell viability was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The percentage of apoptotic cells was analyzed by Annexin V-FITC and PI staining. 5,5',6,6'-Tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to measure Mitochondrial Membrane Potential (ΔΨm). sFasL and Fas/APO-1 protein levels were determined by ELISA method. 2.1 GHz MW radiation was shown to be able to inhibit cell proliferation and induce apoptosis in human breast fibroblast cells. The cell viability of MW-exposed cells was decreased significantly. The percentages of Annexin V-FITC positive cells were higher in MW groups. ΔΨm was decreased significantly due to MW radiation exposure. However, neither sFas nor FasL level was significantly changed in MW-exposed fibroblast cells. The results of this study showed that 2.1 GHz W-CDMA modulated MW radiation-induced apoptotic cell death via the mitochondrial pathway. PMID:23723005

  4. 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.

  5. Mammalian cell viability in electrospun composite nanofiber structures.

    PubMed

    Canbolat, Mehmet Fatih; Tang, Christina; Bernacki, Susan H; Pourdeyhimi, Behnam; Khan, Saad

    2011-10-10

    Incorporation of mammalian cells into nanofibers (cell electrospinning) and multilayered cell-nanofiber structures (cell layering) via electrospinning are promising techniques for tissue engineering applications. We investigate the viability of 3T3-L1 mouse fibroblasts after incorporation into poly(vinyl alcohol) nanofibers and multilayering with poly(caprolactone) nanofibers and analyze the possible factors that affect cell viability. We observe that cells do not survive cell electrospinning but survive cell layering. Assessing the factors involved in cell electrospinning, we find that dehydration and fiber stretching are the main causes of cell death. In cell layering, the choice of solvent is critical, as residual solvent in the electrospun fibers could be detrimental to the cells. PMID:21984502

  6. Phytochemical evaluation, antioxidant assay, antibacterial activity and determination of cell viability (J774 and THP1 alpha cell lines) of P. sylvestris leaf crude and methanol purified fractions.

    PubMed

    Sharma, Dinesh C; Shukla, Ritu; Ali, Jasarat; Sharma, Swati; Bajpai, Priti; Pathak, Neelam

    2016-01-01

    Phoenix sylvestris (Arecaceae family) known as Indian Date Palm has been identified as a component of traditional medicine against various ailments. The present study was focused on phytochemical screening of crude hexane, dichloromethane and methanol leaf extracts. The crude extracts showed the presence of alkaloids, flavonoids, and phenols in the plant leaves. In the study methanol extract was found most potent, so this extract was further fractionated by column chromatography and 9 methanol purified fractions (MPFs) were isolated. Most potential MPF8 (20:80 chloroform: methanol ratio fraction) significantly enhanced free radicals and antibacterial activity. The best MIC (Minimum inhibitory concentration) of MPF8 was investigated against M. luteus and E. coli at 1 mg/ml concentration. However, against other bacteria the MIC ranged from 1 mg/ml to 3 mg/ml. The GC-MS analysis showed the presence of many biologically active compounds such as alcohols, flavonoids, aromatic compounds, aldehydes, terpenoids fatty acid methyl esters, and phenolics. Pentadecanoic acid occupied maximum (52 %) area in GC-MS profiling. MPF8 was assayed for in-vitro cytotoxicity by MTT assay which confirms its less cytotoxicity at lower concentration and also significant ROS determination against J774 and THP1 cell lines after 2 and 4 hours. PMID:27047320

  7. Phytochemical evaluation, antioxidant assay, antibacterial activity and determination of cell viability (J774 and THP1 alpha cell lines) of P. sylvestris leaf crude and methanol purified fractions

    PubMed Central

    Sharma, Dinesh C.; Shukla, Ritu; Ali, Jasarat; Sharma, Swati; Bajpai, Priti; Pathak, Neelam

    2016-01-01

    Phoenix sylvestris (Arecaceae family) known as Indian Date Palm has been identified as a component of traditional medicine against various ailments. The present study was focused on phytochemical screening of crude hexane, dichloromethane and methanol leaf extracts. The crude extracts showed the presence of alkaloids, flavonoids, and phenols in the plant leaves. In the study methanol extract was found most potent, so this extract was further fractionated by column chromatography and 9 methanol purified fractions (MPFs) were isolated. Most potential MPF8 (20:80 chloroform: methanol ratio fraction) significantly enhanced free radicals and antibacterial activity. The best MIC (Minimum inhibitory concentration) of MPF8 was investigated against M. luteus and E. coli at 1 mg/ml concentration. However, against other bacteria the MIC ranged from 1 mg/ml to 3 mg/ml. The GC-MS analysis showed the presence of many biologically active compounds such as alcohols, flavonoids, aromatic compounds, aldehydes, terpenoids fatty acid methyl esters, and phenolics. Pentadecanoic acid occupied maximum (52 %) area in GC-MS profiling. MPF8 was assayed for in-vitro cytotoxicity by MTT assay which confirms its less cytotoxicity at lower concentration and also significant ROS determination against J774 and THP1 cell lines after 2 and 4 hours. PMID:27047320

  8. 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. PMID:25658580

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

    PubMed Central

    2015-01-01

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

  10. 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.

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

    PubMed Central

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

    2013-01-01

    Abstract The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test. PMID:23777253

  12. Salinity effects on viability, metabolic activity and proliferation of three Perkinsus species

    USGS Publications Warehouse

    La, Peyre M.; Casas, S.; La, Peyre J.

    2006-01-01

    Little is known regarding the range of conditions in which many Perkinsus species may proliferate, making it difficult to predict conditions favorable for their expansion, to identify conditions inducing mortality, or to identify instances of potential cross-infectivity among sympatric host species. In this study, the effects of salinity on viability, metabolic activity and proliferation of P. marinus, P. olseni and P. chesapeaki were determined. Specifically, this research examined the effects of 5 salinities (7, 11, 15, 25, 35???), (1) without acclimation, on the viability and metabolic activity of 2 isolates of each Perkinsus species, and (2) with acclimation, on the viability, metabolic activity, size and number of 1 isolate of each species. P. chesapeaki showed the widest range of salinity tolerance of the 3 species, with high viability and cell proliferation at all salinities tested. Although P. chesapeaki originated from low salinity areas (i.e. <15???), several measures (i.e. cell number and metabolic activity) indicated that higher salinities (15, 25???) were more favorable for its growth. P. olseni, originating from high salinity areas, had better viability and proliferation at the higher salinities (15, 25, 35???). Distinct differences in acute salinity response of the 2 P. olseni isolates at lower salinities (7, 11???), however, suggest the need for a more expansive comparison of isolates to better define the lower salinity tolerance. Lastly, P. marinus was more tolerant of the lower salinities (7 and 11???) than P. olseni, but exhibited reduced viability at 7???, even after acclimation. ?? Inter-Research 2006.

  13. Laser viability method for red blood cell-state monitoring

    NASA Astrophysics Data System (ADS)

    Lapotko, Dmitry; Kuchinsky, Georgy; Antonishina, Elena; Scoromnik, Elena

    1996-01-01

    The method for RBC state control is based upon single cell viability control after illumination with laser pulse. Heat shock resulting from absorption of laser energy by a cell is considered as a cell load. This load acts from inside of the cell, is pulsed (10-5) and can be delivered directly to the chosen cells. The result of each illumination as cell survival or damage is controlled optically by monitoring the cells' response on a pulse through photothermal technique. Under fixed laser parameters the percentage of damaged cells is viability index (VI) for a certain cell population. The testing procedure includes consequential illumination of each cell in population and calculation of VI. Experimental set up is based upon optical microscope. Dual laser thermal lens technique is used for cell illumination and monitoring. For cell loading 5 ns pulses, 400 divided by 600 nm with energies up to 20 (mu) J are generated by tunable dye laser. Cell monitoring is realized with cw He-Ne (632.8 nm) laser and photodetector. All data acquisition routines are automated. Up to 3 cell suspensions can be studied in a multisample chamber designed to secure cells. An amount of cell suspension required is 1 (mu) l. One population test at a fixed wavelength takes 2 divided by 3 min. In experiments with rats, treated with LPS E. Coli injection to stimulate fever and a septic stress we found that variation of RBC viability becomes apparent in 20 - 30 min after injection, while the clinical changes (blood pressure, body temperature, skin temperature) become detectable after 1 hour. The results obtained show that the method can reveal additional properties of the cells most abundant for monitoring and diagnostic tasks.

  14. 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. PMID:22934709

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

    PubMed

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

    2010-09-01

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

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

    PubMed

    Bartl, J; Walitza, S; Grünblatt, E

    2014-01-24

    Attention-deficit/hyperactivity disorder (ADHD) is highly prevalent in children and adolescents and both environmental and genetic factors play major roles. Polyunsaturated fatty acids (PUFAs) are postulated to contribute to the development of the infant brain and an imbalance in these may increase the risk of ADHD. In recent clinical studies, supplementation with PUFAs improved symptoms of ADHD in some cases. Similarly, some beneficial effects were observed with PUFA treatment in neuronal cell cultures. Therefore, in this study, we hypothesized that a specific PUFA combination (available on the market as Equazen™ [Vifor Pharma, Switzerland]) along with iron, zinc, or vitamin B5 (vitB5) would produce an additive beneficial effect on the viability of rat pheochromocytoma-12 dopaminergic cells. The specific PUFA combination alone, as well as added to each of the three nutrients, was tested in a dose-response manner. The specific PUFAs significantly improved cell viability, starting at very low doses (100pM) from 60h up to 90h; while the combined treatment with vitB5 and minerals did not provide additional benefit. Our results confirmed the beneficial effect of the specific PUFAs on neuronal cell viability; although supplementation with minerals and vitB5 did not enhance this effect. PMID:24269370

  17. 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. PMID:25782087

  18. Cell viability and angiogenic potential of a bioartificial adipose substitute.

    PubMed

    Panneerselvan, Anitha; Nguyen, Luong T H; Su, Yan; Teo, Wee Eong; Liao, Susan; Ramakrishna, Seeram; Chan, Ching Wan

    2015-06-01

    An implantable scaffold pre-seeded with cells needs to remain viable and encourage rapid angiogenesis in order to replace injured tissues, especially for tissue defect repairs. We created a bioartificial adipose graft composed of an electrospun 3D nanofibrous scaffold and fat tissue excised from New Zealand white rabbits. Cell viability and angiogenesis potential of the bioartificial substitute were examined during four weeks of culture in Dulbecco's Modified Eagle Medium by immunohistochemical staining with LIVE/DEAD® cell kit and PECAM-1 antibody, respectively. In addition, a Matrigel® assay was performed to examine the possibility of blood vessels sprouting from the bioartificial graft. Our results showed that cells within the graft were viable and vascular tubes were present at week 4, while cells in a fat tissue block were dead in vitro. In addition, capillaries were observed sprouting from the graft into the Matrigel, demonstrating its angiogenic potential. We expect that improved cell viability and angiogenesis in the bioartificial substitute, compared to intact autologous graft, could potentially contribute to its survival following implantation. PMID:23166045

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-07-01

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

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

    PubMed

    Gilbert, Daniel F; Boutros, Michael

    2016-01-01

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

  2. Salinity effects on viability, metabolic activity and proliferation of three Perkinsus species.

    PubMed

    La Peyre, Megan; Casas, Sandra; La Peyre, Jerome

    2006-07-11

    Little is known regarding the range of conditions in which many Perkinsus species may proliferate, making it difficult to predict conditions favorable for their expansion, to identify conditions inducing mortality, or to identify instances of potential cross-infectivity among sympatric host species. In this study, the effects of salinity on viability, metabolic activity and proliferation of P. marinus, P. olseni and P. chesapeaki were determined. Specifically, this research examined the effects of 5 salinities (7, 11, 15, 25, 35 per thousand), (1) without acclimation, on the viability and metabolic activity of 2 isolates of each Perkinsus species, and (2) with acclimation, on the viability, metabolic activity, size and number of 1 isolate of each species. P. chesapeaki showed the widest range of salinity tolerance of the 3 species, with high viability and cell proliferation at all salinities tested. Although P. chesapeaki originated from low salinity areas (i.e. <15 per thousand), several measures (i.e. cell number and metabolic activity) indicated that higher salinities (15, 25 per thousand) were more favorable for its growth. P. olseni, originating from high salinity areas, had better viability and proliferation at the higher salinities (15, 25, 35 per thousand). Distinct differences in acute salinity response of the 2 P. olseni isolates at lower salinities (7, 11 per thousand), however, suggest the need for a more expansive comparison of isolates to better define the lower salinity tolerance. Lastly, P. marinus was more tolerant of the lower salinities (7 and 11 per thousand) than P. olseni, but exhibited reduced viability at 7 per thousand, even after acclimation. PMID:16922001

  3. Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro.

    PubMed

    Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Zhou, Ping; Zhang, Xiaolei; Zhang, Wei; Chen, Qingyu; Kou, Dongquan; Ying, Xiaozhou; Shen, Yue; Cheng, Xiaojie; Yu, Ziming; Peng, Lei; Lu, Chuanzhu

    2013-09-01

    Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca(2+) on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca(2+) on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca(2+) (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca(2+) stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca(2+) significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs. PMID:23749732

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

    PubMed Central

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

    2015-01-01

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

  5. Polyphenolic extracts of edible flowers incorporated onto atelocollagen matrices and their effect on cell viability.

    PubMed

    López-García, Jorge; Kuceková, Zdenka; Humpolíček, Petr; Mlček, Jiři; Sáha, Petr

    2013-01-01

    The phenolic extract of chives flowers (Allium schoenoprasum, Liliaceae), introduced Sage (Salvia pratensis, Lamiaceae), European elderberry (Sambucus nigra, Caprifoliaceae) and common dandelion (Taraxacum officinale, Asteraceae) were characterised by High Performance Liquid Chromatography and incorporated in different concentrations onto atelocollagen thin films. In order to assess the biological impact of these phenolic compounds on cell viability, human immortalised non-tumorigenic keratinocyte cell line was seeded on the thin films and cell proliferation was determined by using an MTT assay. In addition, their antimicrobial activity was estimated by using an agar diffusion test. Data indicated the concomitance between cell viability and concentration of polyphenols. These findings suggest that these phenolic-endowed atelocollagen films might be suitable for tissue engineering applications, on account of the combined activity of polyphenols and collagen. PMID:24177700

  6. 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.

  7. Effect of anthralin on cell viability in human prostate adenocarcinoma.

    PubMed

    Raevskaya, A A; Gorbunova, S L; Savvateeva, M V; Severin, S E; Kirpichnikov, M P

    2012-07-01

    The study revealed the key role of serine protease hepsin activity in transition of in situ prostate adenocarcinoma into the metastasizing form. Inhibition of hepsin activity suppresses the invasive growth of the tumor. Hepsin is an convenient target for pharmacological agents, so the study of its inhibitory mechanisms is a promising avenue in drug development. Assay of proteolytic activity in various tumor cell lines in vitro showed that this activity in prostate adenocarcinoma cells significantly surpasses proteolytic activity in other examined tumor cell lines. Selective cytotoxic action of anthralin, an inhibitor of hepsin activity, on human adenocarcinoma cells was demonstrated in comparison with other tumor cell lines. PMID:22866312

  8. 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. PMID:24053141

  9. 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. PMID:26807771

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

    PubMed

    Cantisani, Maria Carmela; Parascandolo, Alessia; Perälä, Merja; Allocca, Chiara; Fey, Vidal; Sahlberg, Niko; Merolla, Francesco; Basolo, Fulvio; Laukkanen, Mikko O; Kallioniemi, Olli Pekka; Santoro, Massimo; Castellone, Maria Domenica

    2016-05-10

    RET, BRAF and other protein kinases have been identified as major molecular players in thyroid cancer. To identify novel kinases required for the viability of thyroid carcinoma cells, we performed a RNA interference screening in the RET/PTC1(CCDC6-RET)-positive papillary thyroid cancer cell line TPC1 using a library of synthetic small interfering RNAs (siRNAs) targeting the human kinome and related proteins. We identified 14 hits whose silencing was able to significantly reduce the viability and the proliferation of TPC1 cells; most of them were active also in BRAF-mutant BCPAP (papillary thyroid cancer) and 8505C (anaplastic thyroid cancer) and in RAS-mutant CAL62 (anaplastic thyroid cancer) cells. These included members of EPH receptor tyrosine kinase family as well as SRC and MAPK (mitogen activated protein kinases) families. Importantly, silencing of the identified hits did not affect significantly the viability of Nthy-ori 3-1 (hereafter referred to as NTHY) cells derived from normal thyroid tissue, suggesting cancer cell specificity. The identified proteins are worth exploring as potential novel druggable thyroid cancer targets. PMID:27058903

  11. Hybrid Enzalutamide Derivatives with Histone Deacetylase Inhibitor Activity Decrease Heat Shock Protein 90 and Androgen Receptor Levels and Inhibit Viability in Enzalutamide-Resistant C4-2 Prostate Cancer Cells.

    PubMed

    Rosati, Rayna; Chen, Bailing; Patki, Mugdha; McFall, Thomas; Ou, Siyu; Heath, Elisabeth; Ratnam, Manohar; Qin, Zhihui

    2016-09-01

    Histone deacetylase inhibitors (HDACIs) can disrupt the viability of prostate cancer (PCa) cells through modulation of the cytosolic androgen receptor (AR) chaperone protein heat shock protein 90 (HSP90). However, toxicities associated with their pleiotropic effects could contribute to the ineffectiveness of HDACIs in PCa treatment. We designed hybrid molecules containing partial chemical scaffolds of enzalutamide and suberoylanilide hydroxamic acid (SAHA), with weakened intrinsic pan-HDACI activities, to target HSP90 and AR in enzalutamide-resistant PCa cells. The potency of the new molecules, compounds 2-75 [4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide] and 1005 [(E)-3-(4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluorophenyl)-N-hydroxyacrylamide], as inhibitors of nuclear and cytosolic histone deacetylases was substantially lower than that of SAHA in cell-free and in situ assays. Compounds 2-75 and 1005 antagonized gene activation by androgen without inducing chromatin association of AR. Enzalutamide had no effect on the levels of AR or HSP90, whereas the hybrid compounds induced degradation of both AR and HSP90, similar to (compound 1005) or more potently than (compound 2-75) SAHA. Similar to SAHA, compounds 2-75 and 1005 decreased the level of HSP90 and induced acetylation in a predicted approximately 55 kDa HSP90 fragment. Compared with SAHA, compound 2-75 induced greater hyperacetylation of the HDAC6 substrate α-tubulin. In contrast with SAHA, neither hybrid molecule caused substantial hyperacetylation of histones H3 and H4. Compounds 2-75 and 1005 induced p21 and caused loss of viability in the enzalutamide-resistant C4-2 cells, with efficacies that were comparable to or better than SAHA. The results suggest the potential of the new compounds as prototype antitumor drugs that would downregulate HSP90 and AR in

  12. 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

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

    PubMed Central

    2014-01-01

    Biosurfactants are molecules with surface activity produced by microorganisms that can be used in many biomedical applications. The anti-tumour potential of these molecules is being studied, although results are still scarce and few data are available regarding the mechanisms underlying such activity. In this work, the anti-tumour activity of a surfactin produced by Bacillus subtilis 573 and a glycoprotein (BioEG) produced by Lactobacillus paracasei subsp. paracasei A20 was evaluated. Both biosurfactants were tested against two breast cancer cell lines, T47D and MDA-MB-231, and a non-tumour fibroblast cell line (MC-3 T3-E1), specifically regarding cell viability and proliferation. Surfactin was found to decrease viability of both breast cancer cell lines studied. A 24 h exposure to 0.05 g l-1 surfactin led to inhibition of cell proliferation as shown by cell cycle arrest at G1 phase. Similarly, exposure of cells to 0.15 g l-1 BioEG for 48 h decreased cancer cells’ viability, without affecting normal fibroblasts. Moreover, BioEG induced the cell cycle arrest at G1 for both breast cancer cell lines. The biosurfactant BioEG was shown to be more active than surfactin against the studied breast cancer cells. The results gathered in this work are very promising regarding the biosurfactants potential for breast cancer treatment and encourage further work with the BioEG glycoprotein. PMID:24949273

  14. Different Cell Viability Assays Reveal Inconsistent Results After Bleomycin Electrotransfer In Vitro.

    PubMed

    Jakštys, Baltramiejus; Ruzgys, Paulius; Tamošiūnas, Mindaugas; Šatkauskas, Saulius

    2015-10-01

    The aim of this study was to compare different and commonly used cell viability assays after CHO cells treatment with anticancer drug bleomycin (20 nM), high voltage (HV) electric pulses (4 pulses, 1200 V/cm, 100 µs, 1 Hz), and combination of bleomycin and HV electric pulses. Cell viability was measured using clonogenic assay, propidium iodide (PI) assay, MTT assay, and employing flow cytometry modality to precisely count cells in definite volume of the sample (flow cytometry assay). Results showed that although clonogenic cell viability drastically decreased correspondingly to 57 and 3 % after cell treatment either with HV pulses or combination of bleomycin and HV pulses (bleomycin electrotransfer), PI assay performed ~15 min after the treatments indicated nearly 100 % cell viability. MTT assay performed at 6-72 h time points after these treatments revealed that MTT cell viability is highly dependent on evaluation time point and decreased with later evaluation time points. Nevertheless, in comparison to clonogenic cell viability, MTT cell viability after bleomycin electrotransfer at all testing time points was significantly higher. Flow cytometry assay if used at later times, 2-3 days after the treatment, allowed reliable evaluation of cell viability. In overall, our results showed that in order to estimate cell viability after cell treatment with combination of the bleomycin and electroporation the most reliable method is clonogenic assay. Improper use of PI and MTT assays can lead to misinterpretation of the experimental results. PMID:26077843

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  18. 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

  19. Synthesis of dental matrix proteins and viability of odontoblast-like cells irradiated with blue LED.

    PubMed

    Alonso, Juliana Rosa Luiz; Turrioni, Ana Paula Silveira; Basso, Fernanda Gonçalves; de Souza Costa, Carlos Alberto; Hebling, Josimeri

    2016-04-01

    To evaluate the effect of irradiation with light-emitting diode (LED; 455 nm) on the viability and synthesis of dentin matrix proteins by odontoblast-like cells, MDPC-23 cells were cultivated (10(4) cells/cm(2)) in 24-well culture plates. After 12 h incubation in Dulbecco's modified Eagle's medium (DMEM), the cells were submitted to nutritional restriction by means of reducing the concentration of fetal bovine serum (FBS) for an additional 12 h. Cells were irradiated one single time with one of the following energy densities (EDs): 0.5, 2, 4, 10, or 15 J/cm(2) and irradiance fixed at 20 mW/cm(2). Non-irradiated cells served as control. After 72 h, cells were evaluated with regard to viability (methylthiazol tetrazolium technique (MTT)), mineralization nodule (MN) formation, total protein (TP) production, alkaline phosphatase activity (ALP), and collagen synthesis (Sircol), n = 8. The data were submitted to Kruskal-Wallis and Mann-Whitney tests (p > 0.05). There was no statistical difference between the viability of cells irradiated or not (control), for all the EDs. However, an increase in TP was observed for all the EDs when compared with the control group. A reduced ALP activity was seen in all irradiated groups, except for the ED of 0.5 J/cm(2), which did not differ from the control. There was no difference between the irradiated groups and control regarding collagen synthesis, with the exception of the ED of 10 J/cm(2), which inhibited this cell function. Significant reduction in MN occurred only for the EDs of 0.5 and 2 J/cm(2). The single irradiation with blue LED (455 nm), irradiance of 20 mW/cm(2), and energy densities ranging from 0.5 to 15 J/cm(2) exerted no effective biostimulatory capacity on odontoblast-like cells. PMID:26873499

  20. Development of a cell viability assay to assess drug metabolite structure-toxicity relationships.

    PubMed

    Rana, Payal; Will, Yvonne; Nadanaciva, Sashi; Jones, Lyn H

    2016-08-15

    Many adverse drug reactions are caused by the cytochrome P450 (CYP)-dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-induced toxicity and to improve the safety of drug candidates, we developed a simple cell viability assay by combining a bioactivation system (human CYP3A4, CYP2D6 and CYP2C9) with Hep3B cells. We screened a series of drugs to explore structural motifs that may be responsible for CYP450-dependent activation caused by reactive metabolite formation, which highlighted specific liabilities regarding certain phenols and anilines. PMID:27397500

  1. In-situ sonosynthesis of nano N-doped ZnO on wool producing fabric with photo and bio activities, cell viability and enhanced mechanical properties.

    PubMed

    Behzadnia, Amir; Montazer, Majid; Rad, Mahnaz Mahmoudi

    2015-08-01

    Here, a simple processing route is introduced for preparation of N-doped nano structure ZnO at 75-80°C using in-situ sonosynthesis method through hydrolysis of zinc acetate at pH≈9-10 adjusting with ammonia. Synthesis and fabrication of nano N-doped ZnO were carried out on the wool fabric through impregnation of the fabric in ultrasound bath using different concentrations of zinc acetate followed by curing. The antibacterial and antifungal activities of the treated fabrics were assessed against two common pathogenic bacteria including Escherichia coli, Staphylococcus aureus and the diploid fungus namely Candida albicans. The photo-catalytic activity of nano N-doped ZnO particles on the wool fabric was determined by degradation of Methylene Blue under daylight irradiation. Increasing zinc acetate and prolonged sonication time led to higher photo-catalytic activity as more dye stain degraded from the stained treated fabric under daylight. Higher photo-catalytic activity was observed on the nano N-doped ZnO sonotreated wool fabric having more hydrophilicity. Finally, the treatment indicated no negative effect on the fabric safety while reduced alkaline solubility and yellowness even enhanced the fabric tensile strength. The response surface methodology was also utilized to optimize the wool fabric treatment conditions. PMID:26057020

  2. Effect of hydrostatic pressure on the viability of non-adherent HL-60 cells

    NASA Astrophysics Data System (ADS)

    Yabuki, Takahiro; Yamanoha, Banri; Shimizu, Akio

    2013-06-01

    We investigated the effect of hydrostatic pressure on the viability of non-adherent HL-60 cell line derived from leukemic cells over a high pressure range. The HL-60 cells are resistant to pressures of up to 100 MPa under pressurization for 20 min at 25°C. However, cell viability decreased markedly between 100 and 200 MPa, and almost all cells died above 200 MPa. In the case of pressures up to 25 MPa at 25°C for four days, the viability of HL-60 cells was inhibited by increasing the pressure above 20 MPa. Although high viability was observed between 1.6 and 2.0 MPa for adherent astrocytes, viability did not change over pressures up to 2.0 MPa in the case of non-adherent HL-60 cells. It is thought that the response of cells to pressure varies among cell types.

  3. Peptidoglycan Crosslinking Relaxation Plays an Important Role in Staphylococcus aureus WalKR-Dependent Cell Viability

    PubMed Central

    Delaune, Aurelia; Poupel, Olivier; Mallet, Adeline; Coic, Yves-Marie; Msadek, Tarek; Dubrac, Sarah

    2011-01-01

    The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl–glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability. PMID:21386961

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

    PubMed Central

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

    2016-01-01

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

  5. 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.

  6. 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

  7. Inorganic fluoride. Divergent effects on human proximal tubular cell viability.

    PubMed Central

    Zager, R. A.; Iwata, M.

    1997-01-01

    Fluoride (F) is a widely distributed nephrotoxin with exposure potentially resulting from environmental pollution and from fluorinated anesthetic use (eg, isoflurane). This study sought to characterize some of the subcellular determinants of fluoride cytotoxicity and to determine whether subtoxic F exposure affects tubular cell vulnerability to superimposed ATP depletion and nephrotoxic attack. Human proximal tubular cells (HK-2) were cultured with differing amounts of NaF (0 to 20 mmol/L, overlapping with clinically relevant intrarenal/urinary levels after fluorinated anesthetic use). After completing 24-hour exposures, cell injury was determined (vital dye uptake). Fluoride effects on cell deacylation ([3]H-C20:4 release) and PLA2 activity were also assessed. To determine whether subtoxic F exposure alters tubular cell susceptibility to superimposed injury, cells were exposed to subtoxic NaF doses for 0 to 24 hours and then challenged with simulated ischemia (ATP depletion plus Ca2+ overload) or a clinically relevant nephrotoxic insult (myoglobin exposure). NaF induced dose-dependent cytotoxicity (up to approximately 90% vital dye uptake and increased [3H]C20:4 release). Extracellular Ca2+ chelation (EGTA) and PLA2 inhibitor therapy (aristolochic acid, dibucaine, or mepacrine) each conferred significant protective effects. When subtoxic NaF doses were applied, partial cytosolic PLA2 depletion rapidly developed (approximately 85% within 3 hours, determined on cell extracts). These partially PLA2-depleted cells were markedly resistant to ATP depletion/Ca2+ ionophore injury and to myoglobin-induced attack (approximately 50% decrease in cell death). We conclude that 1) F induces dose-dependent cytotoxicity in cultured human proximal tubular cells, 2) this occurs, in part, via Ca(2+)- and PLA2-dependent mechanism(s), 3) partial cytosolic PLA2 depletion subsequently results, and 4) subtoxic fluoride exposure can acutely increase cell resistance to further attack

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

    PubMed

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

    2015-06-01

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

  9. Viability, Apoptosis, Proliferation, Activation, and Cytokine Secretion of Human Keratoconus Keratocytes after Cross-Linking

    PubMed Central

    Stachon, Tanja; Wang, Jiong; Seitz, Berthold; Szentmáry, Nóra

    2015-01-01

    Purpose. The purpose of this study was to determine the impact of cross-linking (CXL) on viability, apoptosis, proliferation, activation, and cytokine secretion of human keratoconus (KC) keratocytes, in vitro. Methods. Primary KC keratocytes were cultured in DMEM/Ham's F12 medium supplemented with 10% FCS and underwent UVA illumination (370 nm, 2 J/cm2) during exposure to 0.1% riboflavin and 20% Dextran in PBS. Twenty-four hours after CXL, viability was assessed using Alamar blue assay; apoptosis using APO-DIRECT Kit; proliferation using ELISA-BrdU kit; and CD34 and alpha-smooth muscle actin (α-SMA) expression using flow cytometry. Five and 24 hours after CXL, FGFb, HGF, TGFβ1, VEGF, KGF, IL-1β, IL-6, and IL-8 secretion was measured using enzyme-linked-immunoabsorbent assay (ELISA). Results. Following CXL, cell viability and proliferation decreased (P < 0.05; P = 0.009), the percentage of apoptotic keratocytes increased (P < 0.05) significantly, and CD34 and α-SMA expression remained unchanged (P > 0.06). Five hours after CXL, FGFb secretion increased significantly (P = 0.037); however no other cytokine secretion differed significantly from controls after 5 or 24 hours (P > 0.12). Conclusions. Cross-linking decreases viability, triggers apoptosis, and inhibits proliferation, without an impact on multipotent hematopoietic stem cell transformation and myofibroblastic transformation of KC keratocytes. CXL triggers FGFb secretion of KC keratocytes transiently (5 hours), normalizing after 24 hours. PMID:25699261

  10. D-Limonene modulates T lymphocyte activity and viability.

    PubMed

    Lappas, Courtney M; Lappas, Nicholas T

    2012-09-01

    d-Limonene, a cyclic terpene that is a major component of several plant essential oils, is used widely as an additive in perfumes, soaps, foods and beverages, and has also been shown to possess chemopreventative and chemotherapeutic activity. A limited number of studies have been conducted investigating the effect of d-limonene on immune system function. We show that d-limonene and its metabolites limonene-1-2-diol and perillic acid inhibit the production by CD3(+)CD4(+) T cells of IFN-γ, IL-2, TNF-α, IL-4 and IL-13, and the production by CD3(+)CD8(+) T cells of IFN-γ, IL-2, and TNF-α. Additionally, the upregulation of CD25, CD69 and CD40L by activated T lymphocytes is modulated by d-limonene, limonene-1-2-diol and perillic acid treatment. Furthermore, high concentrations of d-limonene, limonene-1-2-diol and perillic acid induce T lymphocyte cell death. These data suggest that d-limonene possesses immunomodulatory activity that must be considered when utilizing the compound for therapeutic or commercial purposes. PMID:23059811

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

    PubMed Central

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

    2015-01-01

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

  12. Novel Cell Preservation Technique to Extend Bovine In Vitro White Blood Cell Viability

    PubMed Central

    Laurin, Emilie L.; McKenna, Shawn L. B.; Sanchez, Javier; Bach, Horacio; Rodriguez-Lecompte, Juan Carlos; Chaffer, Marcelo; Keefe, Greg P.

    2015-01-01

    Although cell-mediated immunity based diagnostics can be integral assays for early detection of various diseases of dairy cows, processing of blood samples for these tests is time-sensitive, often within 24 hours of collection, to maintain white blood cell viability. Therefore, to improve utility and practicality of such assays, the objective of this study was to assess the use of a novel white blood cell preservation technology in whole bovine blood. Blood samples from ten healthy cows were each divided into an unpreserved control sample and a test sample preserved with commercially-available cell transport medium. Samples were maintained at room temperature and stimulated with the mitogens pokeweed and concanavalinA, as well as with interleukin-12 p40. Stimulation was completed on days 1, 5, and 8 post-sampling. Viability of white blood cells was assessed through interferon gamma production determined with a commercial enzyme linked immunosorbent assay. In addition, mononuclear cell viability was assessed with propidium iodide flow cytometry. Greater interferon gamma production was observed on days 5 and 8 post-collection in preserved samples, with both pokeweed and concanavalinA stimulating positive interferon gamma production on day 5 post-collection. A greater proportion of the amount of interferon gamma produced on day 1 continued to be produced on days 5 and 8 post-collection with concanavalinA stimulation (with or without interleukin 12) as compared to pokeweed stimulation. Additionally, viable mononuclear cells were still present at eight days post-collection, with a higher mean proportion detected at days 5 and 8 in all stimulated preserved samples. This practical and simple method to extend in vitro white blood cell viability could benefit the efficient utilization of cell-based blood tests in ruminants. PMID:26447691

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

    PubMed

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

    2016-01-01

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

  14. The effect of ultrasound-related stimuli on cell viability in microfluidic channels

    PubMed Central

    2013-01-01

    Background In ultrasonic micro-devices, contrast agent micro-bubbles are known to initiate cavitation and streaming local to cells, potentially compromising cell viability. Here we investigate the effects of US alone by omitting contrast agent and monitoring cell viability under moderate-to-extreme ultrasound-related stimuli. Results Suspended H9c2 cardiac myoblasts were exposed to ultrasonic fields within a glass micro-capillary and their viability monitored under different US-related stimuli. An optimal injection flow rate of 2.6 mL/h was identified in which, high viability was maintained (~95%) and no mechanical stress towards cells was evident. This flow rate also allowed sufficient exposure of cells to US in order to induce bioeffects (~5 sec), whilst providing economical sample collection and processing times. Although the transducer temperature increased from ambient 23°C to 54°C at the maximum experimental voltage (29 Vpp), computational fluid dynamic simulations and controls (absence of US) revealed that the cell medium temperature did not exceed 34°C in the pressure nodal plane. Cells exposed to US amplitudes ranging from 0–29 Vpp, at a fixed frequency sweep period (tsw = 0.05 sec), revealed that viability was minimally affected up to ~15 Vpp. There was a ~17% reduction in viability at 21 Vpp, corresponding to the onset of Rayleigh-like streaming and a ~60% reduction at 29 Vpp, corresponding to increased streaming velocity or the potential onset of cavitation. At a fixed amplitude (29 Vpp) but with varying frequency sweep period (tsw = 0.02-0.50 sec), cell viability remained relatively constant at tsw ≥ 0.08 sec, whilst viability reduced at tsw < 0.08 sec and minimum viability recorded at tsw = 0.05 sec. Conclusion The absence of CA has enabled us to investigate the effect of US alone on cell viability. Moderate-to-extreme US-related stimuli of cells have allowed us to discriminate between stimuli that maintain high viability and stimuli that

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

    PubMed Central

    Johnson, M. Brittany; Criss, Alison K.

    2013-01-01

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

  16. 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.

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

    PubMed

    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

  18. 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

  19. Aspirin inhibits cell viability and mTOR downstream signaling in gastroenteropancreatic and bronchopulmonary neuroendocrine tumor cells

    PubMed Central

    Spampatti, Matilde; Vlotides, George; Spöttl, Gerald; Maurer, Julian; Göke, Burkhard; Auernhammer, Christoph J

    2014-01-01

    AIM: To investigate the effect of aspirin on neuroendocrine tumor (NET) cell growth and signaling in vitro. METHODS: Human pancreatic BON1, bronchopulmonary NCI-H727 and midgut GOT1 neuroendocrine tumor cells were treated with different concentrations of aspirin (from 0.001 to 5 mmol/L), and the resulting effects on metabolic activity/cell proliferation were measured using cell proliferation assays and SYBR-DNA-labeling after 72, 144 and 216 h of incubation. The effects of aspirin on the expression and phosphorylation of several critical proteins that are involved in the most common intracellular growth factor signaling pathways (especially Akt protein kinase B) and mammalian target of rapamycin (mTOR) were determined by Western blot analyses. Propidium iodide staining and flow cytometry were used to evaluate changes in cell cycle distribution and apoptosis. Statistical analysis was performed using a 2-tailed Student’s t-test to evaluate the proliferation assays and cell cycle analyses. The results are expressed as the mean ± SD of 3 or 4 independently performed experiments. Statistical significance was set at P < 0.05. RESULTS: Treatment with aspirin suppressed the viability/proliferation of BON1, NCI-H727 and GOT1 cells in a time- and dose-dependent manner. Significant effects were observed at starting doses of 0.5-1 mmol/L and peaked at 5 mmol/L. For instance, after treatment with 1 mmol/L aspirin for 144 h, the viability of pancreatic BON1 cells decreased to 66% ± 13% (P < 0.05), the viability of bronchopulmonary NCI-H727 cells decreased to 53% ± 8% (P < 0.01) and the viability of midgut GOT1 cells decreased to 89% ± 6% (P < 0.01). These effects were associated with a decreased entry into the S phase, the induction of the cyclin-dependent kinase inhibitor p21 and reduced expression of cyclin-dependent kinase 4 and cyclin D3. Aspirin suppressed mTOR downstream signaling, evidenced by the reduced phosphorylation of the mTOR substrates 4E binding protein 1

  20. In situ label-free cell viability assessment of nucleus pulposus tissue.

    PubMed

    Dittmar, Roman; van Dijk, Bart G M; van Zandvoort, Marc A M J; Ito, Keita

    2014-04-01

    Regenerative medicine approaches aiming at treating degenerating intervertebral discs, a major cause of back pain, are increasingly tested in ex-vivo disc explant models mimicking in-vivo conditions. For assessing the efficacy of regenerative therapies, cell viability is commonly measured requiring specific labels to stain cells. Here, we demonstrate and evaluate how cellular auto-fluorescence can be utilized to non-invasively assess viability in disc tissue in-situ using label-free two-photon microscopy. Live and dead bovine disc cells (0% and 100% cell viability) from the nucleus pulposus were seeded into collagen gels and auto-fluorescence was characterized. Subsequently, nucleus pulposus explants were cultured for 6 days in media with different glucose supplementation (0, 0.25, 0.5, and 1 g/L) to induce different degrees of cell death. Then, samples were split and viability was assessed using label-free two-photon microscopy and conventional staining. Results show that live and dead nucleus pulposus cells systematically emit auto-fluorescent light with distinct characteristics. Cell viability values obtained with label-free microscopy did not significantly differ from those acquired with staining. In summary, monitoring auto-fluorescence facilitates accurate cell viability assessment in nucleus tissue requiring no additional dyes. Thus, this technique may be suitable for pre-clinical testing of regenerative therapies in nucleus pulposus cultures. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:545-550, 2014. PMID:24391094

  1. Plant-derived decapeptide OSIP108 interferes with Candida albicans biofilm formation without affecting cell viability.

    PubMed

    Delattin, Nicolas; De Brucker, Katrijn; Craik, David J; Cheneval, Olivier; Fröhlich, Mirjam; Veber, Matija; Girandon, Lenart; Davis, Talya R; Weeks, Anne E; Kumamoto, Carol A; Cos, Paul; Coenye, Tom; De Coninck, Barbara; Cammue, Bruno P A; Thevissen, Karin

    2014-05-01

    We previously identified a decapeptide from the model plant Arabidopsis thaliana, OSIP108, which is induced upon fungal pathogen infection. In this study, we demonstrated that OSIP108 interferes with biofilm formation of the fungal pathogen Candida albicans without affecting the viability or growth of C. albicans cells. OSIP108 displayed no cytotoxicity against various human cell lines. Furthermore, OSIP108 enhanced the activity of the antifungal agents amphotericin B and caspofungin in vitro and in vivo in a Caenorhabditis elegans-C. albicans biofilm infection model. These data point to the potential use of OSIP108 in combination therapy with conventional antifungal agents. In a first attempt to unravel its mode of action, we screened a library of 137 homozygous C. albicans mutants, affected in genes encoding cell wall proteins or transcription factors important for biofilm formation, for altered OSIP108 sensitivity. We identified 9 OSIP108-tolerant C. albicans mutants that were defective in either components important for cell wall integrity or the yeast-to-hypha transition. In line with these findings, we demonstrated that OSIP108 activates the C. albicans cell wall integrity pathway and that its antibiofilm activity can be blocked by compounds inhibiting the yeast-to-hypha transition. Furthermore, we found that OSIP108 is predominantly localized at the C. albicans cell surface. These data point to interference of OSIP108 with cell wall-related processes of C. albicans, resulting in impaired biofilm formation. PMID:24566179

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

    PubMed

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

    2016-06-01

    The effects of an equimolar mixture of l-arginine and l-glutamate (Arg·Glu) on cell viability and cellular stress using in vitro cell culture systems are examined with reference to NaCl, in the context of monoclonal antibody formulation. Cells relevant to subcutaneous administration were selected: the human monocyte cell line THP-1, grown as a single cell suspension, and adherent human primary fibroblasts. For THP-1 cells, the mechanism of cell death caused by relatively high salt concentrations was investigated and effects on cell activation/stress assessed as a function of changes in membrane marker and cytokine (interleukin-8) expression. These studies demonstrated that Arg·Glu does not have any further detrimental effects on THP-1 viability in comparison to NaCl at equivalent osmolalities, and that both salts at higher concentrations cause cell death by apoptosis; there was no significant effect on measures of THP-1 cellular stress/activation. For adherent fibroblasts, both salts caused significant toxicity at ~400 mOsm/kg, although Arg·Glu caused a more precipitous subsequent decline in viability than did NaCl. These data indicate that Arg·Glu is of equivalent toxicity to NaCl and that the mechanism of toxicity is such that cell death is unlikely to trigger inflammation upon subcutaneous injection in vivo. PMID:26873863

  3. 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

  4. Cryopreservation of Chlamydomonas reinhardtii: a cause of low viability at high cell density.

    PubMed

    Piasecki, Brian P; Diller, Kenneth R; Brand, Jerry J

    2009-02-01

    Cryopreservation is a practical method for stabilizing the genetic content of living algae over long periods of time. Yet, Chlamydomonas reinhardtii, the algal species most often utilized in studies requiring genetically defined strains, is difficult to cryopreserve with a consistently high post-thaw viability. Work described here demonstrates that C. reinhardtii retains high viability only when cryopreserved at a low cell density. Low viability at high cell density was caused by the release of an injurious substance into the culture medium. Rapid freezing and thawing under non-cryoprotective conditions released large amounts of the injurious substance. Heat denaturation of cells prevented the release of the injurious substance, but heating did not inactivate it after it was released. Even when concentrated, the injurious substance was non-toxic to cells under normal culture conditions. Reduced viability of cells cryopreserved in the presence of the injurious substance could not be attributed to changes in the tonicity of the medium. A mutant strain of C. reinhardtii (cw10) with a greatly diminished cell wall did not release a substance that reduced the post-thaw viability of wild-type or cw10 cryopreserved cells. Cryopreservation of cw10 cells was achieved with approximately the same post-thaw viability irrespective to the cell concentration at the time of freezing. Acid treatment of the injurious substance was able to partially diminish its injurious effect on cells during cryopreservation. We propose that diminished viability of C. reinhardtii cells cryopreserved at high cell densities is caused by the enzymatic release of a cell-wall component. PMID:19041638

  5. 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. PMID:26988476

  6. Dynamic assessment of cell viability, proliferation and migration using real time cell analyzer system (RTCA).

    PubMed

    Roshan Moniri, Mani; Young, Ada; Reinheimer, Kelsey; Rayat, Jarrett; Dai, Long-Jun; Warnock, Garth L

    2015-03-01

    Cell viability and cell migration capacities are critical parameters for cell culture-related studies. It is essential to monitor the dynamic changes of cell properties under various co-culture conditions to our better understanding of their behaviours and characteristics. The real time cell analyzer (RTCA, xCELLigence, Roche) is an impedance-based technology that can be used for label-free and real-time monitoring of cell properties, such as cell adherence, proliferation, migration and cytotoxicity. The practicality of this system has been proven in our recent cancer studies. In the present method, we intend to use co-cultures of pancreatic cancer cells (HP62) and mesenchymal stem cells to describe in detail, the procedures and benefits of RTCA. PMID:24443077

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

    PubMed Central

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

    2010-01-01

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

  8. 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. PMID:25647735

  9. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2016-05-01

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

  13. Effect of flupirtine on the growth and viability of U373 malignant glioma cells

    PubMed Central

    Panchanathan, Elango; Ramanathan, Gnanasambandan; Lakkakula, Bhaskar Venkata Kameswara Subrahmanya

    2013-01-01

    Objective Flupirtine is a non-opioid analgesic without antipyretic or antiphlogistic properties but with favorable tolerability in humans. This analgesic also exhibits neuroprotective activities. Furthermore, flupirtine antagonizes glutamate- and NMDA-induced intracellular levels of Ca2+ and counteracts the effects of focal cerebral ischemia. Although flupirtine has been used to relieve pain caused by different diseases and clinical procedures, information on the safety and efficacy of flupirtine is limited. The present study was conducted to investigate the neuroprotective effects of flupirtine on U373 malignant glioma (MG) cell lines. Methods Cell viability and cell cycle analysis was performed by MTT assay and flow cytometry, respectively. Results Variations in the growth of U373 MG cells in 5 mM N-methyl-D-aspartate (NMDA), 1 mM flupirtine, and combined treatment indicated the antagonistic effects of NMDA and flupirtine on MG cell lines. The variation in the percentage of gated cell population in different cell cycle phases showed significant variations after 48 h of treatment. Conclusion Flupirtine has neuroprotective effect of on U373 MG cells, which limits its use in the pain management of brain tumors. This property warrants further studies using animal models and large-scale clinical trials. PMID:24379989

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

    PubMed

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

    2016-03-15

    Cancer is a serious threat to human health. Although numerous anti-cancer drugs are available clinically, many have shown toxic side effects due to poor tumor-selectivity, and reduced effectiveness due to cancers rapid development of resistance to treatment. The development of new highly efficient and practical methods to quantify cell viability and its change under drug treatment is thus of significant importance in both understanding of anti-cancer mechanism and anti-cancer drug screening. Here, we present an approach of utilizing a nanomechanical fluctuation based highly sensitive microcantilever sensor, which is capable of characterizing the viability of cells and quantitatively screening (within tens of minutes) their responses to a drug with the obvious advantages of a rapid, label-free, quantitative, noninvasive, real-time and in-situ assay. The microcantilever sensor operated in fluctuation mode was used in evaluating the paclitaxel effectiveness on breast cancer cell line MCF-7. This study demonstrated that the nanomechanical fluctuations of the microcantilever sensor are sensitive enough to detect the dynamic variation in cellular force which is provided by the cytoskeleton, using cell metabolism as its energy source, and the dynamic instability of microtubules plays an important role in the generation of the force. We propose that cell viability consists of two parts: biological viability and mechanical viability. Our experimental results suggest that paclitaxel has little effect on biological viability, but has a significant effect on mechanical viability. This new method provides a new concept and strategy for the evaluation of cell viability and the screening of anti-cancer drugs. PMID:26406457

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-01

    A positive effect of low-level laser irradiation (LLLI) on the proliferation of some cell types has been observed, but little is known about its effect on dental pulp stem cells (DPSCs). The aim of this study was to identify the lowest energy density able to promote the proliferation of DPSCs and to maintain cell viability. Human DPSCs were isolated from two healthy third molars. In the third passage, the cells were irradiated or not (control) with an InGaAlP diode laser at 0 and 48 h using two different energy densities (0.5 and 1.0 J/cm²). Cell proliferation and viability and mitochondrial activity were evaluated at intervals of 24, 48, 72, and 96 h after the first laser application. Apoptosis- and cell cycle-related events were analyzed by flow cytometry. The group irradiated with an energy density of 1.0 J/cm² exhibited an increase of cell proliferation, with a statistically significant difference (p < 0.05) compared to the control group at 72 and 96 h. No significant changes in cell viability were observed throughout the experiment. The distribution of cells in the cell cycle phases was consistent with proliferating cells in all three groups. We concluded that LLLI, particularly a dose of 1.0 J/cm², contributed to the growth of DPSCs and maintenance of its viability. This fact indicates this therapy to be an important future tool for tissue engineering and regenerative medicine involving stem cells. PMID:26341379

  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. PMID:26634866

  18. 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.

  19. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    Kwolek-Mirek, Magdalena; Zadrag-Tecza, Renata

    2014-11-01

    Determination of cell viability is the most commonly used method for assessing the impact of various types of stressors in toxicity research and in industrial microbiology studies. Viability is defined as a percentage of live cells in a whole population. Although cell death is one of the consequences of toxicity, chemical or physical factors may exert their toxic effects through a number of cellular alterations that may compromise cell ability to divide without necessarily leading to cell death. This aspect represents the term 'cell vitality' defined as physiological capabilities of cells. It is important to note that cell viability and cell vitality represent two different aspects of cell functions, and both are required for the estimation of the physiological state of a cell after exposure to various types of stressors and chemical or physical factors. In this paper, we introduced a classification of available methods for estimating both viability and vitality in Saccharomyces cerevisiae yeast cells (wild-type and Δsod1 mutant) in which the effects of selected oxidants causing oxidative stress is evaluated. We present the advantages as well as disadvantages of the selected methods and assess their usefulness in different types of research. PMID:25154541

  2. Crystal Violet Assay for Determining Viability of Cultured Cells.

    PubMed

    Feoktistova, Maria; Geserick, Peter; Leverkus, Martin

    2016-01-01

    Adherent cells detach from cell culture plates during cell death. This characteristic can be used for the indirect quantification of cell death and to determine differences in proliferation upon stimulation with death-inducing agents. One simple method to detect maintained adherence of cells is the staining of attached cells with crystal violet dye, which binds to proteins and DNA. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture. This protocol describes a quick and reliable screening method that is suitable for the examination of the impact of chemotherapeutics or other compounds on cell survival and growth inhibition. However, characterization of the cause of reduced crystal violet staining requires additional methods detailed elsewhere. PMID:27037069

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

    PubMed

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

    2016-01-01

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

  4. 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

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

    PubMed

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

    2014-10-01

    This study investigated the effect of skin viability on its permeability to psoralen delivered by ethosomes, as compared with liposomes. With decreasing skin viability, the amount of liposome-delivered psoralen that penetrated through the skin increased, whereas skin deposition of psoralen from both ethosomes and liposomes reduced. Psoralen delivery to human-immortalized epidermal cells was more effective using liposomes, whereas delivery to human embryonic skin fibroblast cells was more effective when ethosomes were used. These findings agreed with those of in vivo studies showing that skin psoralen deposition from ethosomes and liposomes first increased and then plateaued overtime, which may indicate gradual saturation of intracellular drug delivery. It also suggested that the reduced deposition of ethosome- or liposome-delivered psoralen in skin with reduced viability may relate to reduced cellular uptake. This work indicated that the effects of skin viability should be taken into account when evaluating nanocarrier-mediated drug skin permeation. PMID:25070929

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

    PubMed

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

    2016-05-01

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

  7. Synthesis and anticancer activity of new flavonoid analogs and inconsistencies in assays related to proliferation and viability measurements

    PubMed Central

    FORBES, ALAINA M.; LIN, HUIMIN; MEADOWS, GARY G.; MEIER, G. PATRICK

    2014-01-01

    Flavonoids have been studied intensely for their ability to act as anti-carcinogenic, anti-inflammatory, anti-viral and anti-aging agents and are often marketed as supplements related to their anti-inflammatory activity. Previous studies have primarily focused on the effects of polar natural flavonoids. We examined the activity of novel hydrophobic and lipophilic flavonols against human DU-145 and PC-3 prostate cancer cell lines. All flavonol analogs were more active than the naturally occurring flavonols quercetin, kaempferol, kaempferide and galangin. The most potent analogs were 6.5-fold more active against DU-145 and PC-3 cells than quercetin and fell within the biologically relevant concentration range (low micromolar). We also evaluated the potential toxic effects of flavonol analogs on normal cells, an assessment that has frequently been ignored when studying the anticancer effects of flavonoids. During these analyses, we discovered that various metabolic and DNA staining assays were unreliable methods for assessing cell viability of flavonoids. Flavonoids reduce colorimetric dyes such as MTT and Alamar Blue in the absence of cells. We showed that flavonol-treated prostate cancer cells were stained less intensely with crystal violet than untreated cells at non-toxic concentrations. The trypan blue exclusion assay was selected as a reliable alternative for measuring cell viability. PMID:24859601

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

    PubMed Central

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

    2015-01-01

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

  9. Nrdp1-mediated degradation of BRUCE decreases cell viability and induces apoptosis in human 786-O renal cell carcinoma cells

    PubMed Central

    Chen, Shao-Jun; Lin, Jian-Hai; Yao, Xu-Dong; Peng, Bo; Xu, Yun-Fei; Liu, Min; Zheng, Jun-Hua

    2016-01-01

    Neuregulin receptor degradation protein-1 (Nrdp1) is involved in a plethora of cellular processes and plays an essential role in the development and progression of human cancers. However, its role in renal cell carcinoma (RCC) remains unclear. Therefore, the present study aimed to explore the biological significance of Nrdp1 in RCC. Western blot analyses of tissue samples from 24 patients with primary RCC revealed lower Nrdp1 and higher baculovirus inhibitor of apoptosis repeat-containing ubiquitin-conjugating enzyme (BRUCE) protein levels in RCC tissues compared with adjacent normal tissues. In addition, MTT and apoptosis assays demonstrated that Nrdp1 overexpression resulted in decreased cell viability and enhanced apoptosis in RCC 786-O cells; conversely, Nrdp1 knockdown increased 786-O cell viability and inhibited apoptosis. Further analysis showed that BRUCE downregulation partially attenuated the effects of Nrdp1 knockdown on RCC cell viability and apoptosis. Moreover, an inverse association was obtained between BRUCE and Nrdp1 protein levels. These findings suggest that Nrdp1-mediated degradation of BRUCE decreases cell viability and induces apoptosis in RCC cells, highlighting Nrdp1 as a potential target for RCC treatment.

  10. 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.

  11. 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.

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

    PubMed

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

    2013-01-01

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

  13. 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

  14. WS6 induces both alpha and beta cell proliferation without affecting differentiation or viability

    PubMed Central

    Boerner, Brian P.; George, Nicholas M.; Mir, Shakeel U.R.; Sarvetnick, Nora E.

    2016-01-01

    Agents that stimulate human pancreatic beta cell proliferation are needed to improve diabetes mellitus treatment. Recently, a small molecule, WS6, was observed to stimulate human beta cell proliferation. However, little is known about its other effects on human islets. To better understand the role of WS6 as a possible beta cell regenerative therapy, we carried out in-depth phenotypic analysis of WS6-treated human islets, exploring its effects on non-beta cell proliferation, beta cell differentiation, and islet cell viability. WS6 not only stimulated beta cell proliferation in cultured human islets (in agreement with previous reports), but also human alpha cell proliferation, indicating that WS6 is not a beta cell-specific mitogen. WS6 did not change the proportion of insulin-positive beta cells or the expression of beta cell-specific transcription factors, suggesting that WS6 does not alter beta cell differentiation, and WS6 had no effect on human islet cell apoptosis or viability. In conclusion, WS6 stimulates proliferation of both human beta and alpha cells while maintaining cellular viability and the beta cell differentiated phenotype. These findings expand the literature on WS6 and support the suggestion that WS6 may help increase human islet mass needed for successful treatment of diabetes. PMID:25739404

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

    PubMed

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

    2012-04-20

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

  16. Distinguish on the viability of human umbilical cord mesenchymal stem cells using delayed luminescence

    NASA Astrophysics Data System (ADS)

    Chen, Ping; Li, Xing; Wang, Yan; Bai, Hua; Lin, Lie

    2014-09-01

    In this paper, we report the discrimination of the viability of human umbilical cord mesenchymal stem cells (hUC-MSCs) with photo-induced delayed luminescence (DL). We measure the DL decay kinetics of hUC-MSCs using an ultraweak luminescence detection system, and find the significant difference in the weight distributions of the decay rate for hUC-MSCs with high and low viabilities. Spectral discrimination of hUC-MSCs with high and low viabilities is thus carried out by comparing the DL kinetics parameters, including the initial intensity, the peak decay rate and the peak weight value. Our results show that the novel optical method for the viability diagnosis of hUC-MSCs has a promising prospect.

  17. Insulin and hypoxia-inducible factor-1 cooperate in pancreatic cancer cells to increase cell viability

    PubMed Central

    ZHANG, DAPENG; CUI, LIHUA; LI, SHU SHUN; WANG, FENG

    2015-01-01

    The aim of the present study was to investigate whether interstitial insulin and cancer-induced hypoxia-inducible factor-1 (HIF-1) cooperate in pancreatic cancer cells. A population of 45 nude mice were divided into one intact control group and six pancreatic tumor-carrier groups. Pancreatic tumors were generated using HIF-1-positive wild-type MiaPaCa2 (wt-MiaPaCa2) pancreatic cancer cells in three groups of carriers and MiaPaCa2 cells transfected with small interfering RNA against HIF-1α (si-MiaPaCa2 cells) in the other three carrier groups. To vary the intrapancreatic insulin levels, tumor-carrying mice were subjected to one of the following conditions: i) Untreated, ii) single injection of the β-cell toxin streptozotosin prior to cancer cell transplantation and iii) daily injection of insulin following cancer cell transplantation. After 12 weeks, tumor viability was assessed by histological analysis. Western blotting of the tumor grafts was performed to determine the protein expression levels of insulin receptor (IR) and two downstream proteins, hexokinase-II (HK-II) and vascular endothelial growth factor (VEGF). Histologically, the greatest viability was observed in wt-MiaPaCa2 tumors with carriers that remained untreated. These tumors also exhibited greater IR expression than their si-MiaPaCa2 counterparts, indicating that HIF-1 is necessary for basal expression of IR. However, IR expression was increased in wt-MiaPaCa2 and si-MiaPaCa2 tumors when the carriers were treated with exogenous insulin. This indicates that the insulin-induced IR expression was independent of HIF-1. Notably, the insulin-induced IR expression was associated with increased HK-II and VEGF expression in wt-MiaPaCa2 tumors but not si-MiaPaC2 tumors. Therefore, the present study proposes that insulin and HIF-1 may cooperate to increase pancreatic cancer cell viability. Furthermore, the HIF-1 signaling pathway is required for insulin-induced HK-II and VEGF expression, as well as basal IR

  18. BVES Regulates Intestinal Stem Cell Programs and Intestinal Crypt Viability after Radiation.

    PubMed

    Reddy, Vishruth K; Short, Sarah P; Barrett, Caitlyn W; Mittal, Mukul K; Keating, Cody E; Thompson, Joshua J; Harris, Elizabeth I; Revetta, Frank; Bader, David M; Brand, Thomas; Washington, M Kay; Williams, Christopher S

    2016-06-01

    Blood vessel epicardial substance (BVES/Popdc1) is a junctional-associated transmembrane protein that is underexpressed in a number of malignancies and regulates epithelial-to-mesenchymal transition. We previously identified a role for BVES in regulation of the Wnt pathway, a modulator of intestinal stem cell programs, but its role in small intestinal (SI) biology remains unexplored. We hypothesized that BVES influences intestinal stem cell programs and is critical to SI homeostasis after radiation injury. At baseline, Bves(-/-) mice demonstrated increased crypt height, as well as elevated proliferation and expression of the stem cell marker Lgr5 compared to wild-type (WT) mice. Intercross with Lgr5-EGFP reporter mice confirmed expansion of the stem cell compartment in Bves(-/-) mice. To examine stem cell function after BVES deletion, we used ex vivo 3D-enteroid cultures. Bves(-/-) enteroids demonstrated increased stemness compared to WT, when examining parameters such as plating efficiency, stem spheroid formation, and retention of peripheral cystic structures. Furthermore, we observed increased proliferation, expression of crypt-base columnar "CBC" and "+4" stem cell markers, amplified Wnt signaling, and responsiveness to Wnt activation in the Bves(-/-) enteroids. Bves expression was downregulated after radiation in WT mice. Moreover, after radiation, Bves(-/-) mice demonstrated significantly greater SI crypt viability, proliferation, and amplified Wnt signaling in comparison to WT mice. Bves(-/-) mice also demonstrated elevations in Lgr5 and Ascl2 expression, and putative damage-responsive stem cell populations marked by Bmi1 and TERT. Therefore, BVES is a key regulator of intestinal stem cell programs and mucosal homeostasis. Stem Cells 2016;34:1626-1636. PMID:26891025

  19. Regulative Effect of Nampt on Tumor Progression and Cell Viability in Human Colorectal Cancer

    PubMed Central

    Lv, Xiaoqun; Zhang, Lingyun; Zhu, Yanyan; Said, Harun M.; Shi, Jimin; Xu, Guoxiong

    2015-01-01

    Colorectal cancer (CRC) is the third most common cancer disease. Here we examined Nampt expression in patients with CRC and the effect of Nampt on cell viability in CRC cells. Nampt protein was overexpressed in colorectal adenoma as well as colorectal carcinoma. The immunoreactive staining of Nampt was negative in the adjacent normal colorectal tissue, weak in colorectal adenoma, and strong in colorectal carcinoma, which may represent tumor progression. Further evaluation of clinical data showed that Nampt expression was not correlated with the clinicopathological characteristics of CRC. Additionally, our in vitro studies demonstrated that Nampt promotes CRC cell viability, whereas the Nampt inhibitor FK866 suppressed CRC cell viability, which was in concordance with the previous studies in other cancer cells. Treatment with Nampt-siRNA reduced the Nampt protein expression resulting in the inhibition of the cell viability of HCT116 and Caco2. Thus, the involvement of Nampt in cell growth indicates that Nampt may play an important role in colorectal tumorigenesis. As a consequence, our results suggest that Nampt may be considered as a progression marker of colorectal tumor and a potentially therapeutic target for the treatment of CRC. PMID:26284136

  20. 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. PMID:25991712

  1. In situ screening assay for cell viability using a dimeric cyanine nucleic acid stain.

    PubMed

    Becker, B; Clapper, J; Harkins, K R; Olson, J A

    1994-08-15

    A rapid and sensitive assay is described for the determination of cell viability of adherent and nonadherent cells that can be performed in situ in 96-well microtiter plates using fluorescence plate scanners. The assay, based on dye exclusion, utilizes a plasma membrane-impermeable, dimeric cyanine dye (YOYO-1). YOYO-1 fluoresces brightly only when bound to nucleic acids. Cells are incubated with YOYO-1, and fluorescence is measured before and after the addition of detergent, which allows the dye to enter the cells. The fluorescence before detergent treatment originates from nonviable cells that have membrane damage and take up YOYO-1. The fluorescence after detergent treatment originates from all cells in the sample. The ratio of the two fluorescence values is used as an indicator of cell viability. The cell viability results of this microplate assay closely resemble those of dye exclusion studies by flow cytometry and are similar but not identical to those of the thiazolyl blue assay, which uses a metabolic indicator of cell death. Because the assay can be performed in situ, without removing the medium, disintegrated cells, cell aggregates, and cells that stick to culture vessel walls are all included in the measurement. PMID:7527190

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

    PubMed

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

    2015-01-01

    Various metals produced from human activity are ubiquitously detected in ambient air. The metals may lead to induction and/or exacerbation of respiratory diseases, but the significant metals and factors contributing to such diseases have not been identified. To compare the effects of each metal and different oxidation states of metals on human airway, we examined the viability and production of interleukin (IL)-6 and IL-8 using BEAS-2B cell line, derived from human airway epithelial cells. Airway epithelial cells were exposed to Mn(2+), V(4+), V(5+), Cr(3+), Cr(6+), Zn(2+), Ni(2+), and Pb(2+) at a concentration of 0.5, 5, 50, or 500 μmol/L for 24 hours. Mn and V decreased the cell viability in a concentration-dependent manner, and V(5+) tended to have a greater effect than V(4+). The Cr decreased the cell viability, and (Cr(+6)) at concentrations of 50 and 500 μmol/L was more toxic than (Cr(+3)). Zn at a concentration of 500 μmol/L greatly decreased the cell viability, whereas Ni at the same concentration increased it. Pb produced fewer changes. Mn and Ni at a concentration of 500 μmol/L induced the significant production of IL-6 and IL-8. However, most of the metals including (V(+4), V(+5)), (Cr(+3), Cr(+6)), Zn, and Pb inhibited the production of both IL-6 and IL-8. The present results indicate that various heavy metals have different effects on toxicity and the proinflammatory responses of airway epithelial cells, and those influences also depend on the oxidation states of the metals. PMID:25808165

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

    PubMed Central

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

    2016-01-01

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

  4. Plate reader-based cell viability assays for glioprotection using primary rat optic nerve head astrocytes

    PubMed Central

    Kaja, Simon; Payne, Andrew J.; Naumchuk, Yuliya; Levy, Deborah; Zaidi, Danish H.; Altman, Alexa M.; Nawazish, Saba; Ghuman, Jasleen K.; Gerdes, Bryan C.; Moore, Mark A.; Koulen, Peter

    2015-01-01

    Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute critically to extracellular matrix synthesis during development and throughout life. In glaucoma, and in related disorders affecting the optic nerve and the optic nerve head, pathological changes include altered astrocyte gene and protein expression resulting in their activation and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA in order to slow glaucoma and related pathologies are of high clinical relevance. Herein, we describe the development of standardized methods that will allow for the systematic advancement of such glioprotective strategies. These include isolation, purification and culture of primary adult rat ONHAs, optimized immunocytochemical protocols for cell type validation, as well as plate reader-based assays determining cellular viability, proliferation and the intracellular redox state. We validated and standardized our protocols by performing a glioprotection study using primary ONHAs. Specifically, we measured protection against exogenously-applied oxidative stress using tert-butylhydroperoxide (tBHP) as a model of disease-mediated oxidative stress in the retina and optic nerve head by the prototypic antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Levels of oxidative stress were increased in the response to exogenously applied tBHP and were assessed by 6-carboxy-2′, 7′ dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Normalized DCFDA fluorescence showed a maximal 5.1-fold increase; the half-maximal effect (EC50) for tBHP was 212 ± 25

  5. 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

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

    PubMed Central

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

    2012-01-01

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

  7. Influence of substrate on corneal epithelial cell viability within ocular surface models.

    PubMed

    Feng, Yun; Foster, James; Mi, Shengli; Chen, Bo; Connon, Che John

    2012-08-01

    Corneal tissue engineering has improved dramatically over recent years. It is now possible to apply these technological advancements to the development of superior in vitro ocular surface models to reduce animal testing. We aim to show the effect different substrates can have on the viability of expanded corneal epithelial cells and that those which more accurately mimic the stromal surface provide the most protection against toxic assault. Compressed collagen gel as a substrate for the expansion of a human epithelial cell line was compared against two well-known substrates for modelling the ocular surface (polycarbonate membrane and conventional collagen gel). Cells were expanded over 10 days at which point cell stratification, cell number and expression of junctional proteins were assessed by electron microscopy, immunohistochemistry and RT-PCR. The effect of increasing concentrations of sodium lauryl sulphate on epithelial cell viability was quantified by MTT assay. Results showed improvement in terms of stratification, cell number and tight junction expression in human epithelial cells expanded upon either the polycarbonate membrane or compressed collagen gel when compared to a the use of a conventional collagen gel. However, cell viability was significantly higher in cells expanded upon the compressed collagen gel. We conclude that the more naturalistic composition and mechanical properties of compressed collagen gels produces a more robust corneal model. PMID:22683913

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

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

  9. Impact of nanopore morphology on cell viability on mesoporous polymer and carbon surfaces.

    PubMed

    Chavez, Vicki L; Song, Lingyan; Barua, Sutapa; Li, Xinxin; Wu, Quanyan; Zhao, Dongyuan; Rege, Kaushal; Vogt, Bryan D

    2010-08-01

    Topography at the nanoscale can lead to dramatic changes in the adhesion of cells to surfaces and their subsequent viability. For biological applications, including tissue engineering and cell-based sensing, the large internal surface area of ordered mesoporous carbons provides an opportunity for enhanced sensitivity and performance, but the mesostructure also affects the topography of the material. In this work, we probe the viability and adhesion of osteoblasts on ordered mesoporous materials with different morphologies and matrix chemistries. FDU-15 (hexagonal) and FDU-16 (cubic) films were processed at either 350 degrees C (polymeric) or 800 degrees C (carbon) to provide these different materials. For the films processed at 350 degrees C, the cell adhesion was markedly improved on the mesoporous films in comparison to a dense film analog, consistent with many reports in the literature that nanostructuring of surfaces improves the viability and adhesion of osteoblasts. Conversely, osteoblast adhesion was reduced on the carbonized surfaces processed at 800 degrees C when ordered mesopores were introduced, particularly for the cubic mesostructure (FDU-16). We attribute the decrease in cell adhesion to the propensity of the ordered mesoporous carbon films to sorb organics from aqueous solution, which could lead to removal of adhesion-promoting compounds at the film surface. These results suggest that cell viability on mesoporous polymer and carbon films can be controlled through simple changes in the pyrolysis temperature. PMID:20144750

  10. Loss of cooperativity of secreted CD40L and increased dose-response to IL4 on CLL cell viability correlates with enhanced activation of NF-kB and STAT6.

    PubMed

    Bhattacharya, Nupur; Reichenzeller, Michaela; Caudron-Herger, Maiwen; Haebe, Sarah; Brady, Nathan; Diener, Susanne; Nothing, Maria; Döhner, Hartmut; Stilgenbauer, Stephan; Rippe, Karsten; Mertens, Daniel

    2015-01-01

    Chronic lymphocytic leukemia (CLL) cells fail to enter apoptosis in vivo as opposed to their non-malignant B-lymphocyte counterparts. The ability of CLL cells to escape apoptosis is highly dependent on their microenvironment. Compared to non-malignant B cells, CLL cells are more responsive to complex stimuli that can be reproduced in vitro by the addition of cytokines. To understand the molecular mechanism of the environment-dependent anti-apoptotic signaling circuitry of CLL cells, we quantified the effect of the SDF-1, BAFF, APRIL, anti-IgM, interleukin-4 (IL4) and secreted CD40L (sCD40L) on the survival of in vitro cultured CLL cells and found IL4 and sCD40L to be most efficient in rescuing CLL cells from apoptosis. In quantitative dose-response experiments using cell survival as readout, the binding affinity of IL4 to its receptor was similar between malignant and non-malignant cells. However, the downstream signaling in terms of the amount of STAT6 and its degree of phosphorylation was highly stimulated in CLL cells. In contrast, the response to sCD40L showed a loss of cooperative binding in CLL cells but displayed a largely increased ligand binding affinity. Although a high-throughput microscopy analysis did not reveal a significant difference in the spatial CD40 receptor organization, the downstream signaling showed an enhanced activation of the NF-kB pathway in the malignant cells. Thus, we propose that the anti-apoptotic phenotype of CLL involves a sensitized response for IL4 dependent STAT6 phosphorylation, and an activation of NF-kB signaling due to an increased affinity of sCD40L to its receptor. PMID:24828787

  11. Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability.

    PubMed

    Schapire, Arnaldo L; Voigt, Boris; Jasik, Jan; Rosado, Abel; Lopez-Cobollo, Rosa; Menzel, Diedrik; Salinas, Julio; Mancuso, Stefano; Valpuesta, Victoriano; Baluska, Frantisek; Botella, Miguel A

    2008-12-01

    Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca(2+)-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca(2+)-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness. PMID:19088329

  12. 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

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

    PubMed

    Xia, Bingzhao; Krutkramelis, Kaspars; Oakey, John

    2016-07-11

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

  14. 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. PMID:23719889

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

    PubMed

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

    2007-03-30

    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 alpha (DND1-alpha) and DND1-isoform beta (DND1-beta). Using isoform-specific antibodies, we determined DND1-alpha is expressed in embryos and embryonic gonads whereas DND1-beta expression is restricted to germ cells of the adult testis. Our data implicate DND1-alpha 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. 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.

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

    PubMed

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

    2016-06-01

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

  18. 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.

  19. Effects of Cryopreservation on the Cell Viability, Proliferative Capacity and Neuronal Differentiation Potential of Canine Bone Marrow Stromal Cells

    PubMed Central

    EDAMURA, Kazuya; NAKANO, Rei; FUJIMOTO, Kyohei; TESHIMA, Kenji; ASANO, Kazushi; TANAKA, Shigeo

    2013-01-01

    ABSTRACT We investigated the cell viability, proliferative capacity and neuronal differentiation potential of canine bone marrow stromal cells (BMSCs) after cryopreservation. BMSCs were cryopreserved using cryoprotectant solutions with 10% DMSO and 10% FBS (DF group) or without DMSO and FBS (DF-free group); fresh BMSCs were used as a control. The cell viability and proliferative capacity of BMSCs were similar in the DF-free and control groups, while those in the DF group were lower. In all groups, BMSCs differentiated into neuron-like cells that stained positive against neuron markers, and the mRNA expression levels of neuron markers increased after neuronal induction. In conclusion, cryopreservation with DF-free cryoprotectant solution did not diminish the cell viability, proliferative capacity or neuronal differentiation potential of canine BMSCs. PMID:24334862

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2011-08-01

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

  3. Vitamin D3 regulates cell viability in gastric cancer and cholangiocarcinoma.

    PubMed

    Baek, Sungmin; Lee, Young-Suk; Shim, Hye-Eun; Yoon, Sik; Baek, Sun-Yong; Kim, Bong-Seon; Oh, Sae-Ock

    2011-09-01

    A low serum level of vitamin D has been associated with an increased incidence of gastrointestinal tract cancers. However, the effects of vitamin D3 have not been investigated in gastric cancer and cholangiocarcinoma. In the present study, we found that vitamin D3 treatment significantly suppressed the viability of gastric cancer and cholangiocarcinoma cells. Moreover, vitamin D3 had a synergistic effect with other anti-cancer drugs, such as paclitaxel, adriamycin, and vinblastine, for suppressing cell viability. To determine the underlying mechanism involved in the regulation of viability by vitamin D3, we examined the effects of vitamin D3 on expression of hedgehog signaling target genes, which has been associated with gastric cancer and cholangiocarcinoma. Vitamin D3 treatment decreased the level of mRNA expression of patched1, Gli1, cyclin D1, and Bcl2, suggesting the possibility that vitamin D3 may act through regulation of hedgehog signaling. From the above results, we conclude that vitamin D3 regulates cell viability in gastric cancer and cholangiocarcinoma. PMID:22025972

  4. 2,3,7,8-Tetrachlorodibenzo-p-dioxin alters steroid secretion but does not affect cell viability and the incidence of apoptosis in porcine luteinised granulosa cells.

    PubMed

    Jablonska, Olga; Piasecka-Srader, Joanna; Nynca, Anna; Kołomycka, Agnieszka; Robak, Anna; Wąsowska, Barbara; Ciereszko, Renata E

    2014-09-01

    The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a by-product of human industrial activity, was found to affect ovarian steroidogenesis in animals, but the mechanism of its action is still unclear. The aims of the study were to examine the effect of TCDD on (1) progesterone (P4) and oestradiol (E2) production by granulosa cells isolated from medium (3-6 mm) and preovulatory (≥ 8 mm) porcine follicles, (2) the viability of the cells, and (3) the incidence of apoptosis. Porcine granulosa cells were cultured (48 h) with or without TCDD (100 pM, 100 nM). Steroid hormone concentrations in the medium were determined by radioimmunoassay. The viability of granulosa cells was tested spectrophotometrically (alamarBlue™ assay). Apoptosis was evaluated by flow cytometry using Annexin V and by TUNEL assay. The higher dose of TCDD (100 nM) significantly inhibited P4 and stimulated E2 production by luteinised granulosa cells isolated from medium follicles. The lower dose of TCDD (100 pM) significantly stimulated P4 and inhibited E2 secretion by the cells isolated from preovulatory follicles. None of the two TCDD doses affected cell viability or induced apoptosis in granulosa cells. In conclusion, TCDD directly affected steroid production by granulosa cells obtained from mature pigs, but the effect of TCDD was not due to its cytotoxicity. PMID:25038954

  5. 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

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

    PubMed

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

    2016-03-15

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

  7. 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

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

    PubMed Central

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

    2016-01-01

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

  9. 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. PMID:25406855

  10. FOXL2, GATA4, and SMAD3 Co-Operatively Modulate Gene Expression, Cell Viability and Apoptosis in Ovarian Granulosa Cell Tumor Cells

    PubMed Central

    Anttonen, Mikko; L'Hôte, David; Vattulainen, Sanna; Färkkilä, Anniina; Unkila-Kallio, Leila; Veitia, Reiner A.; Heikinheimo, Markku

    2014-01-01

    Aberrant ovarian granulosa cell proliferation and apoptosis may lead to granulosa cell tumors (GCT), the pathogenesis of which involves transcription factors GATA4, FOXL2, and SMAD3. FOXL2 gene harbors a point mutation (C134W) in a vast majority of GCTs. GATA4 is abundantly expressed in GCTs and its expression correlates with poor prognosis. The TGF-β mediator SMAD3 promotes GCT cell survival through NF-κB activation, and interacts with FOXL2. Here, we find that the expression patterns of these factors overlap in the normal human ovary and 90 GCTs, and positively correlate with each other and with their mutual target gene CCND2, which is a key factor for granulosa cell proliferation. We have explored the molecular interactions of FOXL2, GATA4, and SMAD3 and their roles in the regulation of CCND2 using co-immunoprecipitation, promoter transactivation, and cell viability assays in human GCT cells. We found that not only SMAD3, but also GATA4 physically interact with both wild type and C134W-mutated FOXL2. GATA4 and SMAD3 synergistically induce a 8-fold increase in CCND2 promoter transactivation, which is 50% reduced by both FOXL2 types. We confirmed that wild type FOXL2 significantly decreases cell viability. Interestingly, GATA4 and SMAD3 caused a marked reduction of GCT cell apoptosis induced by wild type FOXL2. Thus, the effects of GATA4 and SMAD3 on both cell viability and apoptosis are distinct from those of wild type FOXL2; a perturbation of this balance due to the oncogenic FOXL2 mutation is likely to contribute to GCT pathogenesis. PMID:24416423

  11. FOXL2, GATA4, and SMAD3 co-operatively modulate gene expression, cell viability and apoptosis in ovarian granulosa cell tumor cells.

    PubMed

    Anttonen, Mikko; Pihlajoki, Marjut; Andersson, Noora; Georges, Adrien; L'hôte, David; Vattulainen, Sanna; Färkkilä, Anniina; Unkila-Kallio, Leila; Veitia, Reiner A; Heikinheimo, Markku

    2014-01-01

    Aberrant ovarian granulosa cell proliferation and apoptosis may lead to granulosa cell tumors (GCT), the pathogenesis of which involves transcription factors GATA4, FOXL2, and SMAD3. FOXL2 gene harbors a point mutation (C134W) in a vast majority of GCTs. GATA4 is abundantly expressed in GCTs and its expression correlates with poor prognosis. The TGF-β mediator SMAD3 promotes GCT cell survival through NF-κB activation, and interacts with FOXL2. Here, we find that the expression patterns of these factors overlap in the normal human ovary and 90 GCTs, and positively correlate with each other and with their mutual target gene CCND2, which is a key factor for granulosa cell proliferation. We have explored the molecular interactions of FOXL2, GATA4, and SMAD3 and their roles in the regulation of CCND2 using co-immunoprecipitation, promoter transactivation, and cell viability assays in human GCT cells. We found that not only SMAD3, but also GATA4 physically interact with both wild type and C134W-mutated FOXL2. GATA4 and SMAD3 synergistically induce a 8-fold increase in CCND2 promoter transactivation, which is 50% reduced by both FOXL2 types. We confirmed that wild type FOXL2 significantly decreases cell viability. Interestingly, GATA4 and SMAD3 caused a marked reduction of GCT cell apoptosis induced by wild type FOXL2. Thus, the effects of GATA4 and SMAD3 on both cell viability and apoptosis are distinct from those of wild type FOXL2; a perturbation of this balance due to the oncogenic FOXL2 mutation is likely to contribute to GCT pathogenesis. PMID:24416423

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

    PubMed Central

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

    2016-01-01

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

  14. Effects of simvastatin on cell viability and proinflammatory pathways in lung adenocarcinoma cells exposed to hydrogen peroxide.

    PubMed

    Gallelli, Luca; Falcone, Daniela; Scaramuzzino, Monica; Pelaia, Girolamo; D'Agostino, Bruno; Mesuraca, Maria; Terracciano, Rosa; Spaziano, Giuseppe; Maselli, Rosario; Navarra, Michele; Savino, Rocco

    2014-01-01

    Lung cancer is characterized by a high mortality rate probably attributable to early metastasis. Oxidative stress is involved in development and progression of lung cancer, through cellular and molecular mechanisms which at least in part overlap with proinflammatory pathways. Simvastatin is a statin with pleiotropic effects that can also act as an anti-oxidant agent, and these pharmacologic properties may contribute to its potential anti-cancer activity. Therefore, the aim of this study was to evaluate, in the human lung adenocarcinoma cell line GLC-82, the effects of a 24-hour treatment with simvastatin on hydrogen peroxide (H2O2)-induced changes in cell viability, ERK phosphorylation, matrix metalloproteinase (MMP) expression, innate immunity signaling, NF-κB activation and IL-8 secretion. Cell counting was performed after trypan blue staining, cell proliferation was assessed using MTT assay, and apoptosis was evaluated through caspase-3 activation and Tunel assay. Western blotting was used to analyze protein extracts, and IL-8 release into cell culture supernatants was assessed by ELISA. Our results show that simvastatin (30 μM) significantly (P <0.01) inhibited the proliferative effect of H2O2 (0.5 mM) and its stimulatory actions on ERK1/2 phosphorylation, NF-κB activation and IL-8 production. Furthermore, simvastatin decreased H2O2-mediated induction of the cellular expression of MMP-2 and MMP-9, as well as of several components of the signaling complex activated by innate immune responses, including MyD88, TRAF2, TRAF6 and TRADD. In conclusion, these findings suggest that simvastatin could play a role in prevention and treatment of lung cancer via modulation of important proinflammatory and tumorigenic events promoted by oxidative stress. PMID:25432084

  15. Monitoring of cell viability and proliferation in hydrogel-encapsulated system by resazurin assay.

    PubMed

    Xiao, Jing; Zhang, Ying; Wang, Jianzheng; Yu, Weiting; Wang, Wei; Ma, Xiaojun

    2010-11-01

    Cell microencapsulation is a promising approach for cell implantation, cell-based gene therapy and large-scale cell culture. For better quality control, it is important to accurately measure the microencapsulated cell viability and proliferation in the culture. A number of assays have been used for this purpose, but limitations arise. In this study, we investigated the feasibility and reliability of resazurin as a cell growth indicator in microencapsulated culture system. According to the experiment data, there was a reversible, time- and dose-dependent growth inhibition as observed for resazurin application in encapsulated cells. A positive relationship was observed between reduction of resazurin and CHO cell number in microcapsule. Moreover, the resazurin assay provided an equivalent result to the commonly used MTT method in determining CHO cell proliferation in APA microcapsule with no notable influence on cell distribution and organization pattern. In conclusion, resazurin assay is offered as a simple, rapid and non-invasive method for in vitro microencapsulated cell viability and proliferation measurement. PMID:20437208

  16. Olive leaf extract activity against Candida albicans and C. dubliniensis - the in vitro viability study.

    PubMed

    Zorić, Nataša; Kopjar, Nevenka; Kraljić, Klara; Oršolić, Nada; Tomić, Siniša; Kosalec, Ivan

    2016-09-01

    Olive leaf extract is characterized by a high content of polyphenols (oleuropein, hydroxytyrosol and their derivatives), which is associated with its therapeutic properties. The objective of the present research was to evaluate the antifungal activity of olive leaf extract against Candida albicans ATCC 10231 and C. dubliniensis CBS 7987 strains. Minimum inhibitory concentrations (MIC) of the extract were determined by several in vitro assays. The extract showed a concentration depended effect on the viability of C. albicans with MIC value of 46.875 mg mL-1 and C. dubliniensis with MIC value 62.5 mg mL-1. Most sensitive methods for testing the antifungal effect of the extracts were the trypan blue exclusion method and fluorescent dye exclusion method while MIC could not be determined by the method according to the EUCAST recommendation suggesting that herbal preparations contain compounds that may interfere with this susceptibility testing. The fluorescent dye exclusion method was also used for the assessment of morphological changes in the nuclei of treated cells. According to the obtained results, olive leaf extract is less effective against the tested strains than hydroxytyrosol, an olive plant constituent tested in our previous study. PMID:27383889

  17. Osteochondral Tissue Cell Viability Is Affected by Total Impulse during Impaction Grafting

    PubMed Central

    Balash, Paul; Kang, Richard W.; Schwenke, Thorsten; Cole, Brian J.; Wimmer, Markus A.

    2010-01-01

    Objective: Osteochondral graft transplantation has garnered significant attention because of its ability to replace the lesion with true hyaline cartilage. However, surgical impaction of the graft to anchor it into the defect site can be traumatic and lead to cell death and cartilage degeneration. This study aimed to test the hypothesis that increasing impulse magnitude during impaction of osteochondral plugs has a direct effect on loss of cell viability. Design: In this controlled laboratory study, the impaction force was kept constant while the impulse was varied. Ninety-six osteochondral plugs were extracted from the trochlea of bovine stifle joints and were randomly assigned into 3 experimental and 1 (nonimpacted) control group. The transferred impulse of the experimental groups reflected the median and the lower and upper quartiles of preceding clinical measurements. Data were obtained at day 0, day 4, and day 8; at each point, cell viability was assessed using the Live/Dead staining kit and histological assessments were performed to visualize matrix structural changes. Results: After impaction, cartilage samples stayed intact and did not show any histological signs of matrix disruption. As expected, higher impulse magnitudes introduced more cell death; however, this relationship was lost at day 8 after impaction. Conclusion: Impulse magnitude has a direct effect on cell viability of the graft. Because impulse magnitude is mostly governed by the press-fit characteristics of the recipient site, this study aids in the definition of optimal insertion conditions for osteochondral grafts. PMID:26069558

  18. Ribosome Modulation Factor, an Important Protein for Cell Viability Encoded by the Polyamine Modulon*

    PubMed Central

    Terui, Yusuke; Tabei, Yuzuru; Akiyama, Mariko; Higashi, Kyohei; Tomitori, Hideyuki; Yamamoto, Kaneyoshi; Ishihama, Akira; Igarashi, Kazuei; Kashiwagi, Keiko

    2010-01-01

    We searched for proteins whose synthesis is enhanced by polyamines at the stationary phase of cell growth using an Escherichia coli polyamine-requiring mutant in which cell viability is greatly decreased by polyamine deficiency. The synthesis of ribosome modulation factor (RMF) was strongly enhanced by polyamines at the level of translation at the stationary phase of cell growth. In rmf mRNA, a Shine-Dalgarno (SD) sequence is located 11 nucleotides upstream of the initiation codon AUG. When the SD sequence was moved to the more common position 8 nucleotides upstream of the initiation codon, the degree of polyamine stimulation was reduced, although the level of RMF synthesis was markedly increased. Polyamine stimulation of RMF synthesis was found to be caused by a selective structural change of the bulged-out region of the initiation site of rmf mRNA. The decrease in cell viability caused by polyamine deficiency was prevented by the addition of a modified rmf gene whose synthesis is not influenced by polyamines. The results indicate that polyamines enhance cell viability of E. coli at least in part by enhancing RMF synthesis. PMID:20628056

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

    PubMed Central

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

    2013-01-01

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

  20. Genome-wide analysis of longevity in nutrient-deprived Saccharomyces cerevisiae reveals importance of recycling in maintaining cell viability.

    PubMed

    Davey, Hazel M; Cross, Emma J M; Davey, Christopher L; Gkargkas, Konstantinos; Delneri, Daniela; Hoyle, David C; Oliver, Stephen G; Kell, Douglas B; Griffith, Gareth W

    2012-05-01

    Although typically cosseted in the laboratory with constant temperatures and plentiful nutrients, microbes are frequently exposed to much more stressful conditions in their natural environments where survival and competitive fitness depend upon both growth rate when conditions are favourable and on persistence in a viable and recoverable state when they are not. In order to determine the role of genetic heterogeneity in environmental fitness we present a novel approach that combines the power of fluorescence-activated cell sorting with barcode microarray analysis and apply this to determining the importance of every gene in the Saccharomyces cerevisiae genome in a high-throughput, genome-wide fitness screen. We have grown > 6000 heterozygous mutants together and exposed them to a starvation stress before using fluorescence-activated cell sorting to identify and isolate those individual cells that have not survived the stress applied. Barcode array analysis of the sorted and total populations reveals the importance of cellular recycling mechanisms (autophagy, pexophagy and ribosome breakdown) in maintaining cell viability during starvation and provides compelling evidence for an important role for fatty acid degradation in maintaining viability. In addition, we have developed a semi-batch fermentor system that is a more realistic model of environmental fitness than either batch or chemostat culture. Barcode array analysis revealed that arginine biosynthesis was important for fitness in semi-batch culture and modelling of this regime showed that rapid emergence from lag phase led to greatly increased fitness. One hundred and twenty-five strains with deletions in unclassified proteins were identified as being over-represented in the sorted fraction, while 27 unclassified proteins caused a haploinsufficient phenotype in semi-batch culture. These methods thus provide a screen to identifying other genes and pathways that have a role in maintaining cell viability. PMID

  1. 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

  2. Biotransformed Soybean Extract (BSE) Inhibits Melanoma Cell Growth and Viability In Vitro: Involvement of Nuclear Factor-Kappa B Signaling

    PubMed Central

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

    2014-01-01

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

  3. Effects of lead on viability and intracellular metal content of C6 rat glioma cells

    SciTech Connect

    Tiffany-Castiglioni, E.; Garcia, D.M.; Wu, J.N.; Zmudzki, J.; Bratton, G.R.

    1988-01-01

    Cultured C6 rat glioma cells were exposed to lead (Pb) acetate (0, 1, 10, or 100 ..mu..M) for 3-4 d. Cells were analyzed for changes in viability and intracellular lead, iron, and copper concentrations after Pb treatment was discontinued. The results were compared with previous findings on astroglia and oligodendroglia in culture in order to evaluate C6 cultures as a model for Pb toxicity in glia. Viability was measured by three methods on the day Pb was removed from the cells (designated d 0), and 2 and 9 d after Pb treatment was discontinued (designated d 2 and 9). The methods used were trypan blue dye exclusion, total cell counts, and incorporation of (/sup 3/H)-L-leucine into proteins. With respect to Pb and Fe uptake, C6 cells closely resembled immature astroglia in culture. Unlike C6 cells, however, astroglia showed elevations of intracellular Fe and Cu after treatment. Thus, Pb effects on C6 cells resembled those on cultured oligodendroglia and astroglia in some respects but not in others. C6 cells appear to be an adequate model for selected events in glial toxicosis, such as PB-stimulated protein synthesis in oligodendroglia and Pb uptake in astroglia, but not Pb-induced alterations of intracellular Cu and Fe in astroglia. Their use as a model for glial progenitor cells in Pb toxicity studies remains to be determined.

  4. 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

  5. Evaluation of cell viability and apoptosis in human amniotic fluid-derived stem cells with natural cryoprotectants.

    PubMed

    Cho, Hyun-Jung; Lee, Seung Hee; Yoo, James J; Shon, Yun-Hee

    2014-04-01

    A previous study demonstrated that disaccharides, antioxidants, and caspase inhibitors can be used in freezing solutions to reduce the concentration of Me2SO from the current standard of 10% (v/v) to 5% (v/v) or 2.5% and to eliminate fetal bovine serum (FBS) for the cryopreservation of human amniotic fluid-derived stem cells (AFSCs). Hence, this study investigated whether an irreversible inhibitor of caspase enzymes, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk), could be used in post-thaw culture media to increase the survival rate of AFSCs. Our results showed that AFSCs cryopreserved in freezing solution containing trehalose, catalase, and 5% (v/v) Me2SO and then supplemented with zVAD-fmk in the post-thaw culture media showed similar post-thawing viability, proliferation, and apoptosis than cells cryopreserved in the control solution (10% (v/v) Me2SO and 20% FBS). The caspase-3 activity in all the cryopreservation solutions tested was similar to that of the control. Caspase-3, caspase-8, caspase-9, and PARP expression was not found in the cryopreserved cells. In addition, no difference was found in the survival rate and apoptosis between short-term (3weeks) and long-term (1year) storage of AFSCs cryopreserved in the solutions used in this study. The results of the present study demonstrate that recovery of cryopreserved cells was enhanced by using a caspase inhibitor in the post-thaw culture media. PMID:24530510

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

    PubMed Central

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

    2016-01-01

    During cancer metastasis, circulating tumor cells constantly experience hemodynamic shear stress in the circulation. Cellular responses to shear stress including cell viability and proliferation thus play critical roles in cancer metastasis. Here, we developed a microfluidic approach to establish a circulatory microenvironment and studied circulating human colon cancer HCT116 cells in response to a variety of magnitude of shear stress and circulating time. Our results showed that cell viability decreased with the increase of circulating time, but increased with the magnitude of wall shear stress. Proliferation of cells survived from circulation could be maintained when physiologically relevant wall shear stresses were applied. High wall shear stress (60.5 dyne/cm2), however, led to decreased cell proliferation at long circulating time (1 h). We further showed that the expression levels of β-catenin and c-myc, proliferation regulators, were significantly enhanced by increasing wall shear stress. The presented study provides a new insight to the roles of circulatory shear stress in cellular responses of circulating tumor cells in a physiologically relevant model, and thus will be of interest for the study of cancer cell mechanosensing and cancer metastasis. PMID:27255403

  7. Flow cytometric lifetime-based cell viability assay using propidium iodide

    NASA Astrophysics Data System (ADS)

    Steinkamp, John A.; Lehnert, Bruce E.; Lehnert, Nancy M.

    1999-05-01

    Assays which discriminate and enumerate dying or dead cells are important in various types of cellular studies. In many instances, there is a need to identify dead cells that interfere with fluorescent probes which are used to measure functional and physiological properties in viable cells. For example, dead cells can introduce analytical errors arising from (1) nonspecific uptake of fluorescent probes, leading to erroneous percentages of positive labeled cells, (2) increased autofluorescence, and (3) altered antigen expression. The ability to detect dead cells is also of importance in determining the effectiveness of cytotoxic agents. Propidium iodide (PPI) exclusion, which is analogous to the non- fluorescent trypan blue dye test for viability, is used extensively in flow cytometry assays. However, the use of PI can potentially limit the application of additional fluorescent probes due to spectral overlap of the probe with PI. In this report we present phase-resolved fluorescence studies on rat and murine thymus cells labeled with phycoerythrin-antiThy 1.1 and phycoerythrin/Texas Red-antiThy 1.2 immunofluorescence markers, respectively, and PI. Overlapping emission spectra are resolved based on differences in fluorescence lifetimes of the probes and PI. These studies demonstrate a new lifetime-based viability method for use in analysis of immunofluorescent probes and for assaying the dynamics of cell killing.

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

    PubMed

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

    2016-01-01

    During cancer metastasis, circulating tumor cells constantly experience hemodynamic shear stress in the circulation. Cellular responses to shear stress including cell viability and proliferation thus play critical roles in cancer metastasis. Here, we developed a microfluidic approach to establish a circulatory microenvironment and studied circulating human colon cancer HCT116 cells in response to a variety of magnitude of shear stress and circulating time. Our results showed that cell viability decreased with the increase of circulating time, but increased with the magnitude of wall shear stress. Proliferation of cells survived from circulation could be maintained when physiologically relevant wall shear stresses were applied. High wall shear stress (60.5 dyne/cm(2)), however, led to decreased cell proliferation at long circulating time (1 h). We further showed that the expression levels of β-catenin and c-myc, proliferation regulators, were significantly enhanced by increasing wall shear stress. The presented study provides a new insight to the roles of circulatory shear stress in cellular responses of circulating tumor cells in a physiologically relevant model, and thus will be of interest for the study of cancer cell mechanosensing and cancer metastasis. PMID:27255403

  9. 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. PMID:23781504

  10. 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

  11. 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.

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

    PubMed

    Wang, Han; Gao, Weiwei

    2016-01-01

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

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

    PubMed Central

    Wang, Han; Gao, Weiwei

    2016-01-01

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

  14. Caffeine inhibits the viability and osteogenic differentiation of rat bone marrow-derived mesenchymal stromal cells

    PubMed Central

    Zhou, Y; Guan, XX; Zhu, ZL; Guo, J; Huang, YC; Hou, WW; Yu, HY

    2010-01-01

    BACKGROUND AND PURPOSE Caffeine is consumed extensively in Europe and North America. As a risk factor for osteoporosis, epidemiological studies have observed that caffeine can decrease bone mineral density, adversely affect calcium absorption and increase the risk of bone fracture. However, the exact mechanisms have not been fully investigated. Here, we examined the effects of caffeine on the viability and osteogenesis of rat bone marrow-derived mesenchymal stromal cells (rBMSCs). EXPERIMENTAL APPROACH Cell viability, apoptosis and necrosis were quantified using thymidine incorporation and flow cytometry. Sequential gene expressions in osteogenic process were measured by real-time PCR. cAMP, alkaline phosphatase and osteocalcin were assessed by immunoassay, spectrophotometry and radioimmunoassay, respectively. Mineralization was determined by calcium deposition. KEY RESULTS After treating BMSCs with high caffeine concentrations (0.1–1 mM), their viability decreased in a concentration-dependent manner. This cell death was primarily due to necrosis and, to a small extent, apoptosis. Genes and protein sequentially expressed in osteogenesis, including Cbfa1/Runx2, collagen I, alkaline phosphatase and its protein, were significantly downregulated except for osteocalcin and its protein. Moreover, caffeine inhibited calcium deposition in a concentration- and time-dependent manner, but increased intracellular cAMP in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS By suppressing the commitment of BMSCs to the osteogenic lineage and selectively inhibiting gene expression, caffeine downregulated some important events in osteogenesis and ultimately affected bone mass. PMID:20726981

  15. Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

    USGS Publications Warehouse

    Hatzinger, P.B.; Palmer, P.; Smith, R.L.; Penarrieta, C.T.; Yoshinari, T.

    2003-01-01

    A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC). In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT). To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3???-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated. Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays. For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude. A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation. Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation. However, one bacterium (Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation. This strain would be considered nonviable based on traditional tetrazolium salt reduction assays. The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may

  16. Intracellular reactive oxygen species are essential for PI3K/Akt/mTOR-dependent IL-7-mediated viability of T-cell acute lymphoblastic leukemia cells.

    PubMed

    Silva, A; Gírio, A; Cebola, I; Santos, C I; Antunes, F; Barata, J T

    2011-06-01

    Interleukin-7 (IL-7) activates phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, thereby mediating viability, proliferation and growth of T-cell acute lymphoblastic leukemia (T-ALL) cells. Reactive oxygen species (ROS) can be upregulated by growth factors and are known to regulate proliferation and viability. Here, we show that IL-7 upregulates ROS in T-ALL cells in a manner that is dependent on PI3K/Akt/mTOR pathway activity and that relies on both NADPH oxidase and mitochondrial respiratory chain. Conversely, IL-7-induced activation of PI3K signaling pathway requires mitochondrial respiration and ROS. We have previously shown that IL-7-mediated activation of PI3K pathway drives the upregulation of the glucose transporter Glut1, promoting glucose uptake in T-ALL cells. Using phloretin to inhibit Glut function, we demonstrate that glucose uptake is mandatory for ROS upregulation in IL-7-treated T-ALL cells, suggesting that IL-7 stimulation leads to increased ROS via PI3K pathway activation and consequent upregulation of Glut1 and glucose uptake. Overall, our data reveal the existence of a critical crosstalk between PI3K/Akt signaling pathway and ROS that is essential for IL-7-mediated T-ALL cell survival, and that may constitute a novel target for therapeutic intervention. PMID:21455214

  17. Multiplexing high-content flow (HCF) and quantitative high-throughput screening (qHTS) to identify compounds capable of decreasing cell viability, activating caspase 3/7, expressing annexin V, and changing mitochondrial membrane integrity.

    PubMed

    Mathews, Lesley A; Keller, Jonathan M; McKnight, Crystal; Michael, Sam; Shinn, Paul; Liu, Dongbo; Staudt, Louis M; Thomas, Craig J; Ferrer, Marc

    2013-01-01

    High-content flow (HCF) screening systems, such as the iQue Screener and HTFC Screening System from IntelliCyt, have facilitated the implementation of flow cytometry assays for high-throughput screening. HCF screening systems enable the use of smaller sample volumes and multiplexed assays to simultaneously assess different cellular parameters from a single well. This becomes invaluable when working with cells or compounds that are available in limited quantities or when conducting large-scale screens. When assays can be miniaturized to a 384- or 1536-well microplate format, it is possible to implement dose-response-based high-throughput screens, also known as quantitative HTS or qHTS. This article describes how qHTS at the new National Center for Advancing Translational Science (NCATS) has been systematically coupled with the HTFC Screening System and Multimetric Apoptosis Screening Kit from IntelliCyt to biologically validate active compounds from primary cell proliferation screens using a model of diffuse large B cell lymphoma (DLBCL). PMID:24391083

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    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. PMID:24995995

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

    PubMed

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

    2015-03-01

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

  1. 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

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

    PubMed Central

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

    2016-01-01

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

  3. Effect of vertebroplasty filler materials on viability and gene expression of human nucleus pulposus cells.

    PubMed

    Lazáry, Aron; Speer, Gábor; Varga, Péter Pál; Balla, Bernadett; Bácsi, Krisztián; Kósa, János P; Nagy, Zsolt; Takács, István; Lakatos, Péter

    2008-05-01

    Consequences of intradiscal cement leakage--often occurring after vertebral cement augmentation for the treatment of vertebral compression fractures--are still unknown. In this study, we have investigated the influences of vertebroplasty filler materials (polymethylmethacrylate-, calcium phosphate- and calcium sulfate-based bone cement) on isolated nucleus pulposus cells. Cell viability of cultured human nucleus pulposus cells were measured after treatment with vertebroplasty filler materials. Gene expression profile of selected genes was determined with quantitative real-time PCR. The widely used polymethylmethacrylate and calcium phosphate cement significantly decreased cell number in a dose- and time-dependent manner while calcium sulfate cement affected cell viability less. Expression of genes involved in matrix metabolism of nucleus pulposus--aggrecan, collagens, small proteoglycans--as well as important transcription factors have also significantly changed due to treatment (e.g., 2.5-fold decrease in aggrecan expression was determined in cultures due to polymethylmethacrylate treatment). Our results suggest that vertebroplasty filler materials--depending on the type of applied material--can accelerate the degeneration of nucleus pulposus cells resulting in a less flexible disc in case of intradiscal cement leakage. This process may increase the risk of a subsequent new vertebral fracture, the main complication of vertebral augmentation. PMID:18176942

  4. 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. PMID:22462453

  5. Drosophila Uri, a PP1α binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity

    PubMed Central

    Kirchner, Jasmin; Vissi, Emese; Gross, Sascha; Szoor, Balazs; Rudenko, Andrey; Alphey, Luke; White-Cooper, Helen

    2008-01-01

    Background Protein phosphatase 1 (PP1) is involved in diverse cellular processes, and is targeted to substrates via interaction with many different protein binding partners. PP1 catalytic subunits (PP1c) fall into PP1α and PP1β subfamilies based on sequence analysis, however very few PP1c binding proteins have been demonstrated to discriminate between PP1α and PP1β. Results URI (unconventional prefoldin RPB5 interactor) is a conserved molecular chaperone implicated in a variety of cellular processes, including the transcriptional response to nutrient signalling and maintenance of DNA integrity. We show that Drosophila Uri binds PP1α with much higher affinity than PP1β, and that this ability to discriminate between PP1c forms is conserved to humans. Most Uri is cytoplasmic, however we found some protein associated with active RNAPII on chromatin. We generated a uri loss of function allele, and show that uri is essential for viability in Drosophila. uri mutants have transcriptional defects, reduced cell viability and differentiation in the germline, and accumulate DNA damage in their nuclei. Conclusion Uri is the first PP1α specific binding protein to be described in Drosophila. Uri protein plays a role in transcriptional regulation. Activity of uri is required to maintain DNA integrity and cell survival in normal development. PMID:18412953

  6. 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.

  7. Hepatocyte-like cells derived from human amniotic epithelial cells can be encapsulated without loss of viability or function in vitro.

    PubMed

    Vaghjiani, Vijesh; Vaithilingam, Vijayaganapathy; Saraswati, Indah; Sali, Adnan; Murthi, Padma; Kalionis, Bill; Tuch, Bernard E; Manuelpillai, Ursula

    2014-04-15

    Placenta derived human amniotic epithelial cells (hAEC) are an attractive source of stem cells for the generation of hepatocyte-like cells (HLC) for therapeutic applications to treat liver diseases. During hAEC differentiation into HLC, they become increasingly immunogenic, which may result in immune cell-mediated rejection upon transplantation into allogeneic recipients. Placing cells within devices such as alginate microcapsules can prevent immune cell-mediated rejection. The aim of this study was to investigate the characteristics of HLC generated from hAEC and to examine the effects of encapsulation on HLC viability, gene expression, and function. hAEC were differentiated for 4 weeks and evaluated for hepatocyte-specific gene expression and function. Differentiated cells were encapsulated in barium alginate microcapsules and cultured for 7 days and the effect of encapsulation on cell viability, function, and hepatocyte related gene expression was determined. Differentiated cells performed key functions of hepatocytes including urea synthesis, drug-metabolizing cytochrome P450 (CYP)3A4 activity, indocyanine green (ICG) uptake, low-density lipoprotein (LDL) uptake, and exhibited glutathione antioxidant capacity. A number of hepatocyte-related genes involved in fat, cholesterol, bile acid synthesis, and xenobiotic metabolism were also expressed showing that the hAEC had differentiated into HLC. Upon encapsulation, the HLC remained viable for at least 7 days in culture, continued to express genes involved in fat, cholesterol, bile acid, and xenobiotic metabolism and had glutathione antioxidant capacity. CYP3A4 activity and urea synthesis by the encapsulated HLC were higher than that of monolayer HLC cultures. Functional HLC can be derived from hAEC, and HLC can be encapsulated within alginate microcapsules without losing viability or function in vitro. PMID:24295364

  8. Hepatocyte-Like Cells Derived from Human Amniotic Epithelial Cells Can Be Encapsulated Without Loss of Viability or Function In Vitro

    PubMed Central

    Vaghjiani, Vijesh; Vaithilingam, Vijayaganapathy; Saraswati, Indah; Sali, Adnan; Murthi, Padma; Kalionis, Bill; Tuch, Bernard E.

    2014-01-01

    Placenta derived human amniotic epithelial cells (hAEC) are an attractive source of stem cells for the generation of hepatocyte-like cells (HLC) for therapeutic applications to treat liver diseases. During hAEC differentiation into HLC, they become increasingly immunogenic, which may result in immune cell-mediated rejection upon transplantation into allogeneic recipients. Placing cells within devices such as alginate microcapsules can prevent immune cell-mediated rejection. The aim of this study was to investigate the characteristics of HLC generated from hAEC and to examine the effects of encapsulation on HLC viability, gene expression, and function. hAEC were differentiated for 4 weeks and evaluated for hepatocyte-specific gene expression and function. Differentiated cells were encapsulated in barium alginate microcapsules and cultured for 7 days and the effect of encapsulation on cell viability, function, and hepatocyte related gene expression was determined. Differentiated cells performed key functions of hepatocytes including urea synthesis, drug-metabolizing cytochrome P450 (CYP)3A4 activity, indocyanine green (ICG) uptake, low-density lipoprotein (LDL) uptake, and exhibited glutathione antioxidant capacity. A number of hepatocyte-related genes involved in fat, cholesterol, bile acid synthesis, and xenobiotic metabolism were also expressed showing that the hAEC had differentiated into HLC. Upon encapsulation, the HLC remained viable for at least 7 days in culture, continued to express genes involved in fat, cholesterol, bile acid, and xenobiotic metabolism and had glutathione antioxidant capacity. CYP3A4 activity and urea synthesis by the encapsulated HLC were higher than that of monolayer HLC cultures. Functional HLC can be derived from hAEC, and HLC can be encapsulated within alginate microcapsules without losing viability or function in vitro. PMID:24295364

  9. Fluorescence-based visualization of autophagic activity predicts mouse embryo viability

    NASA Astrophysics Data System (ADS)

    Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Kito, Seiji; Minami, Naojiro; Kubota, Toshiro; Sato, Ken; Kokubo, Toshiaki

    2014-03-01

    Embryo quality is a critical parameter in assisted reproductive technologies. Although embryo quality can be evaluated morphologically, embryo morphology does not correlate perfectly with embryo viability. To improve this, it is important to understand which molecular mechanisms are involved in embryo quality control. Autophagy is an evolutionarily conserved catabolic process in which cytoplasmic materials sequestered by autophagosomes are degraded in lysosomes. We previously demonstrated that autophagy is highly activated after fertilization and is essential for further embryonic development. Here, we developed a simple fluorescence-based method for visualizing autophagic activity in live mouse embryos. Our method is based on imaging of the fluorescence intensity of GFP-LC3, a versatile marker for autophagy, which is microinjected into the embryos. Using this method, we show that embryonic autophagic activity declines with advancing maternal age, probably due to a decline in the activity of lysosomal hydrolases. We also demonstrate that embryonic autophagic activity is associated with the developmental viability of the embryo. Our results suggest that embryonic autophagic activity can be utilized as a novel indicator of embryo quality.

  10. Metformin and trametinib have synergistic effects on cell viability and tumor growth in NRAS mutant cancer

    PubMed Central

    Vujic, Igor; Sanlorenzo, Martina; Posch, Christian; Esteve-Puig, Rosaura; Yen, Adam J.; Kwong, Andrew; Tsumura, Aaron; Murphy, Ryan; Rappersberger, Klemens; Ortiz-Urda, Susana

    2015-01-01

    Attempts to directly block the mutant neuroblastoma rat sarcoma oncogene (NRAS) protein, a driving mutation in many cancer types, have been unsuccessful. Current treatments focus on inhibition of different components of NRAS' two main downstream cascades: PI3K/AKT/mTOR and MAPK. Here we test a novel dual therapy combination of metformin and trametinib on a panel of 16 NRAS mutant cell lines, including melanoma cells, melanoma cells with acquired trametinib resistance, lung cancer and neuroblastoma cells. We show that both of the main downstream cascades of NRAS can be blocked by this combination: metformin indirectly inhibits the PI3K/AKT/mTOR pathway and trametinib directly impedes the MAPK pathway. This dual therapy synergistically reduced cell viability in vitro and xenograft tumor growth in vivo. We conclude that metformin and trametinib combinations are effective in preclinical models and may be a possible option for treatment of NRAS mutant cancers. PMID:25504439

  11. Human mesenchymal stem cells seeded on extracellular matrix-scaffold: viability and osteogenic potential.

    PubMed

    Penolazzi, Letizia; Mazzitelli, Stefania; Vecchiatini, Renata; Torreggiani, Elena; Lambertini, Elisabetta; Johnson, Scott; Badylak, Stephen F; Piva, Roberta; Nastruzzi, Claudio

    2012-02-01

    The development and the optimization of novel culture systems of mesenchymal osteoprogenitors are some of the most important challenges in the field of bone tissue engineering (TE). A new combination between cells and extracellular matrix (ECM)-scaffold, containing ECM has here been analyzed. As source for osteoprogenitors, mesenchymal stem cells obtained from human umbilical cord Wharton's Jelly (hWJMSCs), were used. As ECM-scaffold, a powder form of isolated and purified porcine urinary bladder matrix (pUBM), was employed. The goals of the current work were: (1) the characterization of the in vitro hWJMSCs behavior, in terms of viability, proliferation, and adhesion to ECM-scaffold; (2) the effectiveness of ECM-scaffold to induce/modulate the osteoblastic differentiation; and (3) the proposal for a possible application of cells/ECM-scaffold construct to the field of cell/TE. In this respect, the properties of the pUBM-scaffold in promoting and guiding the in vitro adhesion, proliferation, and three-dimensional colonization of hWJMSCs, without altering viability and morphological characteristics of the cells, are here described. Finally, we have also demonstrated that pUBM-scaffolds positively affect the expression of typical osteoblastic markers in hWJMSCs. PMID:21830215

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-06-01

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

  14. 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

  15. Lead Toxicity to the Performance, Viability, And Community Composition of Activated Sludge Microorganisms

    SciTech Connect

    Yuan, L; Zhi, W; Liu, YS; Karyala, S; Vikesland, PJ; Chen, X; Zhang, HS

    2015-01-20

    Lead (Pb) is a prominent toxic metal in natural and engineered systems. Current knowledge on Pb toxicity to the activated sludge has been limited to short-term (<= 24 h) toxicity. The effect of extended Pb exposure on process performance, bacterial viability, and community compositions remains unknown. We quantified the 24-h and 7-day Pb toxicity to chemical oxygen demand (COD) and NH3-N removal, bacterial viability, and community compositions using lab-scale experiments. Our results showed that 7-day toxicity was significantly higher than the short-term 24-h toxicity. Ammonia-oxidizing bacteria were more susceptible than the heterotrophs to Pb toxicity. The specific oxygen uptake rate responded quickly to Pb addition and could serve as a rapid indicator for detecting Pb pollutions. Microbial viability decreased linearly with the amount of added Pb at extended exposure. The bacterial community diversity was markedly reduced with elevated Pb concentrations. Surface analysis suggested that the adsorbed form of Pb could have contributed to its toxicity along with the dissolved form. Our study provides for the first time a systematic investigation of the effect of extended exposure of Pb on the performance and microbiology of aerobic treatment processes, and it indicates that long-term Pb toxicity has been underappreciated by previous studies.

  16. Lead toxicity to the performance, viability, and community composition of activated sludge microorganisms.

    PubMed

    Yuan, Li; Zhi, Wei; Liu, Yangsheng; Karyala, Saikumar; Vikesland, Peter J; Chen, Xi; Zhang, Husen

    2015-01-20

    Lead (Pb) is a prominent toxic metal in natural and engineered systems. Current knowledge on Pb toxicity to the activated sludge has been limited to short-term (≤24 h) toxicity. The effect of extended Pb exposure on process performance, bacterial viability, and community compositions remains unknown. We quantified the 24-h and 7-day Pb toxicity to chemical oxygen demand (COD) and NH3–N removal, bacterial viability, and community compositions using lab-scale experiments. Our results showed that 7-day toxicity was significantly higher than the short-term 24-h toxicity. Ammonia-oxidizing bacteria were more susceptible than the heterotrophs to Pb toxicity. The specific oxygen uptake rate responded quickly to Pb addition and could serve as a rapid indicator for detecting Pb pollutions. Microbial viability decreased linearly with the amount of added Pb at extended exposure. The bacterial community diversity was markedly reduced with elevated Pb concentrations. Surface analysis suggested that the adsorbed form of Pb could have contributed to its toxicity along with the dissolved form. Our study provides for the first time a systematic investigation of the effect of extended exposure of Pb on the performance and microbiology of aerobic treatment processes, and it indicates that long-term Pb toxicity has been underappreciated by previous studies. PMID:25536278

  17. Fibronectin-Alginate microcapsules improve cell viability and protein secretion of encapsulated Factor IX-engineered human mesenchymal stromal cells.

    PubMed

    Sayyar, Bahareh; Dodd, Megan; Marquez-Curtis, Leah; Janowska-Wieczorek, Anna; Hortelano, Gonzalo

    2015-01-01

    Continuous delivery of proteins by engineered cells encapsu-lated in biocompatible polymeric microcapsules is of considerable therapeutic potential. However, this technology has not lived up to expectations due to inadequate cell--matrix interactions and subsequent cell death. In this study we hypoth-esize that the presence of fibronectin in an alginate matrix may enhance the viability and functionality of encapsulated human cord blood-derived mesenchymal stromal cells (MSCs) expressing the human Factor IX (FIX) gene. MSCs were encapsulated in alginate-PLL microcapsules containing 10, 100, or 500 μg/ml fibronectin to ameliorate cell survival. MSCs in microcapsules with 100 and 500 μg/ml fibronectin demonstrated improved cell viability and proliferation and higher FIX secretion compared to MSCs in non-supplemented microcapsules. In contrast, 10 μg/ml fibronectin did not significantly affect the viability and protein secretion from the encapsulated cells. Differentiation studies demonstrated osteogenic (but not chondrogenic or adipogenic) differentiation capability and efficient FIX secretion of the enclosed MSCs in the fibronectin-alginate suspension culture. Thus, the use of recombinant MSCs encapsulated in fibronectin-alginate microcapsules in basal or osteogenic cultures may be of practical use in the treatment of hemophilia B. PMID:24564349

  18. 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

  19. 14-3-3γ regulates cell viability and milk fat synthesis in lipopolysaccharide-induced dairy cow mammary epithelial cells

    PubMed Central

    LIU, LIXIN; ZHANG, LI; LIN, YE; BIAN, YANJIE; GAO, XUEJUN; QU, BO; LI, QINGZHANG

    2016-01-01

    Our previous study demonstrated that 14-3-3γ overexpression was able to inhibit the production of lipopolysaccharide (LPS)-induced cytokines in dairy cow mammary epithelial cells (DCMECs) by inhibiting the activation of nuclear factor-κB (NF-κB) signaling pathways. However, the association between 14-3-3γ overexpression and milk fat synthesis in LPS-induced DCMECs remains unclear. Therefore, the present study investigated the effect of 14-3-3γ on cell viability and milk fat synthesis in LPS-induced DCMECs. The results of the MTT assay and lactate dehydrogenase activity assay demonstrated that 14-3-3γ overexpression was able to attenuate LPS-induced cytotoxicity in DCMECs, and increase the viability of the cells. In addition, the results of reverse transcription-quantitative polymerase chain reaction suggested that mRNA expression levels of genes associated with milk fat synthesis, including sterol regulatory element binding protein (SREBP1), peroxisome proliferator-activated receptor-γ (PPARG), cluster of differentiation 36, acetyl-coA carboxylase (ACC), fatty acid synthase (FAS) and fatty acid binding protein-3, were significantly upregulated in cells overexpressing the 14-3-3γ protein. In addition, as compared with the LPS-treated group, the activities of FAS and ACC were significantly increased. Furthermore, western blotting demonstrated that 14-3-3γ overexpression enhanced the protein expression levels of phosphorylated SREBP1 and PPARG. These results suggested that high levels of 14-3-3γ protein were able to attenuate LPS-induced cell damage and promote milk fat synthesis in LPS-induced DCMECs by increasing the cell viability and upregulating the expression levels of transcription factors associated with milk fat synthesis. PMID:27073437

  20. 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

  1. The glial cell modulator ibudilast attenuates neuroinflammation and enhances retinal ganglion cell viability in glaucoma through protein kinase A signaling.

    PubMed

    Cueva Vargas, Jorge L; Belforte, Nicolas; Di Polo, Adriana

    2016-09-01

    Glaucoma is a neurodegenerative disease and the leading cause of irreversible blindness worldwide. Vision deficits in glaucoma result from the selective loss of retinal ganglion cells (RGC). Glial cell-mediated neuroinflammation has been proposed to contribute to disease pathophysiology, but whether this response is harmful or beneficial for RGC survival is not well understood. To test this, we characterized the role of ibudilast, a clinically approved cAMP phosphodiesterase (PDE) inhibitor with preferential affinity for PDE type 4 (PDE4). Here, we demonstrate that intraocular administration of ibudilast dampened macroglia and microglia reactivity in the retina and optic nerve hence decreasing production of proinflammatory cytokines in a rat model of ocular hypertension. Importantly, ibudilast promoted robust RGC soma survival, prevented axonal degeneration, and improved anterograde axonal transport in glaucomatous eyes without altering intraocular pressure. Intriguingly, ocular hypertension triggered upregulation of PDE4 subtype A in Müller glia, and ibudilast stimulated cAMP accumulation in these cells. Co-administration of ibudilast with Rp-cAMPS, a cell-permeable and non-hydrolysable cAMP analog that inhibits protein kinase A (PKA), completely blocked ibudilast-induced neuroprotection. Collectively, these data demonstrate that ibudilast, a safe and well-tolerated glial cell modulator, attenuates gliosis, decreases levels of proinflammatory mediators, and enhances neuronal viability in glaucoma through activation of the cAMP/PKA pathway. This study provides insight into PDE4 signaling as a potential target to counter the harmful effects associated with chronic gliosis and neuroinflammation in glaucoma. PMID:27163643

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

    PubMed

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

    2016-06-01

    Oxidative stress is a risk factor in the pathogenesis of osteoporosis, and plays a major role in bone regeneration of osteoporotic patients. Cerium oxide (CeO2) ceramics have the unique ability to protect various types of cells from oxidative damage, making them attractive for biomedical applications. In this study, we developed a plasma sprayed CeO2 coating with a hierarchical topography where ceria nanoparticles were superimposed in the micro-rough coating surface. The protective effects of the CeO2 coating on the response of osteoblasts to H2O2-induced oxidative stress have been demonstrated in terms of cell viability, apoptosis and differentiation. The CeO2 coating reversed the reduced superoxide dismutase activity, decreased reactive oxygen species production and suppressed malondialdehyde formation in H2O2-treated osteoblasts. It indicated that the CeO2 coating can preserve the intracellular antioxidant defense system. The cytocompatibility of the CeO2 coating was further assessed in vitro by cell viability assay and scanning electron microscopy analysis. Taken together, the CeO2 coating could provide an opportunity to be utilized as a potential candidate for bone regeneration under oxidative stress. PMID:27091042

  3. Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans

    PubMed Central

    Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K.

    2010-01-01

    Background A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO®13, SYTO®24 and SYBR®14 as possible alternatives to FDA. Results We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO®13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. Conclusions From a review of the literature and from our observations on the

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

    PubMed Central

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

    2015-01-01

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

  5. 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)

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

    PubMed

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

    2005-11-28

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

  7. 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

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

    PubMed

    Lestard, Nathalia R; Capella, Marcia A M

    2016-01-01

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

  9. 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

  10. 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. PMID:26252416

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

    PubMed Central

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

    2015-01-01

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

  12. 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

  13. 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. PMID:26221112

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

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

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

  15. 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

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

    PubMed Central

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

    2015-01-01

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

  17. Adaptive response to starvation in the fish pathogen Flavobacterium columnare: cell viability and ultrastructural changes

    PubMed Central

    2012-01-01

    Background The ecology of columnaris disease, caused by Flavobacterium columnare, is poorly understood despite the economic losses that this disease inflicts on aquaculture farms worldwide. Currently, the natural reservoir for this pathogen is unknown but limited data have shown its ability to survive in water for extended periods of time. The objective of this study was to describe the ultrastructural changes that F. columnare cells undergo under starvation conditions. Four genetically distinct strains of this pathogen were monitored for 14 days in media without nutrients. Culturability and cell viability was assessed throughout the study. In addition, cell morphology and ultrastructure was analyzed using light microscopy, scanning electron microscopy, and transmission electron microscopy. Revival of starved cells under different nutrient conditions and the virulence potential of the starved cells were also investigated. Results Starvation induced unique and consistent morphological changes in all strains studied. Cells maintained their length and did not transition into a shortened, coccus shape as observed in many other Gram negative bacteria. Flavobacterium columnare cells modified their shape by morphing into coiled forms that comprised more than 80% of all the cells after 2 weeks of starvation. Coiled cells remained culturable as determined by using a dilution to extinction strategy. Statistically significant differences in cell viability were found between strains although all were able to survive in absence of nutrients for at least 14 days. In later stages of starvation, an extracellular matrix was observed covering the coiled cells. A difference in growth curves between fresh and starved cultures was evident when cultures were 3-months old but not when cultures were starved for only 1 month. Revival of starved cultures under different nutrients revealed that cells return back to their original elongated rod shape upon encountering nutrients. Challenge

  18. Heat shock protein 70 overexpression affects the response to ultraviolet light in murine fibroblasts. Evidence for increased cell viability and suppression of cytokine release.

    PubMed Central

    Simon, M M; Reikerstorfer, A; Schwarz, A; Krone, C; Luger, T A; Jäättelä, M; Schwarz, T

    1995-01-01

    To elucidate cellular concepts for protection against ultraviolet (UV) light we investigated the effect of heat shock protein 70 (hsp70) overexpression on cell viability and on the secretion of UV-inducible immunological cytokines. Transfected murine fibrosarcoma cells (WEHI-S), overexpressing hsp70 or a sham transfected control were used. Overexpression of hsp70 was sufficient to markedly increase cell viability upon treatment with UVB (290-320 nm). Since long wave UV (UVA, 320-400 nm) as well as UVB turned out to stimulate the release of O2- radicals we studied the cell viability upon oxidative stress. Hsp70 overexpression increased viability upon treatment with hydrogen peroxide or menadione, but had no influence on UV-induced O2- release. UV-light is known to upregulate immunologic and proinflammatory cytokines such as IL-1 and IL-6. Oxidative stress appeared to exert a similar effect. Hsp70 overexpression markedly decreased the release of IL-6 induced by UVA, UVB and oxidative stress. To test whether the hsp70 mediated suppression is confined to events caused by UV-light we determined IL-1-mediated effects. IL-1-induced IL-6 release was reduced by hsp70 overexpression, whereas the IL-1 mediated activation of nuclear factor kappa B was not affected. Our data suggests that hsp70 plays a central role not only in cell protection against UV-light, but also in the regulation of proinflammatory cytokine release induced by UV-exposure. Images PMID:7883992

  19. Suppression of viability and acetyl-LDL metabolism in RAW 264 macrophage-like and smooth muscle cells by bisphosphonates in vitro.

    PubMed

    Tuominen, O M; Hollmén, M; Jaakkola, O; Mönkkönen, J; Ylitalo, R

    2002-10-01

    Etidronate and clodronate are bisphosphonates that inhibit the development of experimental atherosclerosis. Etidronate decreases the intimamedia thickness of carotid artery even in man. Liposome-encapsulated bisphosphonates inhibit the cellular metabolism of atherogenic, modified low-density lipoprotein (acetyl-LDL) by cultured macrophages. In the present study, the effects of new bisphosphonate tiludronate and nitrogen-containing bisphosphonate alendronate on cell viability and cellular uptake and degradation of acetyl-LDL were investigated in vitro with macrophages and arterial smooth muscle cells, which have a significant role in atherogenesis. Tiludronate and alendronate decreased the viability of RAW 264 macrophages at high concentration (1,000 microM; p < 0.05), while liposome-encapsulated drugs suppressed the viability at concentrations of 30-300 microM. At concentrations greater than or equal to 10 microM, tiludronate and alendronate inhibited the uptake and degradation of acetyl-LDL by RAW 264 cells in a concentration-dependent manner (p < 0.001). None of the bisphosphonates affected the viability of smooth muscle cells, and none but alendronate at a high concentration (1,000 microM) inhibited the uptake and degradation of acetyl-LDL by smooth muscle cells. The results show that tiludronate and alendronate inhibit the atherogenic activity of macrophages in vitro, as shown previously with etidronate and clodronate, providing further evidence for the antiatherogenic effects of bisphosphonates. PMID:12500427

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

    PubMed

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

    1987-03-01

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

  1. Influence of nanomechanical stress induced by ZnO nanoparticles of different shapes on the viability of cells.

    PubMed

    Matuła, Kinga; Richter, Łukasz; Adamkiewicz, Witold; Åkerström, Bo; Paczesny, Jan; Hołyst, Robert

    2016-05-14

    There is growing interest in nanostructures interacting with living organisms. However, there are still no general rules for the design of biocompatible nanodevices. Here, we present a step towards understanding the interactions between nanostructures and living cells. We study the influence of nanomechanical stress induced by zinc oxide (ZnO) nanostructures of different shapes on the viability of both prokaryotic (Gram-negative bacteria: Escherichia coli and Enterobacter aerogenes, and Gram-positive bacteria: Staphylococcus epidermidis and Corynebacterium glutamicum) and eukaryotic cells (yeast Saccharomyces cerevisiae and liver cancer cell line HepG2). Nanoparticles (NPs) and nanorods (NRs) of matching crystallographic structure (P63mc) and active surface area (in the order of 5 × 10(-2)μm(2)) are almost non-toxic for cells under static conditions. However, under conditions that enable collisions between ZnO nanostructures and cells, NRs appear to be more damaging compared to NPs. This is due to the increased probability of mechanical damage caused by nanorods upon puncturing of the cell wall and membranes. Gram-positive bacteria, which have thicker cell walls, are more resistant to nanomechanical stress induced by NRs compared to Gram-negative strains and eukaryotic cells. The presented results may be exploited to improve the properties of nanotechnology based products such as implants, drug delivery systems, antibacterial emulsions and cosmetics. PMID:27074722

  2. Effects of liposome-encapsulated bisphosphonates on acetylated LDL metabolism, lipid accumulation and viability of phagocyting cells.

    PubMed

    Ylitalo, R; Mönkkönen, J; Ylä-Herttuala, S

    1998-01-01

    Bisphosphonates, the drugs used for the treatment of e.g. osteoporosis, inhibit the development of experimental atherosclerosis. When encapsulated in liposomes, they also inactivate macrophages, which have a key role in atherogenesis. We studied the effects of three clinically used bisphosphonates, i.e. clodronate, etidronate and pamidronate, on 1) the viability of mouse peritoneal macrophages and macrophage-like RAW 264 cells, 2) the degradation of 125I-labeled acetylated LDL by RAW 264 cells, and 3) the formation of LDL-derived foam cells in vitro. Liposome-encapsulated clodronate and pamidronate, but not etidronate, decreased the fraction of viable peritoneal macrophages in a concentration-dependent manner, whereas RAW 264 cells were much more resistant to the cytotoxic effects of bisphosphonates. Preincubation with liposomal clodronate and etidronate inhibited in a concentration-dependent manner the degradation of acetylated LDL in RAW 264 cells, but non-cytotoxic concentrations of liposomal pamidronate had only a weak inhibitory effect. The inhibition was more pronounced by liposomal clodronate than by liposomal etidronate. At high concentrations (500 microg protein/ml) of acetylated and aggregated LDL, RAW 264 cells transformed to foam cells. Preincubation with liposomal clodronate and etidronate reduced the cellular accumulation of acetylated LDL-derived lipids, but the drugs had no effect on the lipid accumulation caused by aggregated LDL. The results suggest that liposomal clodronate and etidronate inhibit the activity of phagocyting cells in internalizing and degrading atherogenic modified LDL. PMID:9449231

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

    PubMed

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

    2013-01-01

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

  4. Cell spreading and viability on zein films may be facilitated by transglutaminase.

    PubMed

    Cui, Hemiao; Liu, Gang L; Padua, Graciela W

    2016-09-01

    Zein is a biocompatible corn protein potentially useful in the development of biomaterials. In this study, the deposition of zein on oxygen plasma treated glass cover slips significantly enhanced cell spreading and viability. The mechanism for cellular response to zein coated surfaces was thought to involve the polyglutamine peptides on the zein structure. We hypothesized that zein was a substrate for tissue transglutaminase (tTG), an extracellular enzyme involved in cell-surface interactions. SDS-PAGE results suggested an interaction between zein and tTG, where zein was the glutamine donor. Cross-linking between zein and tTG may be the first step in successful cell adhesion and spreading. PMID:27315332

  5. 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. PMID:27220915

  6. Pomegranate extract inhibits the proliferation and viability of MMTV-Wnt-1 mouse mammary cancer stem cells in vitro.

    PubMed

    Dai, Zhaoli; Nair, Vidhya; Khan, Maruf; Ciolino, Henry P

    2010-10-01

    Pomegranate (Punica granatum L.) is known to possess anticancer activities. The effects of a standardized extract of pomegranate (PE) on a mouse mammary cancer cell line (designated WA4) derived from mouse MMTV-Wnt-1 mammary tumors were examined in this study. The WA4 cell line has been previously characterized as containing a majority of cells possessing stem cell characteristics. PE inhibited the proliferation of WA4 cells in a time- and concentration-dependent manner. This was due to an arrest of cell cycle progression in the G0/G1 phase. PE was also cytotoxic to quiescent WA4 cells in a concentration-dependent manner at concentrations >10 microg/ml. PE treatment of WA4 cells resulted in an increase in caspase-3 enzyme activity in a time- and concentration-dependent manner, indicating that the cytotoxic effect of PE was due to the induction of apoptosis. We tested the effect of several individual phytochemicals derived from PE on WA4 cells. Ellagic acid, ursolic acid and luteolin caused a time- and concentration-dependent reduction of cell proliferation and viability, suggesting that they contribute to the inhibitory effect of PE, while caffeic acid had no effect. Cancer stem cells, which are highly resistant to conventional chemotherapeutic agents, are thought to be the origin of both primary and secondary breast tumors, and thus are a critical target in both breast cancer therapy and prevention. These data suggest that PE, which is a proven and safe dietary supplement, has promise as an treatment against breast cancer by preventing proliferation of cancer stem cells. PMID:20811693

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

    PubMed

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

    2015-11-01

    The information about adverse effects of emerging contaminants on aquatic protozoa is very scarce. The growth inhibition effect, cell viability, genotoxicity and multixenobiotic resistance (MXR) responses of two commonly used antimicrobial agents, triclosan (TCS) and triclocarban (TCC) to protozoan Tetrahymena thermophila were investigated in this study. The results revealed that TCS and TCC can inhibit the growth of T. thermophila with 24h EC50 values of 1063 and 295μgL(-1), respectively. The impairment of plasma membrane was observed after 2h exposure of TCS or TCC at the level of mg/L. Furthermore, it is noticeable that at environmentally relevant concentration (1.0μgL(-1)), both TCS and TCC can lead to statistically significant DNA damage in T. thermophila, while the inhibition of growth and change of cell viability cannot be observed. Our results firstly provide the evidence for genotoxic effects of TCS and TCC on the freshwater protozoan. Additionally, both TCS and TCC were found to inhibit the efflux transporter activities, with the inhibitory potencies of 39% and 40% (using verapamil as a model inhibitor), respectively. Particularly, TCC could significantly down-regulate the expression of MXR related gene Abcb15, which encodes the membrane efflux protein that acting as P-gp in T. thermophila. The results raise the awareness of potential aquatic ecological and human health risks from the exposure of TCS and TCC, as they might potentiate the toxic effects by chemosensitizing with co-existing toxicants. PMID:26246462

  8. 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. PMID:21647502

  9. Trehalose-Based Eye Drops Preserve Viability and Functionality of Cultured Human Corneal Epithelial Cells during Desiccation

    PubMed Central

    Hill-Bator, Aneta; Misiuk-Hojło, Marta; Marycz, Krzysztof; Grzesiak, Jakub

    2014-01-01

    This paper presents the evaluation of cytoprotective ability of trehalose-based eye drops in comparison with commercially available preparations during the experimental desiccation of cultured human corneal epithelial cells. Cultured human corneal epithelial cells (hCEC) underwent incubation with 7 different, commercially available medicaments used commonly in dry eye syndrome treatment, followed by desiccation trial performed on air under the flow hood for 5, 15, 30, and 45 minutes. Cell viability was quantified by live/dead fluorescent assay, while the presence of apoptotic cells was estimated by immunofluorescent staining for active caspase 3 protein. The preservation of membrane functions was evaluated using neutral red staining, while the preservation of proper morphology and phenotype was determined by fluorescent staining for actin filaments, nuclei, and p63 protein. The trehalose-based eye drops showed the highest efficiency in prevention of cell death from desiccation; moreover, this preparation preserved the normal cellular morphology, functions of cell membrane, and proliferative activity more effectively than other tested medicaments. PMID:24995283

  10. Recombinant hirudin suppresses the viability, adhesion, migration and invasion of Hep-2 human laryngeal cancer cells.

    PubMed

    Lu, Qian; Lv, Mei; Xu, Erdong; Shao, Fangyu; Feng, Ya; Yang, Jingru; Shi, Lin

    2015-03-01

    Recombinant hirudin (rH) is a highly potent and specific inhibitor of thrombin, and has been shown to inhibit the growth and metastasis of several types of cancers in experimental tumor models. The objective of this study was to evaluate the antitumor effects and explore the underlying mechanisms of rH in Hep-2 human laryngeal carcinoma (LC) cells. Hep-2 cells were treated with various concentrations of rH for 24 h. The cell viability was evaluated by a water-soluble tetrazolium salt (WST) assay. The adhesion ability of the cells was evaluated by cell adhesion to fibronectin. Cell migration and invasion were measured with the Boyden chamber assay. Cell apoptosis was detected by Hoechst 33324 fluorescence staining. A chicken chorioallantoic membrane (CAM) assay was used to assess the effects of rH on angiogenesis in vivo. Western blotting was used to detect the expression levels of vascular endothelial growth factor receptor (VEGF-R), focal adhesion kinase (FAK), Bcl-2-associated agonist of cell death (Bad) and B-cell CLL/lymphoma 2 (Bcl-2) proteins. rH significantly inhibited the cell viability and induced apoptosis in LC Hep-2 cells in a dose-dependent manner, as compared with phosphate-buffered saline (PBS) as control. These results were accompanied by a decrease in the anti-apoptotic protein Bcl-2 and an increase in the pro-apoptotic protein Bad. Moreover, rH dose-dependently inhibited the adhesion, migration and invasion of the Hep-2 cells, compared to the vehicle PBS. In addition, rH robustly suppressed angiogenesis in the CAM assay. Importantly, the expression of adhesion and angiogenesis-associated proteins FAK and VEGF-R was significantly downregulated by rH in a dose-dependent manner. The present findings demonstrate that rH exerts antitumor effects in Hep-2 human laryngeal cancer cells via multiple mechanisms and suggests that targeting thrombin by rH is a potential strategy for the treatment of LC. PMID:25592110

  11. Single cell adhesion force measurement for cell viability identification using an AFM cantilever-based micro putter

    NASA Astrophysics Data System (ADS)

    Shen, Yajing; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Kojima, Masaru; Fukuda, Toshio

    2011-11-01

    Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level.

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

    PubMed

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

    2016-05-01

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

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

    PubMed

    Stepanenko, A A; Dmitrenko, V V

    2015-12-15

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

  14. High Glucose and Glucose Deprivation Modulate Müller Cell Viability and VEGF Secretion

    PubMed Central

    Vellanki, S; Ferrigno, A; Alanis, Y; Betts-Obregon, BS; Tsin, AT

    2016-01-01

    Purpose Diabetic retinopathy is manifested by excessive angiogenesis and high level of vascular endothelial growth factor (VEGF) in the eye. Methods Human (MIO-M1) and rat (rMC-1) Müller cells were treated with 0, 5.5, or 30mM glucose for 24 hours. Viable cell counts were obtained by Trypan Blue Dye Exclusion Method. ELISA was used to determine VEGF levels in cell medium. Results Compared to 24 hour treatment by 5.5mM glucose, MIO-M1 and rMC-1 in 30mM glucose increased in viable cell number by 38% and 24% respectively. In contrast, viable cells in 0mM glucose decreased by 28% and 50% respectively. Compared to 5.5mM, MIO-M1 and rMC-1 in 30mM glucose had increased levels of VEGF in cell medium (pg/ml by 24% and 20%) and also VEGF concentration in cells held in 0mM increased by 47% and 10% respectively. In both MIO-M1 and rMC-1, the amount of VEGF secreted per cell increased by about 100% when glucose was changed from 5.5 to 0mM but decreased slightly (17% in MIO-M1 and 11% in rMC-1) when glucose was increased from 5.5 to 30mM. Conclusions Our results show that MIO-M1 and rMC-1 are highly responsive to changes in glucose concentrations. 30mM compared to 5.5mM significantly increased cell viability but induced a significant change in VEGF secretion per cell in rMC-1 only. At 0, 5.5, and 30mM glucose, MIO-M1 secreted about 5-7-fold higher level of VEGF (pg/cell) than rMC-1. The mechanism of glucose-induced changes in rMC-1 and MIO-M1 cell viability and VEGF secretion remains to be elucidated. PMID:27347496

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

    PubMed

    Bunik, Victoria I; Mkrtchyan, Garik; Grabarska, Aneta; Oppermann, Henry; Daloso, Danilo; Araujo, Wagner L; Juszczak, Malgorzata; Rzeski, Wojciech; Bettendorff, Lucien; Fernie, Alisdair R; Meixensberger, Jürgen; Stepulak, Andrzej; Gaunitz, Frank

    2016-05-01

    2-Oxoglutarate dehydrogenase (OGDH) of the tricarboxylic acid (TCA) cycle is often implied to be inactive in cancer, but this was not experimentally tested. We addressed the question through specific inhibition of OGDH by succinyl phosphonate (SP). SP action on different cancer cells was investigated using indicators of cellular viability and reactive oxygen species (ROS), metabolic profiling and transcriptomics. Relative sensitivity of various cancer cells to SP changed with increasing SP exposure and could differ in the ATP- and NAD(P)H-based assays. Glioblastoma responses to SP revealed metabolic sub-types increasing or decreasing cellular ATP/NAD(P)H ratio under OGDH inhibition. Cancer cell homeostasis was perturbed also when viability indicators were SP-resistant, e.g. in U87 and N2A cells. The transcriptomics database analysis showed that the SP-sensitive cells, such as A549 and T98G, exhibit the lowest expression of OGDH compared to other TCA cycle enzymes, associated with higher expression of affiliated pathways utilizing 2-oxoglutarate. Metabolic profiling confirmed the dependence of cellular SP reactivity on cell-specific expression of the pathways. Thus, oxidative decarboxylation of 2-oxoglutarate is significant for the interdependent homeostasis of NAD(P)H, ATP, ROS and key metabolites in various cancer cells. Assessment of cell-specific responses to OGDH inhibition is of diagnostic value for anticancer strategies. PMID:27027236

  16. 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.

  17. HIF prolyl hydroxylase inhibition increases cell viability and potentiates dopamine release in dopaminergic cells.

    PubMed

    Johansen, Jens Leander; Sager, Thomas Nikolaj; Lotharius, Julie; Witten, Louise; Mørk, Arne; Egebjerg, Jan; Thirstrup, Kenneth

    2010-10-01

    Hypoxia-inducible factor (HIF) controls the expression of genes that adapts the cellular condition to accommodate oxidative stress. The potential beneficial effect of HIF up-regulation in ischemia has recently gained interest substantiated by the known HIF-regulation of erythropoietin and other hypoxia accommodating genes. So far the perspectives for HIF up-regulation has been focused on anemia and ischemia related diseases but little information is available about the relevance of HIF biology for neurodegenerative disease like Parkinson's disease. We therefore sought out to characterize the effect of HIF-up-regulation on survival and dopamine homeostasis in dopaminergic cells. We used a low molecular weight HIF prolyl hydroxylase (HPH) inhibitor and lentiviral based shRNA knockdown of HPH subtypes as molecular tools to increase HIF protein level and downstream HIF-regulated genes. We show that HIF induction results in protection against oxidative stress in cellular models based on PC12 cells and LUHMES cells. In addition, HPH inhibition elevates tyrosine hydroxylase expression and activity, which causes increased dopamine synthesis and release in both PC12 cells and a primary rat ventral mesencephalic cell culture. All together these findings suggest that prolyl hydroxylases may represent novel targets for therapeutic intervention in disorders characterized by dopamine homeostasis dysregulation like Parkinson's disease. PMID:20649842

  18. Importance of viability and attachment to an ascites tumor in the release of plasminogen activator.

    PubMed Central

    Dong, Q.; Zhou, M.; Subbarao, V.; Ts'ao, C.

    1991-01-01

    Tumor plasminogen activator (PA) has been alleged to play a role in the growth and metastasis of tumors. Before such a role can be realized, PA first must be released from tumor cells. Having determined intra- and extracellular PA and PA-inhibitor activities in an experimental pancreatic ascites tumor grown in hamsters, the release of PA from these cells was investigated. No PA activity was detected in the suspension medium of freshly isolated tumor cells; inclusion of plasminogen, fibrinogen, or collagen in the medium yielded similar negative results. On the other hand, PA activity was demonstrated to be released in a time-dependent manner from these tumor cells embedded in fibrin clots. Plasminogen activator activity also was not found in the suspension medium of frozen-thawed tumor cells, despite the fact that most of them had breaks on their cell membrane. Unlike freshly isolated tumor cells, PA was not released from frozen-thawed cells embedded in fibrin clots. Full PA activity was demonstrated in frozen-thawed cells treated with Triton X-100, however. Frozen-thawed cells exhibited signs of severe damage, and more than 80% of them failed to exclude trypan blue. Obviously PA is released from viable tumor cells embedded in fibrin clots but not suspended in artificial medium. The PA-release mechanism, not PA itself, is destroyed in cells rendered nonviable by freeze thawing. Images Figure 1 Figure 5 Figure 6 Figure 7 PMID:1902626

  19. Human Cerebrospinal Fluid Promotes Neuronal Viability and Activity of Hippocampal Neuronal Circuits In Vitro

    PubMed Central

    Perez-Alcazar, Marta; Culley, Georgia; Lyckenvik, Tim; Mobarrez, Kristoffer; Bjorefeldt, Andreas; Wasling, Pontus; Seth, Henrik; Asztely, Frederik; Harrer, Andrea; Iglseder, Bernhard; Aigner, Ludwig; Hanse, Eric; Illes, Sebastian

    2016-01-01

    For decades it has been hypothesized that molecules within the cerebrospinal fluid (CSF) diffuse into the brain parenchyma and influence the function of neurons. However, the functional consequences of CSF on neuronal circuits are largely unexplored and unknown. A major reason for this is the absence of appropriate neuronal in vitro model systems, and it is uncertain if neurons cultured in pure CSF survive and preserve electrophysiological functionality in vitro. In this article, we present an approach to address how human CSF (hCSF) influences neuronal circuits in vitro. We validate our approach by comparing the morphology, viability, and electrophysiological function of single neurons and at the network level in rat organotypic slice and primary neuronal cultures cultivated either in hCSF or in defined standard culture media. Our results demonstrate that rodent hippocampal slices and primary neurons cultured in hCSF maintain neuronal morphology and preserve synaptic transmission. Importantly, we show that hCSF increases neuronal viability and the number of electrophysiologically active neurons in comparison to the culture media. In summary, our data indicate that hCSF represents a physiological environment for neurons in vitro and a superior culture condition compared to the defined standard media. Moreover, this experimental approach paves the way to assess the functional consequences of CSF on neuronal circuits as well as suggesting a novel strategy for central nervous system (CNS) disease modeling. PMID:26973467

  20. Vitronectin absorbed on nanoparticles mediate cell viability/proliferation and uptake by 3T3 Swiss albino mouse fibroblasts: in vitro study.

    PubMed

    Rosso, F; Marino, G; Grimaldi, A; Cafiero, G; Chiellini, E; Chiellini, F; Barbarisi, M; Barbarisi, A

    2013-01-01

    We study the interaction of 3T3 Swiss albino mouse fibroblasts with polymeric nanoparticles (NPs) and investigate cellular behaviour in terms of viability/cytotoxicity, cell cycle, NPs uptake, MAP kinase (ERK1/2), and focal adhesion kinase (FAK) activation. After incubation of NPs with cell culture media, western blot analysis showed that Vitronectin is retained by NPs, while Fibronectin is not detected. From cytotoxicity studies (MTT and BrdU methods) an LD50 of about 1.5 mg/mL results for NPs. However, NPs in the range 0.01-0.30 mg/mL are able to trigger a statistically significant increase in proliferation and cell cycle progression in dose and time depending manner. Also, biochemical evaluation of ERK1/2 and FAK clearly shows an increasing phosphorylation in a dose and time depending manner. Finally, we found by transmission electron microscopy that NPs are internalised by cells. Competitively blocking VN-integrin receptors with echistatin (1 μg/mL) results in a decrease of viability/proliferation, cell cycle progression, cellular uptake, and FAK/ERK activation showing the involvement of Vitronectin receptors in signal transduction. In conclusion, our results show that cell surface NPs interactions are mediated by absorbed plasma proteins (i.e., Vitronectin) that represent an external stimuli, switched to the nucleus by FAK enzyme, which in turn modulate fibroblasts viability/proliferation. PMID:23710450

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

    PubMed Central

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

    2016-01-01

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

  2. A comparative study on the effect of algal and fish oil on viability and cell proliferation of Caco-2 cells.

    PubMed

    van Beelen, Vincent A; Roeleveld, Johannes; Mooibroek, Hans; Sijtsma, Lolke; Bino, Raoul J; Bosch, Dirk; Rietjens, Ivonne M C M; Alink, Gerrit M

    2007-05-01

    Polyunsaturated fatty acid (PUFA) rich micro-algal oil was tested in vitro and compared with fish oil for antiproliferative properties on cancer cells in vitro. Oils derived from Crypthecodinium cohnii, Schizochytrium sp. and Nitzschia laevis, three commercial algal oil capsules, and menhaden fish oil were used in cell viability and proliferation tests with human colon adenocarcinoma Caco-2 cells. With these tests no difference was found between algal oil and fish oil. The nonhydrolysed algal oils and fish oil showed a much lower toxic effect on cell viability, and cell proliferation in Caco-2 cells than the hydrolysed oils and the free fatty acids (FFAs). Eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3) were used as samples for comparison with the tested hydrolysed and nonhydrolysed oils. The hydrolysed samples showed comparative toxicity as the free fatty acids and no difference between algal and fish oil. Oxidative stress was shown to play a role in the antiproliferative properties of EPA and DHA, as alpha-tocopherol could partially reverse the EPA/DHA-induced effects. The results of the present study support a similar mode of action of algal oil and fish oil on cancer cells in vitro, in spite of their different PUFA content. PMID:17141934

  3. Nanofibrous Chitosan-Polyethylene Oxide Engineered Scaffolds: A Comparative Study between Simulated Structural Characteristics and Cells Viability

    PubMed Central

    Dilamian, Mandana; Mirian, Mina; Sadeghi-Aliabadi, Hojjat; Maleknia, Laleh; 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. PMID:24995296

  4. Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser.

    PubMed

    Alexsandra da Silva Neto Trajano, Larissa; da Silva, Camila Luna; de Carvalho, Simone Nunes; Cortez, Erika; Mencalha, André Luiz; de Souza da Fonseca, Adenilson; Stumbo, Ana Carolina

    2016-07-01

    Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells. PMID:26886589

  5. 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

  6. 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. PMID:27286857

  7. Isolation, characterization and targeted disruption of mouse ppia: cyclophilin A is not essential for mammalian cell viability.

    PubMed

    Colgan, J; Asmal, M; Luban, J

    2000-09-01

    Cyclophilins (CyPs) are a family of proteins found in organisms ranging from prokaryotes to humans. These molecules exhibit peptidyl-prolyl isomerase activity in vitro, suggesting that they influence the conformation of proteins in cells. CyPs also bind with varying affinities to the immunosuppressive drug cyclosporin A (CsA), a compound used clinically to prevent allograft rejection. The founding member of the family, cyclophilin A (CyPA), is an abundant, ubiquitously expressed protein of unknown function that binds with nanomolar affinity to CsA. Here, we describe the isolation and characterization of mouse Ppia (mPpia), the gene encoding CyPA. Ppia was isolated using a PCR screen that distinguishes the expressed gene from multiple pseudogenes present in the mouse genome. mPpia consists of 5 exons and 4 introns spanning roughly 4.5 kb and maps to chromosome 11 near the centromere. Sequence analysis of a 369-bp fragment from the proximal promoter region of mPpia revealed the presence of a TATA box and sites recognized by several transcriptional regulators, including Sp1, AP-2, GATA factors, c-Myb, and NF-IL-6. This region is sufficient to drive high-level reporter gene expression in transfected cells. Both copies of Ppia were disrupted in murine embryonic stem (ES) cells via gene targeting. Ppia(-/-) ES cells grow normally and differentiate into hematopoeitic precursor cells in vitro, indicating that CyPA is not essential for mammalian cell viability. PMID:10964515

  8. 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

  9. A small molecule focal adhesion kinase (FAK) inhibitor, targeting Y397 site: 1-(2-hydroxyethyl)-3, 5, 7-triaza-1-azoniatricyclo [3.3.1.1(3,7)]decane; bromide effectively inhibits FAK autophosphorylation activity and decreases cancer cell viability, clonogenicity and tumor growth in vivo.

    PubMed

    Golubovskaya, Vita M; Figel, Sheila; Ho, Baotran T; Johnson, Christopher P; Yemma, Michael; Huang, Grace; Zheng, Min; Nyberg, Carl; Magis, Andrew; Ostrov, David A; Gelman, Irwin H; Cance, William G

    2012-05-01

    Focal adhesion kinase (FAK) is a protein tyrosine kinase that is overexpressed in most solid types of tumors and plays an important role in the survival signaling. Recently, we have developed a novel computer modeling combined with a functional assay approach to target the main autophosphorylation site of FAK (Y397). Using these approaches, we identified 1-(2-hydroxyethyl)-3, 5, 7-triaza-1-azoniatricyclo [3.3.1.1(3,7)]decane; bromide, called Y11, a small molecule inhibitor targeting Y397 site of FAK. Y11 significantly and specifically decreased FAK autophosphorylation, directly bound to the N-terminal domain of FAK. In addition, Y11 decreased Y397-FAK autophosphorylation, inhibited viability and clonogenicity of colon SW620 and breast BT474 cancer cells and increased detachment and apoptosis in vitro. Moreover, Y11 significantly decreased tumor growth in the colon cancer cell mouse xenograft model. Finally, tumors from the Y11-treated mice demonstrated decreased Y397-FAK autophosphorylation and activation of poly (ADP ribose) polymerase and caspase-3. Thus, targeting the major autophosphorylation site of FAK with Y11 inhibitor is critical for development of cancer therapeutics and carcinogenesis field. PMID:22402131

  10. Long term cryopreservation in 5% DMSO maintains unchanged CD34(+) cells viability and allows satisfactory hematological engraftment after peripheral blood stem cell transplantation.

    PubMed

    Abbruzzese, L; Agostini, F; Durante, C; Toffola, R T; Rupolo, M; Rossi, F M; Lleshi, A; Zanolin, S; Michieli, M; Mazzucato, M

    2013-07-01

    Peripheral blood stem cell cryopreservation is associated with cell damage and decreased viability. We evaluated the impact of up to 10 years of cryopreservation (5% DMSO) on viability of CD34(+) cells utilizing graft samples of consecutive patients (2002-2012) with different malignancies who underwent stem cell collection and transplantation. Viability of CD34(+) cells from oncohaematological patients measured after 5 weeks (97·2 ± 0·6%) or after 9-10 years of cryopreservation (95·9 ± 0·5%) was unaffected. Haemoglobin, granulocyte and platelet recovery after transplantation of long-term cryopreserved grafts occurred within 8-13 days. CD34(+) stem cells can be safely stored up to 9-10 years, without affecting cell viability and clinical effectiveness. PMID:23384290

  11. Acute and chronic responses of activated sludge viability and performance to silica nanoparticles.

    PubMed

    Zheng, Xiong; Su, Yinglong; Chen, Yinguang

    2012-07-01

    Recently, the potential health and environmental risks of silica nanoparticles (SiO(2) NPs) are attracting great interest. However, little is known about their possible impacts on wastewater biological nitrogen and phosphorus removal. In this study, the acute and chronic effects of SiO(2) NPs on activated sludge viability and biological nutrient removal performance were investigated. It was found that the presence of environmentally relevant concentration (1 mg/L) of SiO(2) NPs caused no adverse acute and chronic effects on sludge viability and wastewater nitrogen and phosphorus removal. However, chronic exposure to 50 mg/L SiO(2) NPs induced the increase of effluent nitrate concentration, and thus depressed the total nitrogen (TN) removal efficiency from 79.6% to 51.6% after 70 days of exposure, which was due to the declined activities of denitrifying enzymes, nitrate reductase and nitrite reductase. Wastewater phosphorus removal was insensitive to 1 and 50 mg/L SiO(2) NPs after either the acute or chronic exposure, because the critical factors closely related to biological phosphorus removal were not significantly changed, such as the activities of exopolyphosphatase and polyphosphate kinase and the intracellular transformations of polyhydroxyalkanoates and glycogen. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that the bacterial community structure was changed after long-term exposure to 50 mg/L SiO(2) NPs, and the quantitative PCR assays indicated that the abundance of denitrifying bacteria was decreased, which was consistent with the declined wastewater nitrogen removal. PMID:22697807

  12. Glycosylated cell-penetrating peptides and their conjugates to a proapoptotic peptide: preparation by click chemistry and cell viability studies

    PubMed Central

    Dutot, Laurence; Lécorché, Pascaline; Burlina, Fabienne; Marquant, Rodrigue; Point, Vanessa; Sagan, Sandrine; Chassaing, Gérard; Mallet, Jean-Maurice

    2009-01-01

    Cell-penetrating peptides (CPPs), which are usually short basic peptides, are able to cross cell membranes and convey bioactive cargoes inside cells. CPPs have been widely used to deliver inside cells peptides, proteins, and oligonucleotides; however, their entry mechanisms still remain controversial. A major problem concerning CPPs remains their lack of selectivity to target a specific type of cell and/or an intracellular component. We have previously shown that myristoylation of one of these CPPs affected the intracellular distribution of the cargo. We report here on the synthesis of glycosylated analogs of the cell-penetrating peptide (R6/W3): Ac-RRWWRRWRR-NH2. One, two, or three galactose(s), with or without a spacer, were introduced into the sequence of this nonapeptide via a triazole link, the Huisgen reaction being achieved on a solid support. Four of these glycosylated CPPs were coupled via a disulfide bridge to the proapoptotic KLAK peptide, (KLAKLAKKLAKLAK), which alone does not enter into cells. The effect on cell viability and the uptake efficiency of different glycosylated conjugates were studied on CHO cells and were compared to those of the nonglycosylated conjugates: (R6/W3)S-S-KLAK and penetratinS-S-KLAK. We show that glycosylation significantly increases the cell viability of CHO cells compared to the nonglycosylated conjugates and concomitantly decreases the internalization of the KLAK cargo. These results suggest that glycosylation of CPP may be a key point in targeting specific cells. Electronic supplementary material The online version of this article (doi:10.1007/s12154-009-0031-9) contains supplementary material, which is available to authorized users. PMID:19899012

  13. 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

  14. Application of viability theory for road vehicle active safety during cornering manoeuvres

    NASA Astrophysics Data System (ADS)

    Vandanjon, P.-O.; Coiret, A.; Lorino, T.

    2014-02-01

    Viability theory proposes geometric metaphors in addition to classical ordinary differential equation analysis. In this paper, advantages of applying viability theory to road safety domain are presented. The exact issue is to determine if, from an initial state of a vehicle/road/driver system, a soft controls strategy is compatible with a safe driving sequence. The case of a car negotiating a curve is considered. The application of the viability theory to this issue offers the advantage to avoid classical full computing of the system. Instead of that, it consists on verifying that the states and the controls belong to a subset called the viability kernel. The construction and the use of the viability kernel for a vehicle system dynamic is proposed by using support vector machines algorithm. Then, the applicability of this theory is demonstrated through experimental tests. This innovative application of the viability theory to vehicle dynamics with road safety concerns could benefit to robust embedded warning systems.

  15. Effects of nano-scaled particles on endothelial cell function in vitro: studies on viability, proliferation and inflammation.

    PubMed

    Peters, Kirsten; Unger, Ronald E; Kirkpatrick, C James; Gatti, Antonietta M; Monari, Emanuela

    2004-04-01

    Recent studies give support for a connection between the presence of inorganic particles (of microm and nm size) in different organs and tissues and the development of inflammatory foci, called granulomas. As the potential source of particles (e.g. porcelain dental bridges) and the location of particle detection were topographically far apart, a distribution via the blood stream appears highly probable. Thus, endothelial cells, which line the inner surface of blood vessels, would come into direct contact with these particles, making particle-endothelial interactions potentially pathogenically relevant. The objective of this study was to evaluate the effects that five different nano-scaled particles (PVC, TiO2, SiO2, Co, Ni) have on endothelial cell function and viability. Therefore, human endothelial cells were exposed to different amounts of the above-mentioned particles. Although most particle types are shown to be internalised (except Ni-particles), only Co-particles possessed cytotoxic effects. Furthermore, an impairment of the proliferative activity and a pro-inflammatory stimulation of endothelial cells were induced by exposure to Co- and, to a lesser extent, by SiO2-particles. If a pro-inflammatory stimulation of endothelial cells occurs in vivo, a chronic inflammation could be a possible consequence. PMID:15332593

  16. Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds.

    PubMed

    Gantenbein-Ritter, Benjamin; Potier, Esther; Zeiter, Stephan; van der Werf, Marije; Sprecher, Christoph M; Ito, Keita

    2008-12-01

    Several different assays are commonly used to evaluate survival of cells inside tissues or three-dimensional carriers, but their accuracy and reliability have not been evaluated. Here, we compare three methods for cell viability (CV) determination: (i) lactate dehydrogenase (LDH) staining on cryosections, (ii) calcein AM/ethidium homodimer-1 (CaAM/EthH) staining, and (iii) carrier digestion and trypan blue (TB) assay. Living and dead cell populations were generated from bovine chondrocytes and combined to produce approximately 0%, 25%, 50%, 75%, and 100% CV mixtures. CV ratios were measured with TB assay (MIX) before seeding cells into fibrin carriers. CV was then determined using the three methods (n = 5/method). Custom-written macros were used to process LDH- and CaAM/EthH-stained images, and hand counting with hemocytometer was used for the TB method. Absolute error and intraclass correlation (ICC) were used for accuracy and reliability evaluation. All methods estimated CV values close to MIX values. TB method was the most accurate (ICC = 0.99) followed by CaAM/EthH (ICC = 0.98) and LDH (ICC = 0.97). As for absolute quantification of living and dead cells, TB and LDH methods performed well (ICC = 0.75-0.96), whereas CaAM/EthH largely overestimated cell numbers (living, ICC = 0.30; dead, ICC = 0.30). Although TB was the most accurate, LDH and CaAM/EthH provide valuable information on cell shape and spatial distribution of cells in tissue or a scaffold. PMID:18800876

  17. Inhibition of SIRT6 in prostate cancer reduces cell viability and increases sensitivity to chemotherapeutics.

    PubMed

    Liu, Yewei; Xie, Qian Reuben; Wang, Boshi; Shao, Jiaxiang; Zhang, Tingting; Liu, Tengyuan; Huang, Gang; Xia, Weiliang

    2013-09-01

    SIRT6 is an important histone modifying protein that regulates DNA repair, telomere maintenance, energy metabolism, and target gene expression. Recently SIRT6 has been identified as a tumor suppressor and is down-regulated in certain cancer types, but not in other cancers. From deposited gene profiling studies we found that SIRT6 was overexpressed in prostate tumors, compared with normal or paratumor prostate tissues. Tissue micro-array studies confirmed the higher levels of SIRT6 in both prostate tumor tissues and prostate cancer cells than in their normal counterparts. Knockdown of SIRT6 in human prostate cancer cells led to sub-G1 phase arrest of cell cycle, increased apoptosis, elevated DNA damage level and decrease in BCL2 gene expression. Moreover, SIRT6-deficiency reduced cell viability and enhanced chemotherapeutics sensitivity. Taken together, this study provides the first evidence of SIRT6 overexpression in human prostate cancer, and SIRT6 regulation could be exploited for prostate cancer therapy. PMID:23982738

  18. Flexible Bioimpedance Sensor for Label-Free Detection of Cell Viability and Biomass.

    PubMed

    Fernandez, Renny Edwin; Lebiga, Elise; Koklu, Anil; Sabuncu, Ahmet Can; Beskok, Ali

    2015-10-01

    We introduce a flexible microfluidic bioimpedance sensor that is capable of detecting biomass and cell viability variations in a cell suspension. The sensor is developed on indium tin oxide (ITO) coated polyethylene terephthalate (PET) substrate and is devoid of gold, silicon, PDMS, or glass. In conjugation with a custom built PCB read-out module, the impedance characteristics of a cell suspension can be measured within one minute of sample introduction using liquid volumes less than 5 μL. The portable sensor system occupies very little bench space and has the potential to be developed as a disposable electrical bioimpedance probe for rapid detection of dielectric variations in a biological suspension. The sensor is designed to generate a differential impedance spectra exclusive to a cell suspension with a dual-electrode-pair system. The potential of the sensor to discriminate between live and heat treated Saccharomyces cerevisiae is demonstrated in this study. The disposable sensor along with the distance variation technique is touted to be an inexpensive alternative to some of the existing online disposable biomass detection probes and electrochemical sensors. PMID:26415205

  19. Thiostrepton is an Inducer of Oxidative and Proteotoxic Stress that Impairs Viability of Human Melanoma Cells but not Primary Melanocytes

    PubMed Central

    Qiao, Shuxi; Lamore, Sarah D.; Cabello, Christopher M.; Lesson, Jessica L.; Muñoz-Rodriguez, José L.; Wondrak, Georg T.

    2012-01-01

    Pharmacological induction of oxidative and proteotoxic stress has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Guided by a differential phenotypic drug screen for novel lead compounds that selectively induce melanoma cell apoptosis without compromising viability of primary human melanocytes, we have focused on the cyclic pyridinyl-polythiazolyl peptide-antimicrobial thiostrepton. Using comparative gene expression-array analysis, the early cellular stress response induced by thiostrepton was examined in human A375 metastatic melanoma cells and primary melanocytes. Thiostrepton displayed selective antimelanoma activity causing early induction of proteotoxic stress with massive upregulation of heat shock (HSPA6, HSPA1A, DNAJB4, HSPB1, HSPH1, HSPA1L, CRYAB, HSPA5, DNAJA1), oxidative stress (HMOX1, GSR, SOD1), and ER stress response (DDIT3) gene expression, confirmed by immunodetection (Hsp70, Hsp70B′, HO-1, phospho-eIF2α). Moreover, upregulation of p53, proapoptotic modulation of Bcl-2 family members (Bax, Noxa, Mcl-1, Bcl-2), and induction of apoptotic cell death were observed. Thiostrepton rapidly induced cellular oxidative stress followed by inactivation of chymotrypsin-like proteasomal activity and melanoma cell-directed accumulation of ubiquitinated proteins, not observed in melanocytes that were resistant to thiostrepton-induced apoptosis. Proteotoxic and apoptogenic effects were fully antagonized by antioxidant intervention. In RPMI 8226 multiple myeloma cells, known to be exquisitely sensitive to proteasome inhibition, early proteotoxic and apoptogenic effects of thiostrepton were confirmed by array analysis indicating pronounced upregulation of heat shock response gene expression. Our findings demonstrate that thiostrepton displays dual activity as a selective prooxidant and proteotoxic chemotherapeutic, suggesting feasibility of experimental intervention targeting metastatic melanoma and other

  20. Quantitative proteomic analysis of the inhibitory effects of CIL-102 on viability and invasiveness in human glioma cells

    SciTech Connect

    Teng, Chih-Chuan; Kuo, Hsing-Chun; Sze, Chun-I

    2013-11-01

    CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone), the major active agent of the alkaloid derivative, has been demonstrated to exert anticancer effects. Herein, we present an investigation focused on the identification of the target(s) of CIL-102's action and the mechanism of its action in apoptotic and anti-invasive pathways. Proteomic approaches were used to purify and identify the protein substrates using 2D difference gel electrophoresis (2D SDS-PAGE) to assess changes in the expression of relevant protein treatment with CIL-102 that resulted in the inhibition of viability and invasion. Our results demonstrate that CIL-102 treatment of U87 cells decreased cell proliferation and invasiveness. CIL-102 dose-dependent induction of apoptosis and inhibitory invasiveness were accompanied by sustained phosphorylation of JNK1/2 and p70S6K as well as generation of the reactive oxygen species. In addition, differential proteins displayed between CIL-102-treated and untreated U87 were determined and validated. There were 11 differentially expressed proteins between the CIL-102-treated and untreated groups. Furthermore, we demonstrated that CIL-102 inhibited cancer cell proliferation and reduced anti-invasion properties by up-regulating the levels of FUMH (Fumarate hydratase). The investigation demonstrated that there was an increase in the cellular levels of FUMH in the CIL-102 reduction in viability and invasion via the activation of JNK1/2 and mTOR signaling modules. NAC administration and shRNA FUMH conferred resistance to CIL-102-inhibited HIF1α and MMP-2 levels via inhibition of JNK1/2 and mTOR activation. We concluded that CIL-102-induced an apoptosis cascade and decreased aggressiveness in astrocytoma cells by modulation of mitochondria function, providing a new mechanism for CIL-102 treatment. - Highlights: • We found the effect of CIL-102 on neuroblastoma cells. • Fumarate hydratase as a CIL-102's target by proteomic differential displays. • CIL

  1. A new antiviral screening method that simultaneously detects viral replication, cell viability, and cell toxicity.

    PubMed

    Matza-Porges, Sigal; Eisen, Kobi; Ibrahim, Hadeel; Haberman, Adva; Fridlender, Bertold; Joseph, Gili

    2014-11-01

    Viruses cause a variety of illnesses in humans, yet only a few antiviral drugs have been developed; thus, new antiviral drugs are urgently needed. Plants could be a good source of antiviral drugs, they do not have mobility and can only defend themselves by producing compounds against pathogens such as viruses in their own fix environment. These compounds may have the potential to inhibit animal and human viruses as well. In this study, a fast and reliable method for screening plant extracts for specific antiviral activity against Herpes simplex virus type-1 (HSV-1) was developed. This method distinguishes between host cell death due to infectivity and multiplicity of the virus versus toxicity of the plant extract. Extracts from 80 plant and plant organs were screened using this approach. Six plant extracts showed potential to exert specific HSV-1 growth inhibition activity. In two cases, different organs from the same plant showed similar active results. With this method it is possible to screen a large number of extracts in a rapid and accurate way to detect antiviral substances against HSV-I and other viruses. PMID:25152527

  2. 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

  3. 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. PMID:26945926

  4. Cell viability and inflammatory response in hydrogel sponges implanted in the rabbit cornea.

    PubMed

    Vijayasekaran, S; Fitton, J H; Hicks, C R; Chirila, T V; Crawford, G J; Constable, I J

    1998-12-01

    In the quest for the development of a functional keratoprosthesis, the biocompatibility of the porous skirt material in the Chirila keratoprosthesis (KPro) was investigated. The population of live and dead cells within, and the inflammatory response to, a tissue-integrating poly(2-hydroxyethyl methacrylate) (PHEMA) sponge were studied. Samples of the hydrogel sponge were implanted in rabbit corneas and explanted at predetermined time points up to 12 weeks. The explanted sponges were subjected to cell viability assay using two types of fluoroprobes, 5-chloromethylfluorescein diacetate and ethidium homodimer-1. A semiquantitative analysis was performed to assess the number of dead cells within the sponge and in the area of corneal stroma proximal to the sponge. Five rabbits were used for each end point (2, 4 and 12 weeks). To investigate the inflammatory response to the sponge, immunocytochemistry, using specific antibodies to rabbit macrophages, enzyme histochemistry of chloroacetate esterase (to detect neutrophils) and transmission electron microscopy (TEM) were also employed at 24 h, 2, 4 and 12 weeks after implantation. Four weeks after implantation, fewer viable cells were observed in the sponge when compared to the 2-week implant. However, the proportion of viable cells increased dramatically by 12 weeks. The proportion of nonviable cells decreased gradually with time; central sponge contained 34+/-11 % dead cells after 2 weeks, and 15+/-4.3% after 12 weeks. The staining of inflammatory cells demonstrated the presence of macrophages and neutrophils up to 12 weeks after implantation. TEM confirmed the presence of these cell types and others. including eosinophils and myofibroblasts, as well as blood capillaries. The presence of a significant number of viable cells at each time point and the uniform reduction of the nonviable cell proportion with time suggests that the sponge is a conducive environment supporting a prolific, viable cellular colonization. Dead

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

    PubMed

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

    2008-04-01

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

  6. Overexpression of the Insulin-Like Growth Factor II Receptor Increases β-Amyloid Production and Affects Cell Viability

    PubMed Central

    Wang, Y.; Buggia-Prévot, V.; Zavorka, M. E.; Bleackley, R. C.; MacDonald, R. G.; Thinakaran, G.

    2015-01-01

    Amyloid β (Aβ) peptides originating from amyloid precursor protein (APP) in the endosomal-lysosomal compartments play a critical role in the development of Alzheimer's disease (AD), the most common type of senile dementia affecting the elderly. Since insulin-like growth factor II (IGF-II) receptors facilitate the delivery of nascent lysosomal enzymes from the trans-Golgi network to endosomes, we evaluated their role in APP metabolism and cell viability using mouse fibroblast MS cells deficient in the murine IGF-II receptor and corresponding MS9II cells overexpressing the human IGF-II receptors. Our results show that IGF-II receptor overexpression increases the protein levels of APP. This is accompanied by an increase of β-site APP-cleaving enzyme 1 levels and an increase of β- and γ-secretase enzyme activities, leading to enhanced Aβ production. At the cellular level, IGF-II receptor overexpression causes localization of APP in perinuclear tubular structures, an increase of lipid raft components, and increased lipid raft partitioning of APP. Finally, MS9II cells are more susceptible to staurosporine-induced cytotoxicity, which can be attenuated by β-secretase inhibitor. Together, these results highlight the potential contribution of IGF-II receptor to AD pathology not only by regulating expression/processing of APP but also by its role in cellular vulnerability. PMID:25939386

  7. Aptamer targeting of the elongation factor 1A impairs hepatocarcinoma cells viability and potentiates bortezomib and idarubicin effects.

    PubMed

    Scaggiante, Bruna; Farra, Rosella; Dapas, Barbara; Baj, Gabriele; Pozzato, Gabriele; Grassi, Mario; Zanconati, Fabrizio; Grassi, Gabriele

    2016-06-15

    The high morbidity and mortality of hepatocellular carcinoma (HCC) is mostly due to the limited efficacy of the available therapeutic approaches. Here we explore the anti-HCC potential of an aptamer targeting the elongation factor 1A (eEF1A), a protein implicated in the promotion of HCC. As delivery methods, we have compared the effectiveness of cationic liposome and cholesterol-mediated approaches. A75 nucleotide long aptamer containing GT repetition (GT75) was tested in three HCC cell lines, HepG2, HuH7 and JHH6. When delivered by liposomes, GT75 was able to effectively reducing HCC cells viability in a dose and time dependent fashion. Particular sensitive were JHH6 where increased apoptosis with no effects on cell cycle were observed. GT75 effect was likely due to the interference with eEF1A activity as neither the mRNA nor the protein levels were significantly affected. Notably, cholesterol-mediated delivery of GT75 abrogated its efficacy due to cellular mis-localization as proven by fluorescence and confocal microscopic analysis. Finally, liposome-mediated delivery of GT75 improved the therapeutic index of the anticancer drugs bortezomib and idarubicin. In conclusion, liposome but not cholesterol-mediated delivery of GT75 resulted in an effective delivery of GT75, causing the impairment of the vitality of a panel of HCC derived cells. PMID:27094354

  8. Biased Type 1 Cannabinoid Receptor Signaling Influences Neuronal Viability in a Cell Culture Model of Huntington Disease.

    PubMed

    Laprairie, Robert B; Bagher, Amina M; Kelly, Melanie E M; Denovan-Wright, Eileen M

    2016-03-01

    Huntington disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder with limited treatment options. Prior to motor symptom onset or neuronal cell loss in HD, levels of the type 1 cannabinoid receptor (CB1) decrease in the basal ganglia. Decreasing CB1 levels are strongly correlated with chorea and cognitive deficit. CB1 agonists are functionally selective (biased) for divergent signaling pathways. In this study, six cannabinoids were tested for signaling bias in in vitro models of medium spiny projection neurons expressing wild-type (STHdh(Q7/Q7)) or mutant huntingtin protein (STHdh(Q111/Q111)). Signaling bias was assessed using the Black and Leff operational model. Relative activity [ΔlogR (τ/KA)] and system bias (ΔΔlogR) were calculated relative to the reference compound WIN55,212-2 for Gαi/o, Gαs, Gαq, Gβγ, and β-arrestin1 signaling following treatment with 2-arachidonoylglycerol (2-AG), anandamide (AEA), CP55,940, Δ(9)-tetrahydrocannabinol (THC), cannabidiol (CBD), and THC+CBD (1:1), and compared between wild-type and HD cells. The Emax of Gαi/o-dependent extracellular signal-regulated kinase (ERK) signaling was 50% lower in HD cells compared with wild-type cells. 2-AG and AEA displayed Gαi/o/Gβγ bias and normalized CB1 protein levels and improved cell viability, whereas CP55,940 and THC displayed β-arrestin1 bias and reduced CB1 protein levels and cell viability in HD cells. CBD was not a CB1 agonist but inhibited THC-dependent signaling (THC+CBD). Therefore, enhancing Gαi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD. In contrast, cannabinoids that are β-arrestin-biased--such as THC found at high levels in modern varieties of marijuana--may be detrimental to CB1 signaling, particularly in HD where CB1 levels are already reduced. PMID:26700564

  9. Dopamine agonist resistance-related endocan promotes angiogenesis and cells viability of prolactinomas.

    PubMed

    Cai, Lin; Leng, Zhi Gen; Guo, Yu Hang; Lin, Shao Jian; Wu, Ze Rui; Su, Zhi Peng; Lu, Jiang Long; Wei, Li Fei; Zhuge, Qi Chuan; Jin, Kunlin; Wu, Zhe Bao

    2016-06-01

    Dopamine agonists (DAs) are the first-line treatment of prolactinomas. They function through the dopamine 2 receptor (D2R) in the tumor cells. Endocan, also called endothelial cell-specific molecule-1 (ESM1), has been described as a marker of neoangiogenesis. However, whether ESM1 promotes the resistance of prolactinomas to DA therapy is largely unknown. In our study, 25 patients with prolactinomas were divided into resistant- and sensitive- groups according to the clinical response to bromocriptine. We found that ESM1-microvessel density of resistant prolactinomas was significantly higher than that of sensitive prolactinomas (47.9 ± 11.6, n = 8, vs 13.1 ± 2.8, n = 17, p = 0.0006), indicating that ESM1 was a DA resistance-related gene. Immunostaining showed that ESM1 was expressed in tumor vessels and sporadic tumor cells, and ESM1 was overlapped with the Smooth Muscle Actin (SMA) and von Willebrand Factor (VWF) in the tumor vessels. Silencing of ESM1 markedly suppressed the viability of GH3 and MMQ cells in vitro, and furthermore, significantly increased the sensitivity of GH3 and MMQ cells to DA treatment. Additionally, silencing of ESM1 down-regulated the angiogenesis-associated genes, such as VEGFR2, FGF2, CD34, CD31, VWF, and EGFR. Knockdown of ESM1 decreased endothelial tube formation of HUVECs, and significantly increased the sensitivity of HUVECs to Avastin treatment. Therefore, we first demonstrate that DA resistance-related ESM1 promotes the angiogenesis and tumor cells growth of prolactinomas, suggesting that ESM1 may be a novel therapeutic target for prolactinomas. PMID:26662185

  10. 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

  11. Two-dimensional and three-dimensional viability measurements of adult stem cells with optical coherence phase microscopy

    NASA Astrophysics Data System (ADS)

    Bagnaninchi, Pierre O.; Holmes, Christina; Drummond, Nicola; Daoud, Jamal; Tabrizian, Maryam

    2011-08-01

    Cell viability assays are essential tools for cell biology. They assess healthy cells in a sample and enable the quantification of cellular responses to reagents of interest. Noninvasive and label-free assays are desirable in two-dimensional (2D) and three-dimensional (3D) cell culture to facilitate time-course viability studies. Cellular micromotion, emanating from cell to substrate distance variations, has been demonstrated as a marker of cell viability with electric cell-substrate impedance sensing (ECIS). In this study we investigated if optical coherence phase microscopy (OCPM) was able to report phase fluctuations of adult stem cells in 2D and 3D that could be associated with cellular micromotion. An OCPM has been developed around a Thorlabs engine (λo = 930 nm) and integrated in an inverted microscope with a custom scanning head. Human adipose derived stem cells (ADSCs, Invitrogen) were cultured in Mesenpro RS medium and seeded either on ECIS arrays, 2D cell culture dishes, or in 3D highly porous microplotted polymeric scaffolds. ADSC micromotion was confirmed by ECIS analysis. Live and fixed ADSCs were then investigated in 2D and 3D with OCPM. Significant differences were found in phase fluctuations between the different conditions. This study indicated that OCPM could potentially assess cell vitality in 2D and in 3D microstructures.

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

    PubMed Central

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

    2016-01-01

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

  13. Anti-Giardia activity of Syzygium aromaticum essential oil and eugenol: effects on growth, viability, adherence and ultrastructure.

    PubMed

    Machado, M; Dinis, A M; Salgueiro, L; Custódio, José B A; Cavaleiro, C; Sousa, M C

    2011-04-01

    The present work evaluates the anti-Giardia activity of Syzygium aromaticum and its major compound eugenol. The effects were evaluated on parasite growth, adherence, viability and ultrastructure. S. aromaticum essential oil (IC(50)=134 μg/ml) and eugenol (IC(50)=101 μg/ml) inhibited the growth of G. lamblia. The essential oil inhibited trophozoites adherence since the first hour of incubation and was able to kill almost 50% of the parasites population in a time dependent manner. The eugenol inhibited G. lamblia trophozoites adherence since the third hour and not induce cell lyses. The main morphological alterations were modifications on the cell shape, presence of precipitates in the cytoplasm, autophagic vesicles, internalization of flagella and ventral disc, membrane blebs, and intracellular and nuclear clearing. Taken together, our findings lead us to propose that eugenol was responsible for the anti-giardial activity of the S. aromaticum essential oil and both have potential for use as therapeutic agents against giardiasis. PMID:21272580

  14. 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.

  15. 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. PMID:25463682

  16. 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

  17. 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

  18. Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty

    PubMed Central

    Bhogal, Maninder; Balda, Maria S; Matter, Karl; Allan, Bruce D

    2016-01-01

    Purpose To describe a novel method of global cell viability assessment for Descemet membrane endothelial keratoplasty (DMEK) and the comparison of two contemporary methods of donor tissue preparation. Methods DMEK transplants were prepared using two different methods: liquid bubble separation and manual peeling (n=8 each group). Samples were incubated with Hoechst, calcein-AM and ethidium homodimer prior to mounting on a curved imaging chamber. Z-stacked fluorescence microscopy images were combined to produce an in-focus global image capable of resolving all cell nuclei. Image processing software was used to define a calcein-positive live cell area, count all cell nuclei within this area and subtract ethidium-positive dead cells to derive the total viable endothelial cell count. Corrected global cell density was calculated by dividing the number of viable cells by the graft area, which had been corrected for imaging a curved surface. Results Corrected global cell density was lower than the central endothelial cell density in both groups: 85.5% of the pre-preparation central endothelial cell density in the peel group and 75.8% in the bubble group. Corrected global cell density was significantly lower in the liquid bubble separation group than in the peel group (p=0.04). Conclusions Eye bank estimations of central endothelial cell density overestimate true cell density after graft preparation in DMEK. A peel method is less damaging and more consistent than a liquid bubble method. Cell loss correlated strongly with the degree of stromal hydration prior to bubble separation in the liquid bubble group. PMID:26740609

  19. A stress assembly that confers cell viability by preserving ERES components during amino-acid starvation

    PubMed Central

    Zacharogianni, Margarita; Aguilera Gomez, Angelica; Veenendaal, Tineke; Smout, Jan; Rabouille, Catherine

    2014-01-01

    Nutritional restriction leads to protein translation attenuation that results in the storage and degradation of free mRNAs in cytoplasmic assemblies. In this study, we show in Drosophila S2 cells that amino-acid starvation also leads to the inhibition of another major anabolic pathway, the protein transport through the secretory pathway, and to the formation of a novel reversible non-membrane bound stress assembly, the Sec body that incorporates components of the ER exit sites. Sec body formation does not depend on membrane traffic in the early secretory pathway, yet requires both Sec23 and Sec24AB. Sec bodies have liquid droplet-like properties, and they act as a protective reservoir for ERES components to rebuild a functional secretory pathway after re-addition of amino-acids acting as a part of a survival mechanism. Taken together, we propose that the formation of these structures is a novel stress response mechanism to provide cell viability during and after nutrient stress. DOI: http://dx.doi.org/10.7554/eLife.04132.001 PMID:25386913

  20. 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

  1. Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability[W

    PubMed Central

    Schapire, Arnaldo L.; Voigt, Boris; Jasik, Jan; Rosado, Abel; Lopez-Cobollo, Rosa; Menzel, Diedrik; Salinas, Julio; Mancuso, Stefano; Valpuesta, Victoriano; Baluska, Frantisek; Botella, Miguel A.

    2008-01-01

    Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca2+-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca2+-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness. PMID:19088329

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

    PubMed

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

    2016-03-29

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

  3. GSK-3α Is a Novel Target of CREB and CREB-GSK-3α Signaling Participates in Cell Viability in Lung Cancer

    PubMed Central

    Herbst, Roy S.; Koo, Ja Seok

    2016-01-01

    Overexpression or activation of cyclic AMP-response element-binding protein (CREB) has been known to be involved in several human malignancies, including lung cancer. Genes regulated by CREB have been reported to suppress apoptosis, induce cell proliferation, inflammation, and tumor metastasis. However, the critical target genes of CREB in lung cancer have not been well understood. Here, we identified GSK-3α as one of the CREB target genes which is critical for the viability of lung cancer cells. The CREB knockdown significantly reduced the expression of GSK-3α and the direct binding of CREB on the promoter of GSK3A was identified. Kaplan-Meier analysis with a public database showed a prognostic significance of aberrant GSK-3α expression in lung cancer. Inhibition of GSK-3α suppressed cell viability, colony formation, and tumor growth. For the first time, we demonstrated that GSK-3α is regulated by CREB in lung cancer and is required for the cell viability. These findings implicate CREB-GSK-3α axis as a novel therapeutic target for lung cancer treatment. PMID:27049759

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

    PubMed Central

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

    2016-01-01

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

  5. 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. PMID:23038647

  6. Impact of alginate concentration on the viability, cryostorage, and angiogenic activity of encapsulated fibroblasts.

    PubMed

    Mohanty, Swetaparna; Wu, Yang; Chakraborty, Nilay; Mohanty, Pravansu; Ghosh, Gargi

    2016-08-01

    Cryopreservation or cryostorage of tissue engineered constructs can enhance the off-the shelf availability of these products and thus can potentially facilitate the commercialization or clinical translation of tissue engineered products. Encapsulation of cells within hydrogel matrices, in particular alginate, is widely used for fabrication of tissue engineered constructs. While previous studies have explored the cryopreservation response of cells encapsulated within alginate matrices, systematic investigation of the impact of alginate concentration on the metabolic activity and functionality of cryopreserved cells is lacking. The objective of the present work is to determine the metabolic and angiogenic activity of cryopreserved human dermal fibroblasts encapsulated within 1.0%, 1.5% and 2.0% (w/v) alginate matrices. In addition, the goal is to compare the efficacy of dimethyl sulfoxide (DMSO) and trehalose as cryoprotectant. Our study revealed that the concentration of alginate plays a significant role in the cryopreservation response of encapsulated cells. The lowest metabolic activity of the cryopreserved cells was observed in 1% alginate microspheres. When higher concentration of alginate was utilized for cell encapsulation, the metabolic and angiogenic activity of the cells frozen in the absence of cryoprotectants was comparable to that observed in the presence of DMSO or trehalose. PMID:27157752

  7. Rosiglitazone induces the unfolded protein response, but has no significant effect on cell viability, in monocytic and vascular smooth muscle cells

    SciTech Connect

    Caddy, J.; Isa, S.; Mainwaring, L.S.; Adam, E.; Roberts, A.; Lang, D.; Morris, R.H.K.; Thomas, A.W.; Webb, R.

    2010-10-01

    Research highlights: {yields} Rosiglitazone rapidly (30 min) inhibited microsomal Ca{sup 2+}ATPase activity (IC{sub 50} {approx}2 {mu}M). {yields} After 4 h rosiglitazone exposure, the UPR transcription factor XBP-1 was activated. {yields} Within 24-72 h, UPR target genes were upregulated, enhancing ER Ca{sup 2+} sequestration. {yields} Replenishment of ER Ca{sup 2+} stores appeared to restore normal cell physiology. {yields} Monocyte/VSMC viability was not decreased during 2 weeks' rosiglitazone treatment. -- Abstract: Given the safety concerns expressed over negative cardiovascular outcomes resulting from the clinical use of rosiglitazone, and the view that rosiglitazone exerts PPAR{gamma}-independent effects alongside its insulin-sensitising PPAR{gamma}-dependent effects, we hypothesised that rosiglitazone may trigger Unfolded Protein Responses (UPRs) due to disruptions in [Ca{sup 2+}]{sub i} homeostasis within two cardiovascular cell types: monocytic (MM6) and vascular smooth muscle (A7r5) cells. In microsomal samples derived from both cell types, pre-incubation with rosiglitazone rapidly (30 min) brought about concentration-dependent PPAR{gamma}-independent inhibition of Ca{sup 2+}ATPase activity (IC{sub 50} {approx}2 {mu}M). Fluo-3 fluorimetric data demonstrated in intact cells that 1 h treatment with 1 or 10 {mu}M rosiglitazone caused Ca{sup 2+} ions to leak into the cytoplasm. Gene expression analysis showed that within 4 h of rosiglitazone exposure, the UPR transcription factor XBP-1 was activated (likely due to corresponding ER Ca{sup 2+} depletion), and the UPR target genes BiP and SERCA2b were subsequently upregulated within 24-72 h. After 72 h 1 or 10 {mu}M rosiglitazone treatment, microsomal Ca{sup 2+}ATPase activity increased to >2-fold of that seen in control microsomes, while [Ca{sup 2+}]{sub i} returned to basal, indicating that UPR-triggered SERCA2b upregulation was responsible for enhanced enzymatic Ca{sup 2+} sequestration within the ER. This

  8. Prioritizing conservation activities using reserve site selection methods and population viability analysis.

    PubMed

    Newbold, Stephen C; Siikamäki, Juha

    2009-10-01

    In recent years a large literature on reserve site selection (RSS) has developed at the interface between ecology, operations research, and environmental economics. Reserve site selection models use numerical optimization techniques to select sites for a network of nature reserves for protecting biodiversity. In this paper, we develop a population viability analysis (PVA) model for salmon and incorporate it into an RSS framework for prioritizing conservation activities in upstream watersheds. We use spawner return data for three closely related salmon stocks in the upper Columbia River basin and estimates of the economic costs of watershed protection from NOAA to illustrate the framework. We compare the relative cost-effectiveness of five alternative watershed prioritization methods, based on various combinations of biological and economic information. Prioritization based on biological benefit-economic cost comparisons and accounting for spatial interdependencies among watersheds substantially outperforms other more heuristic methods. When using this best-performing prioritization method, spending 10% of the cost of protecting all upstream watersheds yields 79% of the biological benefits (increase in stock persistence) from protecting all watersheds, compared to between 20% and 64% for the alternative methods. We also find that prioritization based on either costs or benefits alone can lead to severe reductions in cost-effectiveness. PMID:19831069

  9. 3D printing of HEK 293FT cell-laden hydrogel into macroporous constructs with high cell viability and normal biological functions.

    PubMed

    Ouyang, Liliang; Yao, Rui; Chen, Xi; Na, Jie; Sun, Wei

    2015-01-01

    3D printing has evolved into a versatile technology for fabricating tissue-engineered constructs with spatially controlled cells and biomaterial distribution to allow biomimicking of in vivo tissues. In this paper, we reported a novel study of 3D printing of cell lines derived from human embryonic kidney tissue into a macroporous tissue-like construct. Nozzle temperature, chamber temperature and the composition of the matrix material were studied to achieve high cell viability (>90%) after 3D printing and construct formation. Long-term construct stability with a clear grid structure up to 30 days was observed. Cells continued to grow as cellular spheroids with strong cell-cell interactions. Two transfected cell lines of HEK 293FT were also 3D printed and showed normal biological functions, i.e. protein synthesis and gene activation in responding to small molecule stimulus. With further refinement, this 3D cell printing technology may lead to a practical fabrication of functional embryonic tissues in vitro. PMID:25691496

  10. 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.

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

    PubMed

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

    2010-01-01

    Ethylene glycol ethers (EGEs) are a class of chemicals used extensively in the manufacture of a wide range of domestic and industrial products, which may result in human exposure and toxicity. Hematologic and reproductive toxicity of EGEs are well known whereas their action on neuronal cell viability has not been studied so far. In the present study, we investigated the effects of some EGEs on cell viability and on the hydrogen peroxide-induced damage in the human neuroblastoma (SH-SY5Y) cells. It has been found that 2-phenoxyethanol in a concentration-dependent manner (5-25 mM, 24 h) increased the basal and H(2)O(2)-induced lactate dehydrogenase (LDH) release and 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT) reduction. 2-Butoxyethanol given alone did not affect LDH release and MTT reduction but concentration-dependently enhanced the cytotoxic effect of H(2)O(2). 2-Isopropoxyethanol significantly and concentration-dependently (1-25 mM) increased the basal LDH release and attenuated MTT reduction, but did not potentiate the cytotoxic effect of H(2)O(2). Contrary to this, 2-methoxyethanol did not show a cytotoxic effect while 2-ethoxyethanol at high concentrations intensified the hydrogen peroxide action. This study demonstrated that among the EGEs studied, 2-phenoxyethanol showed the most consistent cytotoxic effect on neurons in in vitro conditions and enhanced the hydrogen peroxide action. 2-Isopropoxyethanol had also a potent cytotoxic effect, but it did not enhance the hydrogen peroxide action, whereas 2-butoxyethanol only potentiated cytotoxic effect of H(2)O(2). It is concluded that the results of the present study should be confirmed in in vivo conditions and that some EGEs, especially 2-phenoxyethanol, 2-butoxyethanol and 2-isopropoxyethanol, may be responsible for initiation or exacerbation of neuronal cell damage. PMID:21273685

  12. 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

  13. Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability.

    PubMed

    Munkley, Jennifer; Vodak, Daniel; Livermore, Karen E; James, Katherine; Wilson, Brian T; Knight, Bridget; Mccullagh, Paul; Mcgrath, John; Crundwell, Malcolm; Harries, Lorna W; Leung, Hing Y; Robson, Craig N; Mills, Ian G; Rajan, Prabhakar; Elliott, David J

    2016-06-01

    Steroid androgen hormones play a key role in the progression and treatment of prostate cancer, with androgen deprivation therapy being the first-line treatment used to control cancer growth. Here we apply a novel search strategy to identify androgen-regulated cellular pathways that may be clinically important in prostate cancer. Using RNASeq data, we searched for genes that showed reciprocal changes in expression in response to acute androgen stimulation in culture, and androgen deprivation in patients with prostate cancer. Amongst 700 genes displaying reciprocal expression patterns we observed a significant enrichment in the cellular process glycosylation. Of 31 reciprocally-regulated glycosylation enzymes, a set of 8 (GALNT7, ST6GalNAc1, GCNT1, UAP1, PGM3, CSGALNACT1, ST6GAL1 and EDEM3) were significantly up-regulated in clinical prostate carcinoma. Androgen exposure stimulated synthesis of glycan structures downstream of this core set of regulated enzymes including sialyl-Tn (sTn), sialyl Lewis(X) (SLe(X)), O-GlcNAc and chondroitin sulphate, suggesting androgen regulation of the core set of enzymes controls key steps in glycan synthesis. Expression of each of these enzymes also contributed to prostate cancer cell viability. This study identifies glycosylation as a global target for androgen control, and suggests loss of specific glycosylation enzymes might contribute to tumour regression following androgen depletion therapy. PMID:27428423

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

    PubMed

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

    2016-01-01

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

  15. 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

  16. 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.

  17. Viability and Recovery of Peripheral Blood Mononuclear Cells Cryopreserved for up to 12 Years in a Multicenter Study

    PubMed Central

    Kleeberger, Cynthia A.; Lyles, Robert H.; Margolick, Joseph B.; Rinaldo, Charles R.; Phair, John P.; Giorgi, Janis V.

    1999-01-01

    The Multicenter AIDS Cohort Study (MACS), an ongoing prospective study of the natural history of human immunodeficiency virus (HIV), has stored biologic specimens, including peripheral blood mononuclear cells (PBMC), from 5,622 participants for up to 12 years. The purpose of the present analysis was to evaluate the quality of the PBMC in the MACS repository in order to test the validity and feasibility of nested retrospective studies and to guide the planning of future repositories. PBMC were collected from MACS participants at four centers at 6-month intervals from 1984 to 1995, cryopreserved, and transported to a central repository for storage. A total of 596 of these specimens were subsequently tested for viability and used to evaluate cell function, to conduct immunophenotype analysis, or to isolate HIV. Simple linear regression models were applied to evaluate trends in recovery and viability over time and by center. Results indicated that from a nominal 107 cells cryopreserved per vial at all four centers, the median number of viable cells recovered was at least 5 × 106 (50% of the number stored) and the median viability was at least 90%. Results suggested that cryopreserved cells can be stored for at least 12 years with no general tendency toward cell loss over time. Furthermore, there were no statistically significant changes in the percent cell viability according to the length of time frozen, regardless of HIV serostatus or the level of CD4+ lymphocytes. Storing 107 PBMC per vial yields sufficient viable cells for phenotypic and/or functional analysis. Results from the MACS provide the basis for the planning of future repositories for use by investigators with similar research goals. PMID:9874657

  18. Preparation, Physicochemical Characterization, and Cell Viability Evaluation of Long-Circulating and pH-Sensitive Liposomes Containing Ursolic Acid

    PubMed Central

    Caldeira de Araújo Lopes, Sávia; Vinícius Melo Novais, Marcus; Salviano Teixeira, Cláudia; Honorato-Sampaio, Kinulpe; Tadeu Pereira, Márcio; Ferreira, Lucas Antônio Miranda; Braga, Fernão Castro; Cristina Oliveira, Mônica

    2013-01-01

    Cancer is one of the leading causes of death worldwide. Although several drugs are used clinically, some tumors either do not respond or are resistant to the existing pharmacotherapy, thus justifying the search for new drugs. Ursolic acid (UA) is a triterpene found in different plant species that has been shown to possess significant antitumor activity. However, UA presents a low solubility in aqueous medium, which presents a barrier to its biological applications. In this context, the use of liposomes presents a promising strategy to deliver UA and allow for its intravenous administration. In this work, long-circulating and pH-sensitive liposomes containing UA (SpHL-UA) were developed, and their chemical and physicochemical properties were evaluated. SpHL-UA presented adequate properties, including a mean diameter of 191.1 ± 6.4 nm, a zeta potential of 1.2 ± 1.4 mV, and a UA entrapment of 0.77 ± 0.01 mg/mL. Moreover, this formulation showed a good stability after having been stored for 2 months at 4°C. The viability studies on breast (MDA-MB-231) and prostate (LNCaP) cancer cell lines demonstrated that SpHL-UA treatment significantly inhibited cancer cell proliferation. Therefore, the results of the present work suggest the applicability of SpHL-UA as a new and promising anticancer formulation. PMID:23984367

  19. Comparison of Cell Viability and Embryoid Body Size of Two Embryonic Stem Cell Lines After Different Exposure Times to Bone Morphogenetic Protein 4

    PubMed Central

    Zarei Fard, Nehleh; Talaei-Khozani, Tahereh; Bahmanpour, Soghra; Esmaeilpour, Tahereh

    2015-01-01

    Background Activation of bone morphogenetic protein 4 (BMP4) signaling pathway in embryonic stem (ES) cells plays an important role in controlling cell proliferation, differentiation, and apoptosis. Adverse effects of BMP4 occur in a time dependent manner; however, little is known about the effect of different time exposure of this growth factor on cell number in culture media. In this study, we investigated the role of two different exposure times to BMP4 in cell viability, embryoid body (EB), size, and cavitation of ES cells. Methods Embryonic stem cells (R1 and B1 lines) were released from the feeder cell layers and were cultured using EBs protocol by using the hanging drop method and monolayer culture system. The cells were cultured for 5 days with 100 ng/mL BMP4 from the beginning (++BMP4) or after 48 h (+BMP4) of culture and their cell number were counted by trypan blue staining. The data were analyzed using non-parametric two-tailed Mann-Whitney test. P<0.05 was considered as significant. Results In EB culture protocol, cell number significantly decreased in +BMP4 culture condition with greater cavity size compared to the ++BMP4 condition at day 5 (P=0.009). In contrast, in monolayer culture system, there was no significant difference in the cell number between all groups (P=0.91). Conclusion The results suggest that short-term exposure of BMP4 is required to promote cavitation in EBs according to lower cell number in +BMP4 condition. Different cell lines showed different behavior in cavitation formation. PMID:25821290

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2015-05-01

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

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

    SciTech Connect

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

    1993-12-01

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

  3. Hippuristanol reduces the viability of primary effusion lymphoma cells both in vitro and in vivo.

    PubMed

    Ishikawa, Chie; Tanaka, Junichi; Katano, Harutaka; Senba, Masachika; Mori, Naoki

    2013-09-01

    Primary effusion lymphoma (PEL) caused by Kaposi's sarcoma-associated herpesvirus (also known as human herpesvirus-8) shows serious lymphomatous effusion in body cavities. PEL is difficult to treat and there is no standard treatment strategy. Hippuristanol is extracted from Okinawan coral Isis hippuris, and inhibits translational initiation by blocking eukaryotic initiation factor 4A, an ATP-dependent RNA helicase, binding to mRNA. Recently, there has been much interest in targeting translation initiation as an anticancer therapy. Here, we show that treatment of PEL cell lines with hippuristanol resulted in cell cycle arrest at G1 phase, and induced caspases activation and apoptosis. Hippuristanol also reduced the expression of cyclin D2, CDK2, CDK4, CDK6 and prosurvival XIAP and Mcl-1 proteins. Activation of activator protein-1, signal transducers and activators of transcription protein 3 and Akt pathways plays a critical role in the survival and growth of PEL cells. Hippuristanol suppressed the activities of these three pathways by inhibiting the expression of JunB, JunD, c-Fos, signal transducers and activators of transcription protein 3 and Akt proteins. In a xenograft mouse model that showed ascites and diffused organ invasion of PEL cells, treatment with hippuristanol significantly inhibited the growth and invasion of PEL cells compared with untreated mice. The results of the in vitro and in vivo experiments underline the potential usefulness of hippuristanol in the treatment of PEL. PMID:24018901

  4. Ginseng (Panax quinquefolius) and Licorice (Glycyrrhiza uralensis) Root Extract Combinations Increase Hepatocarcinoma Cell (Hep-G2) Viability

    PubMed Central

    Popovich, David G.; Yeo, Shi Yun; Zhang, Wei

    2011-01-01

    The combined cytoactive effects of American ginseng (Panax quinquefolius) and licorice (Glycyrrhiza uralensis) root extracts were investigated in a hepatocarcinoma cell line (Hep-G2). An isobolographic analysis was utilized to express the possibility of synergistic, additive or antagonistic interaction between the two extracts. Both ginseng and licorice roots are widely utilized in traditional Chinese medicine preparations to treat a variety of ailments. However, the effect of the herbs in combination is currently unknown in cultured Hep-G2 cells. Ginseng (GE) and licorice (LE) extracts were both able to reduce cell viability. The LC50 values, after 72 h, were found to be 0.64 ± 0.02 mg/mL (GE) and 0.53 ± 0.02 mg/mL (LE). An isobologram was plotted, which included five theoretical LC50s calculated, based on the fixed fraction method of combination ginseng to licorice extracts to establish a line of additivity. All combinations of GE to LE (1/5, 1/3, 1/2, 2/3, 4/5) produced an effect on Hep-G2 cell viability but they were all found to be antagonistic. The LC50 of fractions 1/3, 1/2, 2/3 were 23%, 21% and 18% above the theoretical LC50. Lactate dehydrogenase release indicated that as the proportion of GE to LE increased beyond 50%, the influence on membrane permeability increased. Cell-cycle analysis showed a slight but significant arrest at the G1 phase of cell cycle for LE. Both GE and LE reduced Hep-G2 viability independently; however, the combinations of both extracts were found to have an antagonistic effect on cell viability and increased cultured Hep-G2 survival. PMID:19617200

  5. Dietary apigenin potentiates the inhibitory effect of interferon-α on cancer cell viability through inhibition of 26S proteasome-mediated interferon receptor degradation

    PubMed Central

    Li, Sheng; Yang, Li-juan; Wang, Ping; He, Yu-jiao; Huang, Jun-mei; Liu, Han-wei; Shen, Xiao-fei; Wang, Fei

    2016-01-01

    Background Type I interferons (IFN-α/β) have broad and potent immunoregulatory and antiproliferative activities. However, it is still known whether the dietary flavonoids exhibit their antiviral and anticancer properties by modulating the function of type I IFNs. Objective This study aimed at determining the role of apigenin, a dietary plant flavonoid abundant in common fruits and vegetables, on the type I IFN-mediated inhibition of cancer cell viability. Design Inhibitory effect of apigenin on human 26S proteasome, a known negative regulator of type I IFN signaling, was evaluated in vitro. Molecular docking was conducted to know the interaction between apigenin and subunits of 26S proteasome. Effects of apigenin on JAK/STAT pathway, 26S proteasome-mediated interferon receptor stability, and cancer cells viability were also investigated. Results Apigenin was identified to be a potent inhibitor of human 26S proteasome in a cell-based assay. Apigenin inhibited the chymotrypsin-like, caspase-like, and trypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Results from computational modeling of the potential interactions of apigenin with the chymotrypsin site (β5 subunit), caspase site (β1 subunit), and trypsin site (β2 subunit) of the proteasome were consistent with the observed proteasome inhibitory activity. Apigenin enhanced the phosphorylation of signal transducer and activator of transcription proteins (STAT1 and STAT2) and promoted the endogenous IFN-α-regulated gene expression. Apigenin inhibited the IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Apigenin also sensitized the inhibitory effect of IFN-α on viability of cervical carcinoma HeLa cells. Conclusion These results suggest that apigenin potentiates the inhibitory effect of IFN-α on cancer cell viability by activating JAK/STAT signaling pathway through inhibition of 26S proteasome

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

    PubMed

    Janakiraman, Narayanan; Johnson, Marimuthu

    2016-06-01

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

  7. Super-competitor status of Drosophila Myc cells requires p53 as a fitness sensor to reprogram metabolism and promote viability

    PubMed Central

    de la Cova, Claire; Senoo-Matsuda, Nanami; Ziosi, Marcello; Wu, D. Christine; Bellosta, Paola; Quinzii, Catarina M.; Johnston, Laura A.

    2014-01-01

    Summary In growing tissues, cell fitness disparities provoke interactions that promote stronger cells at the expense of the weaker in a process called cell competition. The mechanistic definition of cell fitness remains unclear, as does how differences are recognized. In Drosophila cells with extra Myc activity acquire “super-competitor” status upon confrontation with wild-type (WT) cells, prompting the latters’ elimination via apoptosis. Confrontation enhances Myc cell fitness by increasing glycolytic flux and promoting expansion of the population. p53 is induced in these cells and promotes their enhanced metabolism. Whereas p53 loss in noncompeting Myc cells is inconsequential, it impairs metabolism, reduces viability and prevents the killing activity of Myc super-competitor cells. We propose that p53 acts as a general sensor of competitive confrontation to enhance the fitness of “winner” cells. Our findings suggest that the initial confrontation between pre-cancerous and WT cells could enhance cancer cell fitness and promote tumor progression. PMID:24561262

  8. Chidamide, a novel histone deacetylase inhibitor, inhibits the viability of MDS and AML cells by suppressing JAK2/STAT3 signaling

    PubMed Central

    Zhao, Sida; Guo, Juan; Zhao, Youshan; Fei, Chengming; Zheng, Qingqing; Li, Xiao; Chang, Chunkang

    2016-01-01

    Many studies have indicated that histone deacetylase (HDAC) activity is always increased in a lot of human tumors, and inhibition of HDAC activity is a promising new strategy in the treatment of cancers. Chidamide, a novel HDAC inhibitor of the benzamide class, is currently under clinical trials. In this study, we aimed to investigate the antitumor activity of Chidamide on myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) cell lines and explore the possible mechanism. Chidamide exhibited efficient anti-proliferative activity on MDS and AML cells in a time- and dose-dependent manner, accompanied by cell cycle arrest at G0/G1 phase and cell apoptosis. Importantly, Chidamide possessed potent HDAC inhibition property, as evaluated by HDAC activity analysis and acetylation of histone H3 and H4. Moreover, Chidamide significantly increased the expression of Suppressors of cytokine signaling 3 (SOCS3), reduced the expression of Janus activated kinases 2 (JAK2) and Signal transducer and activator of transcription 3 (STAT3), and inhibited STAT3 downstream genes, including c-Myc, Bcl-xL, and Mcl-1, which are involved in cell cycle progression and anti-apoptosis. Therefore, we demonstrate that Chidamide exhibits potent inhibitory effect on cell viability of MDS and AML cells, and the possible mechanism may lie in the downregulation of JAK2/STAT3 signaling through SOCS3 upregulation. Our data provide rationale for clinical investigations of Chidamide in MDS and AML. PMID:27508038

  9. [Effect of preservation conditions on the viability and activity of Streptomyces griseus--producer of the antibiotic grizin].

    PubMed

    Trenina, G A; Lavrova, L N; Iustratova, L S; Kuklin, V V

    1983-03-01

    Strain AURIGenetics- 1552 of Str. griseus producing grisin used as a feed additive was obtained in the course of stepped selection. It is deposited at the Central Collection of Industrial Microorganisms of the USSR and designated as strain S-248. The strain was stored under varying conditions and their effect on its viability, morphological variation and antibiotic potency was studied. Investigation of the strain variation with respect to the cultural and morphological properties and the antibiotic production revealed definite correlation between the morphological properties of the culture and its capacity for the synthesis of the antibiotic. The medium G-1 containing KNO3 as the only source of nitrogen was found to be advantageous for maintaining the strain potency on storage with the method of subcultures or under a layer of mineral oil. It was shown that the strain can be successfully stored by lyophilization. This method in combination with selection of active variants from populations of lyophilized cells provided not only maintenance of the culture potency at the required level but also its increase. PMID:6407387

  10. 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.

  11. Detection of GNAQ mutations and reduction of cell viability in uveal melanoma cells with functionalized gold nanoparticles

    PubMed Central

    Latorre, Alfonso; Crosby, Michelle B.; Celli, Anna; Latorre, Ana; Vujic, Igor; Sanlorenzo, Martina; Green, Gary A.; Weier, Jingly; Zekhtser, Mitchell; Ma, Jeffrey; Monico, Gabriela; Char, Devron H.; Jusufbegovic, Denis; Rappersberger, Klemens; Somoza, Álvaro; Ortiz-Urda, Susana

    2015-01-01

    Background Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Early treatment may improve any chances of preventing metastatic disease, but diagnosis of small UM is challenging. Up to 95 % of all UMs carry somatic mutations in the G-coupled proteins GNAQ and GNA11 promoting anchorage-independent growth and proliferation. About 50%of UMs are fatal. Once metastatic, patients have limited options for successful therapy. Methods We have developed functionalized gold nanoparticles (AuNPs) to visualize transcripts of mutant GNAQ mRNA in living cells. In addition to their suitability as a specific tool for GNAQ mutation detection, we have developed a novel linker that enables conjugation of siRNAs to AuNPs allowing for greater and more rapid intracellular release of siRNAs compared to previously described approaches. Results Binding of modified AuNPs to matching target mRNA leads to conformational changes, resulting in a detectable fluorescent signal that can be used for mutation detection in living cells. Knockdown of GNAQ with siRNA-AuNPs effectively reduced downstream signals and decreased cell viability in GNAQ mutant uveal melanoma cells. Conclusion AuNPs may in future be developed to serve as sensors for mutations of vital importance. The new release system for siRNA-AuNP improves previous systems, which conceivably will be useful for future therapeutic gene regulatory approaches. PMID:25653058

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-07-01

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

  15. Evaluation of cell viability and T2 relaxivity of fluorescein conjugated SPION-PAMAM third generation nanodendrimers for bioimaging.

    PubMed

    Khosroshahi, Mohammad E; Rezvani, Hamideh Alanagh; Keshvari, Hamid; Bonakdar, Shahin; Tajabadi, Maryam

    2016-05-01

    This study has investigated the possibility of using fluorescent dendronized magnetic nanoparticles (FDMNPs) for potential applications in drug delivery and imaging. FDMNPs were first synthesized, characterized and then the effect of Polyamidoamine (PAMAM) dendrimer functionalization and fluorescein isothiocyanate (FITC) conjugation on biocompatibility of superparamagnetic iron oxide nanoparticles (SPIONs) was evaluated. The nanostructures' cytotoxicity tests were performed at different concentrations from 10 to 500μg/mL using MCF-7 and L929 cell lines. IC50 in MTT assay were 139.22 and 201.88μg/mL for DMNP incubated L929 and MCF-7 cell lines respectively, whereas the cell viability for FDMNPs did not decrease to 50%. The results showed that FITC conjugation diminishes the toxicity of dendronized magnetic nanoparticles (DMNPs) mainly due to the reduction of surface charge. DMNP appears to be cytotoxic at the concentration levels being used for both cell lines. On the contrary, FDMNPs showed more biocompatibility and cell viability of MCF-7 and L929 cell lines at all concentrations. The fluorescence microscopy of FDMNPs incubated with MCF-7 cells showed a successful localization of cells indicating their ability for applications such as a magnetic fluorescent probe in cell studies and imaging purposes. T2 relaxivity measurements demonstrated the applicability of the synthesized nanostructures as the contrast agents in tissue differential assessment by altering their relaxation times. In our case, the r2 relaxivity of FDMNPs was measured as 103.67mM(-1)S(-1). PMID:26952457

  16. Effects of different detachment procedures on viability, nitroxide reduction kinetics and plasma membrane heterogeneity of V-79 cells.

    PubMed

    Batista, Urska; Garvas, Maja; Nemec, Marjana; Schara, Milan; Veranic, Peter; Koklic, Tilen

    2010-06-01

    Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V-79 cells in the plateau phase of growth (arrested in G1). We have measured cell viability by a dye-exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin-probe MeFASL(10,3) (5-doxylpalmitoyl-methylester), which partitions mainly in cell membranes and the hydrophilic spin-probe TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin-probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin-treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V-79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the

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

    PubMed

    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

  18. 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

  19. 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.

  20. 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. PMID:23936178

  1. 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

  2. Rhomboid Enhancer Activity Defines a Subset of Drosophila Neural Precursors Required for Proper Feeding, Growth and Viability

    PubMed Central

    Gresser, Amy L.; Gutzwiller, Lisa M.; Gauck, Mackenzie K.; Hartenstein, Volker; Cook, Tiffany A.; Gebelein, Brian

    2015-01-01

    Organismal growth regulation requires the interaction of multiple metabolic, hormonal and neuronal pathways. While the molecular basis for many of these are well characterized, less is known about the developmental origins of growth regulatory structures and the mechanisms governing control of feeding and satiety. For these reasons, new tools and approaches are needed to link the specification and maturation of discrete cell populations with their subsequent regulatory roles. In this study, we characterize a rhomboid enhancer element that selectively labels four Drosophila embryonic neural precursors. These precursors give rise to the hypopharyngeal sensory organ of the peripheral nervous system and a subset of neurons in the deutocerebral region of the embryonic central nervous system. Post embryogenesis, the rhomboid enhancer is active in a subset of cells within the larval pharyngeal epithelium. Enhancer-targeted toxin expression alters the morphology of the sense organ and results in impaired larval growth, developmental delay, defective anterior spiracle eversion and lethality. Limiting the duration of toxin expression reveals differences in the critical periods for these effects. Embryonic expression causes developmental defects and partially penetrant pre-pupal lethality. Survivors of embryonic expression, however, ultimately become viable adults. In contrast, post-embryonic toxin expression results in fully penetrant lethality. To better define the larval growth defect, we used a variety of assays to demonstrate that toxin-targeted larvae are capable of locating, ingesting and clearing food and they exhibit normal food search behaviors. Strikingly, however, following food exposure these larvae show a rapid decrease in consumption suggesting a satiety-like phenomenon that correlates with the period of impaired larval growth. Together, these data suggest a critical role for these enhancer-defined lineages in regulating feeding, growth and viability. PMID

  3. Rhomboid Enhancer Activity Defines a Subset of Drosophila Neural Precursors Required for Proper Feeding, Growth and Viability.

    PubMed

    Gresser, Amy L; Gutzwiller, Lisa M; Gauck, Mackenzie K; Hartenstein, Volker; Cook, Tiffany A; Gebelein, Brian

    2015-01-01

    Organismal growth regulation requires the interaction of multiple metabolic, hormonal and neuronal pathways. While the molecular basis for many of these are well characterized, less is known about the developmental origins of growth regulatory structures and the mechanisms governing control of feeding and satiety. For these reasons, new tools and approaches are needed to link the specification and maturation of discrete cell populations with their subsequent regulatory roles. In this study, we characterize a rhomboid enhancer element that selectively labels four Drosophila embryonic neural precursors. These precursors give rise to the hypopharyngeal sensory organ of the peripheral nervous system and a subset of neurons in the deutocerebral region of the embryonic central nervous system. Post embryogenesis, the rhomboid enhancer is active in a subset of cells within the larval pharyngeal epithelium. Enhancer-targeted toxin expression alters the morphology of the sense organ and results in impaired larval growth, developmental delay, defective anterior spiracle eversion and lethality. Limiting the duration of toxin expression reveals differences in the critical periods for these effects. Embryonic expression causes developmental defects and partially penetrant pre-pupal lethality. Survivors of embryonic expression, however, ultimately become viable adults. In contrast, post-embryonic toxin expression results in fully penetrant lethality. To better define the larval growth defect, we used a variety of assays to demonstrate that toxin-targeted larvae are capable of locating, ingesting and clearing food and they exhibit normal food search behaviors. Strikingly, however, following food exposure these larvae show a rapid decrease in consumption suggesting a satiety-like phenomenon that correlates with the period of impaired larval growth. Together, these data suggest a critical role for these enhancer-defined lineages in regulating feeding, growth and viability. PMID

  4. Improving Viability and Transfection Efficiency with Human Umbilical Cord Wharton's Jelly Cells Through Use of a ROCK Inhibitor

    PubMed Central

    Mellott, Adam J.; Godsey, Megan E.; Shinogle, Heather E.; Moore, David S.; Forrest, M. Laird

    2014-01-01

    Abstract Differentiating stem cells using gene delivery is a key strategy in tissue engineering and regenerative medicine applications. Nonviral gene delivery bypasses several safety concerns associated with viral gene delivery; however, leading nonviral techniques, such as electroporation, subject cells to high stress and can result in poor cell viabilities. Inhibition of Rho-associated coiled-coil kinase (ROCK) has been shown to mitigate apoptotic mechanisms associated with detachment and freezing of induced pluripotent stem cells and embryonic stem cells; however, inhibiting ROCK in mesenchymal stromal cells (MSCs) for improving gene delivery applications has not been reported previously. In this study, we hypothesized that ROCK Inhibitor (RI) would improve cell viability and gene expression in primary human umbilical cord mesenchymal stromal cells (hUCMSCs) when transfected via Nucleofection™. As hypothesized, the pre-treatment and post-treatment of hUCMSCs transfected via nucleofection with Y-27632-RI significantly improved survival rates of hUCMSCs and gene expression as measured by green fluorescent protein intensity. This study provides the first comparative look at the effect of Y-27632-RI on hUCMSCs that underwent transfection via nucleofection and shows that using Y-27632-RI in concert with nucleofection could greatly enhance the utility of differentiating and reprogramming hUCMSCs for tissue engineering applications. PMID:24552552

  5. Improving viability and transfection efficiency with human umbilical cord wharton's jelly cells through use of a ROCK inhibitor.

    PubMed

    Mellott, Adam J; Godsey, Megan E; Shinogle, Heather E; Moore, David S; Forrest, M Laird; Detamore, Michael S

    2014-04-01

    Differentiating stem cells using gene delivery is a key strategy in tissue engineering and regenerative medicine applications. Nonviral gene delivery bypasses several safety concerns associated with viral gene delivery; however, leading nonviral techniques, such as electroporation, subject cells to high stress and can result in poor cell viabilities. Inhibition of Rho-associated coiled-coil kinase (ROCK) has been shown to mitigate apoptotic mechanisms associated with detachment and freezing of induced pluripotent stem cells and embryonic stem cells; however, inhibiting ROCK in mesenchymal stromal cells (MSCs) for improving gene delivery applications has not been reported previously. In this study, we hypothesized that ROCK Inhibitor (RI) would improve cell viability and gene expression in primary human umbilical cord mesenchymal stromal cells (hUCMSCs) when transfected via Nucleofection™. As hypothesized, the pre-treatment and post-treatment of hUCMSCs transfected via nucleofection with Y-27632-RI significantly improved survival rates of hUCMSCs and gene expression as measured by green fluorescent protein intensity. This study provides the first comparative look at the effect of Y-27632-RI on hUCMSCs that underwent transfection via nucleofection and shows that using Y-27632-RI in concert with nucleofection could greatly enhance the utility of differentiating and reprogramming hUCMSCs for tissue engineering applications. PMID:24552552

  6. Development of a new oxygen consumption rate assay in cultures of Acanthamoeba (Protozoa: Lobosea) and its application to evaluate viability and amoebicidal activity in vitro.

    PubMed

    Heredero-Bermejo, I; Criado-Fornelio, A; Soliveri, J; Díaz-Martín, J A; Matilla-Fuentes, J; Sánchez-Arias, J A; Copa-Patiño, J L; Pérez-Serrano, J

    2015-08-01

    A new fluorometric method has been developed for measuring the oxygen consumption rate (OCR) of Acanthamoeba cultures in microplates and for screening molecules with amoebicidal activity against this microorganism. The use of a biofunctional matrix (containing an oxygen-sensitive fluorogenic probe) attached to the microplate wells allowed continuous measurement of OCR in the medium, hence assessment of amoebic growth. The new OCR method applied to cell viability yielded a linear relationship and monitoring was much quicker than with indirect viability assays previously used. In addition, two drugs were tested in a cytotoxicity assay monitored by the new OCR viability test. With this procedure, the standard amoebicidal drug chlorhexidine digluconate showed an IC50 of 3.53 + 1.3 mg/l against Acanthamoeba polyphaga and 3.19 + 1.2 mg/l against Acanthamoeba castellanii, whereas a cationic dendrimer [G1Si(NMe3+)4] showed an IC50 of 6.42 + 1.3 mg/l against A. polyphaga. These data agree with previous studies conducted in our laboratory. Therefore, the new OCR method has proven powerful and quick for amoebicidal drug screening and is likely to be applied in biochemical studies concerning protozoa respiration and metabolism. PMID:25956947

  7. Increased viability of odontoblast-like cells subjected to low-level laser irradiation

    NASA Astrophysics Data System (ADS)

    Oliveira, C. F.; Basso, F. G.; Lins, E. C.; Kurachi, C.; Hebling, J.; Bagnato, V. S.; de Souza Costa, C. A.

    2010-07-01

    Studies have shown that the increase of cell metabolism depends on the low level laser therapy (LLLT) parameters used to irradiate the cells. However, the optimal laser dose to up-regulate pulp cell activity remains unknown. Consequently, the aim of this study was to evaluate the metabolic response of odontoblast-like cells (MDPC-23) exposed to different LLLT doses. Cells at 20000 cells/cm2 were seeded in 24-well plates using plain culture medium (DMEM) and were incubated in a humidified incubator with 5% CO2 at 37°C. After 24 h, the culture medium was replaced by fresh DMEM supplemented with 5% (stress by nutritional deficit) or 10% fetal bovine serum (FBS). The cells were exposed to different laser doses from a near infrared diode laser prototype designed to provide a uniform irradiation of the wells. The experimental groups were: G1: 1.5 J/cm2 + 5% FBS; G2: 1.5 J/cm2 + 10% FBS; G3: 5 J/cm2 + 5% FBS; G4: 5 J/cm2 + 10% FBS; G5: 19 J/cm2 + 5% FBS; G6: 19 J/cm2 + 10% FBS. LLLT was performed in 3 consecutive irradiation cycles with a 24-hour interval. Non-irradiated cells cultured in DMEM supplemented with either 5 or 10% FBS served as control groups. The analysis of the metabolic response was performed by the MTT assay 3 h after the last irradiation. G1 presented an increase in SDH enzyme activity and differed significantly (Mann-Whitney test, p < 0.05) from the other groups. Analysis by scanning electron microscopy showed normal cell morphology in all groups. Under the tested conditions, LLLT stimulated the metabolic activity of MDPC-23 cultured in DMEM supplemented with 5% FBS and exposed to a laser dose of 1.5 J/cm2. These findings are relevant for further studies on the action of near infrared lasers on cells with odontoblast phenotype.

  8. Neutrophil apoptosis: impact of granulocyte macrophage colony stimulating factor on cell survival and viability in chronic kidney disease and hemodialysis patients

    PubMed Central

    Zahran, Nariman; Sayed, Azza; William, Iman; Sabry, Omar; Rafaat, Manar

    2013-01-01

    Introduction Altered neutrophil apoptosis might be responsible for recurrent bacterial infections encountered in hemodialysis (HD) and chronic kidney disease (CKD) patients. This work was designed to assess the neutrophil apoptotic activity and the impact of implementation of granulocyte macrophage colony stimulating factor (GM-CSF), as a survival factor, on neutrophil apoptosis among these patients. Material and methods Twenty-five patients on regular HD along with 34 CKD patients on conservative treatment, as well as 15 healthy controls, were investigated for apoptotic rate via assessment of neutrophil expression of Annexin-V by flow cytometry, before and after 20 h culture in absence and presence of GM-CSF. Neutrophil viability was determined using light microscopy. The preservation of neutrophil activation in these patients was analyzed by flow cytometric CD18 neutrophil expression. Chronic inflammatory state was evaluated by estimating C-reactive protein (CRP) and soluble intercellular adhesion molecule-1 (sICAM-1). Obtained data were statistically analyzed. Results Compared to controls, both HD and CKD groups had a significant increase of Annexin-V and CD18 expression and significant decrease in neutrophil viability. Culture of their neutrophils with GM-CSF showed significant decrease of apoptosis accompanied by improvement of neutrophil viability compared to their cultured cells without GM-CSF. These patients also showed significant elevation of CRP and sICAM-1. Conclusions Granulocyte macrophage colony stimulating factor demonstrated an evident impact on improving in vitro neutrophil survival and viability in HD and CKD patients. Therefore, this may represent promising preventive and/or therapeutic strategies against infection frequently observed in these patients and causing morbidity and mortality. PMID:24482640

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

    PubMed

    Koo, YoungWon; Kim, GeunHyung

    2016-06-01

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

  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. PMID:25923294

  11. In vitro morphology, viability and cytokine secretion of uterine telocyte-activated mouse peritoneal macrophages.

    PubMed

    Chi, Chi; Jiang, Xiao-Juan; Su, Lei; Shen, Zong-Ji; Yang, Xiao-Jun

    2015-12-01

    Telocytes (TCs), a distinct interstitial cell population, have been identified in the uterus, oviduct and placenta, with multiple proposed potential biological functions. Their unique structure allows them to form intercellular junctions with various immunocytes, both in normal and diseased tissues, suggesting a potential functional relationship with the local immune response. It has been hypothesized that through direct heterocellular junctions or indirect paracrine effects, TCs influence the activity of local immunocytes that are involved in the inflammatory process and in immune-mediated reproductive abnormalities. However, no reliable cytological evidence for this hypothesis is currently available. In this study, we cultured primary murine uterine TCs and collected TC conditioned media (TCM). Mouse peritoneal macrophages (pMACs) were co-cultured for 48 hrs with TCM or with DMEM/F12 or lipopolysaccharide (LPS) as negative and positive controls, respectively. Normal uterine TCs with a typical structure and a CD-34-positive/vimentin-positive/c-kit-negative immunophenotype were observed during culture. Morphologically, TCM-treated pMACs displayed an obvious activation/immunoresponse, in contrast to over-stimulation and cell death after LPS treatment and no sign of activation in the presence of DMEM/F12. Accordingly, a cell counting kit 8 (CCK-8) assay indicated significant activation of pMACs by TCM and LPS compared to DMEM/F12, thus supporting the marked morphological differences among these groups of cells. Furthermore, within a panel of macrophage-derived cytokines/enzymes, interleukin-6 (IL-6) and inducible nitric oxide synthase were significantly elevated in TCM-treated pMACs; tumour necrosis factor α, IL1-R1, and IL-10 were slightly, but significantly, up-regulated; and no changes were observed for transforming growth factor-β1, IL-1β, IL-23α and IL-18. Our results indicate that TCs are not simply innocent bystanders but are rather functional players in

  12. 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

  13. Slashing the timelines: Opting to generate high-titer clonal lines faster via viability-based single cell sorting.

    PubMed

    Misaghi, Shahram; Shaw, David; Louie, Salina; Nava, Adrian; Simmons, Laura; Snedecor, Brad; Poon, Chungkee; Paw, Jonathan S; Gilmour-Appling, Laurie; Cupp, James E

    2016-01-01

    Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 - 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry-based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines. PMID:26587808

  14. Membrane-based electrochemical nanobiosensor for Escherichia coli detection and analysis of cells viability.

    PubMed

    Cheng, Ming Soon; Lau, Suk Hiang; Chow, Vincent T; Toh, Chee-Seng

    2011-08-01

    A sensitive and selective membrane-based electrochemical nanobiosensor is developed for specific quantitative label-free detection of Escherichia coli (E. coli) cells and analysis of viable but nonculturable (VBNC) E. coli cells which remain mostly undetected using current methods. The sensing mechanism relies on the blocking of nanochannels of a nanoporous alumina-membrane modified electrode, upon the formation of immune complexes at the nanoporous membrane. The resulting obstacle to diffusive mass transfer of a redox probe in the analysis solution to the underlying platinum electrode reduces the Faradaic signal response of the biosensor, measured using cyclic voltammetry. Antibody loading under conditions of varying antibody concentrations and pHs are optimized. The biosensor gives a low detection limit of 22 cfu mL(-1) (R(2) = 0.999) over a wide linear working range of 10 to 10(6) cfu mL(-1). It is specific toward E. coli with minimal cross-reactivity to two other pathogenic bacteria (commonly found in waters). Relative standard deviation (RSD) for triplicate measurements of 2.5% indicates reasonably useful level of reproducibility. Differentiation of live, VBNC, and dead cells are carried out after the cell capture and quantitation step, by simple monitoring of the cells' enzyme activity using the same redox probe in the analysis solution, in the presence of glucose. PMID:21688778

  15. Evaluation of Osteoblast-Like Cell Viability and Differentiation on the Gly-Arg-Gly-Asp-Ser Peptide Immobilized Titanium Dioxide Nanotube via Chemical Grafting.

    PubMed

    Kim, Ga-Hyun; Kim, Il-Shin; Park, Sang-Won; Lee, Kwangmin; Yun, Kwi-Dug; Kim, Hyun-Seung; Oh, Gye-Jeong; Ji, Min-Kyung; Lim, Hyun-Pil

    2016-02-01

    This study examined the effect of the immobilization of the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide on titanium dioxide (TiO2) nanotube via chemical grafting on osteoblast-like cell (MG-63) viability and differentiation. The specimens were divided into two groups; TiO2 nanotubes and GRGDS-immobilized TiO2 nanotubes. The surface characteristics of GRGDS-immobilized TiO2 nanotubes were observed by using X-ray photoelectron spectroscopy (XPS) and a field emission scanning electron microscope (FE-SEM). The morphology of cells on specimens was observed by FE-SEM after 2 hr and 24 hr. The level of cell viability was investigated via a tetrazolium (XTT) assay after 2 and 4 days. Alkaline phosphatase (ALP) activity was evaluated to measure the cell differentiation after 4 and 7 days. The presence of nitrogen up-regulation or C==O carbons con- firmed that TiO2 nanotubes were immobilized with GRGDS peptides. Cell adhesion was enhanced on the GRGDS-immobilized TiO2 nanotubes compared to TiO2 nanotubes. Furthermore, significantly increased cell spreading and proliferation were observed with the cells grown on GRGDS-immobilized TiO2 nanotubes (P < .05). However, there was no significant difference in ALP activity between GRGDS-immobilized TiO2 nanotubes and TiO2 nanotubes. These results suggest that the GRGDS-immobilized TiO2 nanotubes might be effective in improving the osseointegration of dental implants. PMID:27433593

  16. 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

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

    PubMed

    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

  18. EFFECT OF AROCLOR 1254 ON THE TRANSCRIPTION FACTOR CREB AND CELL VIABILITY IN A PRIMARY CULTURE OF IMMATURE CORTICAL CELLS.

    EPA Science Inventory

    Considerable work indicates that elevations in Ca2+ levels and kinase activity are sensitive responses to polychlorinated biphenyls (PCBs), which are developmental neurotoxicants. In cortical cells in vitro the PCB mixture Aroclor 1254 (A1254) induces temporally and mechanistica...

  19. Novel role of KCNQ2/3 channels in regulating neuronal cell viability.

    PubMed

    Zhou, X; Wei, J; Song, M; Francis, K; Yu, S P

    2011-03-01

    Overactivation of certain K(+) channels can mediate excessive K(+) efflux and intracellular K(+) depletion, which are early ionic events in apoptotic cascade. The present investigation examined a possible role of the KCNQ2/3 channel or M-channel (also named Kv7.2/7.3 channels) in the pro-apoptotic process. Whole-cell recordings detected much larger M-currents (212 ± 31 pA or 10.5 ± 1.5 pA/pF) in cultured hippocampal neurons than that in cultured cortical neurons (47 ± 21 pA or 2.4 ± 0.8 pA/pF). KCNQ2/3 channel openers N-ethylmaleimide (NEM) and flupirtine caused dose-dependent K(+) efflux, intracellular K(+) depletion, and cell death in hippocampal cultures, whereas little cell death was induced by NEM in cortical cultures. The NEM-induced cell death was antagonized by co-applied KCNQ channel inhibitor XE991 (10 μM), or by elevated extracellular K(+) concentration. Supporting a mediating role of KCNQ2/3 channels in apoptosis, expression of KCNQ2 or KCNQ2/3 channels in Chinese hamster ovary (CHO) cells initiated caspase-3 activation. Consistently, application of NEM (20 μM, 8 h) in hippocampal cultures similarly caused caspase-3 activation assessed by immunocytochemical staining and western blotting. NEM increased the expression of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), induced mitochondria membrane depolarization, cytochrome c release, formation of apoptosome complex, and apoptosis-inducing factor (AIF) translocation into nuclear. All these events were attenuated by blocking KCNQ2/3 channels. These findings provide novel evidence that KCNQ2/3 channels could be an important regulator in neuronal apoptosis. PMID:20885443

  20. LY303366 exhibits rapid and potent fungicidal activity in flow cytometric assays of yeast viability.

    PubMed

    Green, L J; Marder, P; Mann, L L; Chio, L C; Current, W L

    1999-04-01

    LY303366 is a semisynthetic analog of the antifungal lipopeptide echinocandin B that inhibits (1,3)-beta-D-glucan synthase and exhibits efficacy in animal models of human fungal infections. In this study, we utilized flow cytometric analysis of propidium iodide uptake, single-cell sorting, and standard microbiological plating methods to study the antifungal effect of LY303366 on Saccharomyces cerevisiae and Candida albicans. Our data indicate that an initial 5-min pulse treatment with LY303366 caused yeasts to take up propidium iodide and lose their ability to grow. Amphotericin B and cilofungin required longer exposure periods (30 and 180 min, respectively) and higher concentrations to elicit these fungicidal effects. These two measurements of fungicidal activity by LY303366 were highly correlated (r > 0.99) in concentration response and time course experiments. As further validation, LY303366-treated yeasts that stained with propidium iodide were unable to grow in single-cell-sorted cultures. Our data indicate that LY303366 is potent and rapidly fungicidal for actively growing yeasts. The potency and rapid action of this new fungicidal compound suggest that LY303366 may be useful for antifungal therapy. PMID:10103187

  1. Clumping and Viability of Bone Marrow Derived Mesenchymal Stromal Cells under Different Preparation Procedures: A Flow Cytometry-Based In Vitro Study

    PubMed Central

    Cui, Li-li; Kinnunen, Tuure; Boltze, Johannes; Nystedt, Johanna

    2016-01-01

    Complications of microocclusions have been reported after intra-arterial delivery of mesenchymal stromal cells. Hence, quantification and efficient limitation of cell clumps in suspension before transplantation is important to reduce the risk. We used a flow cytometry-based pulse-width assay to assess the effects of different cell suspension concentrations (0.2–2.0 × 106/mL), storage solutions (complete growth medium, Dulbecco's phosphate-buffered saline, and normal saline), storage time in suspension (0–9 h), and freeze-thawing procedure on the clumping of rat bone marrow derived mesenchymal stromal cells (BMMSCs) and also evaluated cell viability at the same time. Surprisingly, increasing the cell concentration did not result in more cell clumps in vitro. Freshly harvested (fresh) cells in normal saline had significantly fewer cell clumps and also displayed high viability (>90%). A time-dependent reduction in viability was observed for cells in all three storage solutions, without any significant change in the clumping tendency except for cells in medium. Fresh cells were more viable than their frozen-thawed counterparts, and fresh cells in normal saline had fewer cell clumps. In conclusion, cell clumping and viability could be affected by different cell preparation procedures, and quantification of cell clumping can be conducted using the flow cytometry-based pulse-width assay before intra-arterial cell delivery. PMID:27022399

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

    PubMed

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

    2015-08-01

    The objective of the present study was to determine the role of different vehicles in carotenoids delivery and their influence on cell viability, cell cycle progression and induction of apoptosis in HeLa cells. Cells (5 × 10(3)) were treated with different concentrations (25-100 µM) of β-carotene (BC) or lutein (L) or astaxanthin (AST) dissolved in 0.5% of tetrahydrofuran (THF), dimethylsulfoxide (DMSO), and fetal bovine serum (FBS), respectively. The effect of delivery vehicle on carotenoids uptake, cytotoxicity, oxidative status, cell cycle distribution, and apoptosis was examined after 48 h of incubation. The results shown that, cell viability reduced significantly in a dose- and time-dependent manner irrespective of carotenoid delivered in vehicles. Cellular uptake of BC delivered in THF was higher by 49.1, 29.7% and L delivered through THF was higher by 41.7 and 37.5% than DMSO and FBS, respectively. While, AST delivered through DMSO was higher by 36.1 and 43.7% than the THF and FBS, respectively. In case of cells treated either with BC or L delivered through THF and AST in DMSO decreased the glutathione and increased the malondialdehyde levels. The net increase in the G 2/M phase percentage of cell cycle progression was observed in carotenoid-treated cells. The % induction of apoptosis by BC or L delivered with THF and AST in DMSO was higher than other treated groups. In conclusion, choice of suitable vehicle for specific carotenoids delivery is essential that in turn may influence on cell proliferation and cell-based assays. PMID:25998494

  3. Viability and Effects on Bacterial Proteins by Oral Rinses with Hypochlorous Acid as Active Ingredient.

    PubMed

    Castillo, Diana Marcela; Castillo, Yormaris; Delgadillo, Nathaly Andrea; Neuta, Yineth; Jola, Johana; Calderón, Justo Leonardo; Lafaurie, Gloria Inés

    2015-10-01

    This study investigated the effect of hypochlorous acid (HOCl) rinses and chlorhexidine (CHX) on the bacterial viability of S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae and E. cloacae. The percentage of live bacteria was tested by fluorescence method using Live/Dead kit(r) and BacLight (Molecular Probes(r)) and compared between groups by the Kruskal-Wallis and U Mann-Whitney tests with Bonferroni correction (p value<0.012). The effect of HOCl and CHX on total proteins of P. gingivalis and S. mutans was determined by SDS-PAGE. CHX showed a higher efficacy than HOCl against S. mutans, A. israelii, E. corrodens and E. cloacae (p<0.001) while HOCl was more effective than CHX against P. gingivalis, A. actinomycetemcomitans, C. rectus and K. oxytoca (p=0.001). CHX and HOCl had similar efficacy against K. pneumoniae. Proteins of P. gingivalis and S. mutans were affected similarly by HOCl and CHX. HOCl reduced the bacterial viability especially in periodontopathic bacteria, which may support its use in the control of subgingival biofilm in periodontal patients. PMID:26647939

  4. Effects of a discoloration-resistant calcium aluminosilicate cement on the viability and proliferation of undifferentiated human dental pulp stem cells.

    PubMed

    Niu, Li-na; Watson, Devon; Thames, Kyle; Primus, Carolyn M; Bergeron, Brian E; Jiao, Kai; Bortoluzzi, Eduardo A; Cutler, Christopher W; Chen, Ji-hua; Pashley, David H; Tay, Franklin R

    2015-01-01

    Discoloration-resistant calcium aluminosilicate cement has been formulated to overcome the timely problem of tooth discoloration reported in the clinical application of bismuth oxide-containing hydraulic cements. The present study examined the effects of this experimental cement (Quick-Set2) on the viability and proliferation of human dental pulp stem cells (hDPSCs) by comparing the cellular responses with commercially available calcium silicate cement (white mineral trioxide aggregate; WMTA) after different aging periods. Cell viability and proliferation were examined using assays that examined plasma membrane integrity, leakage of cytosolic enzyme, caspase-3 activity for early apoptosis, oxidative stress, mitochondrial metabolic activity and intracellular DNA content. Results of the six assays indicated that both Quick-Set2 and WMTA were initially cytotoxic to hDPSCs after setting for 24 h, with Quick-Set2 being comparatively less cytotoxic than WMTA at this stage. After two aging cycles, the cytotoxicity profiles of the two hydraulic cements were not significantly different and were much less cytotoxic than the positive control (zinc oxide-eugenol cement). Based on these results, it is envisaged that any potential beneficial effect of the discoloration-resistant calcium aluminosilicate cement on osteogenesis by differentiated hDPSCs is more likely to be revealed after outward diffusion and removal of its cytotoxic components. PMID:26617338

  5. Effects of a discoloration-resistant calcium aluminosilicate cement on the viability and proliferation of undifferentiated human dental pulp stem cells

    PubMed Central

    Niu, Li-na; Watson, Devon; Thames, Kyle; Primus, Carolyn M.; Bergeron, Brian E.; Jiao, Kai; Bortoluzzi, Eduardo A.; Cutler, Christopher W.; Chen, Ji-hua; Pashley, David H.; Tay, Franklin R.

    2015-01-01

    Discoloration-resistant calcium aluminosilicate cement has been formulated to overcome the timely problem of tooth discoloration reported in the clinical application of bismuth oxide-containing hydraulic cements. The present study examined the effects of this experimental cement (Quick-Set2) on the viability and proliferation of human dental pulp stem cells (hDPSCs) by comparing the cellular responses with commercially available calcium silicate cement (white mineral trioxide aggregate; WMTA) after different aging periods. Cell viability and proliferation were examined using assays that examined plasma membrane integrity, leakage of cytosolic enzyme, caspase-3 activity for early apoptosis, oxidative stress, mitochondrial metabolic activity and intracellular DNA content. Results of the six assays indicated that both Quick-Set2 and WMTA were initially cytotoxic to hDPSCs after setting for 24 h, with Quick-Set2 being comparatively less cytotoxic than WMTA at this stage. After two aging cycles, the cytotoxicity profiles of the two hydraulic cements were not significantly different and were much less cytotoxic than the positive control (zinc oxide–eugenol cement). Based on these results, it is envisaged that any potential beneficial effect of the discoloration-resistant calcium aluminosilicate cement on osteogenesis by differentiated hDPSCs is more likely to be revealed after outward diffusion and removal of its cytotoxic components. PMID:26617338

  6. Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

    PubMed Central

    Schilz, Jodi R.; Reddy, K. J.; Nair, Sreejayan; Johnson, Thomas E.; Tjalkens, Ronald B.; Krueger, Kem P.; Clark, Suzanne

    2015-01-01

    In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic however; this method could have a broader use assessing CuO-NP treatment in more neutral waters. PMID:26132311

  7. Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability.

    PubMed

    Schilz, Jodi R; Reddy, K J; Nair, Sreejayan; Johnson, Thomas E; Tjalkens, Ronald B; Krueger, Kem P; Clark, Suzanne

    2015-01-01

    In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic however; this method could have a broader use assessing CuO-NP treatment in more neutral waters. PMID:26132311

  8. Differential eosinophil and mast cell regulation: mast cell viability and accumulation in inflammatory tissue are independent of proton-sensing receptor GPR65.

    PubMed

    Zhu, Xiang; Mose, Eucabeth; Hogan, Simon P; Zimmermann, Nives

    2014-06-01

    Extracellular acidification has been observed in allergic inflammatory diseases. Recently, we demonstrated that the proton-sensing receptor G protein-coupled receptor 65 (GPR65) regulates eosinophil survival in an acidic environment in vitro and eosinophil accumulation in an allergic lung inflammation model. For mast cells, another inflammatory cell type critical for allergic responses, it remains unknown whether GPR65 is expressed and/or regulates mast cell viability. Thus, in the present study, we employed in vitro experiments and an intestinal anaphylaxis model in which both mastocytosis and eosinophilia can be observed, particularly in the gastrointestinal tract, to enable us to directly compare the effect of GPR65 expression on these two cell types. We identified GPR65 expression on mast cells; however, unlike eosinophil viability, mast cell viability in vitro is not affected by acidification or GPR65 expression. Mechanistically, we determined that mast cells do not respond to extracellular acidification with increased cAMP levels. Furthermore, in the intestinal anaphylaxis model, we observed a significant reduction of eosinophils (59.1 ± 9.2% decrease) in the jejunum of allergen-challenged GPR65-deficient mice compared with allergen-challenged wild-type mice, despite the degree of antigen sensitization and the expression levels of Th2 cytokines (Il4, Il13) and eosinophil chemokines (Ccl11, Ccl24) in the jejunum being comparable. In contrast, the accumulation of mast cells in allergen-challenged mice was not affected by GPR65 deficiency. In conclusion, our study demonstrates differential regulation of eosinophils and mast cells in inflammatory tissue, with mast cell viability and accumulation being independent of GPR65. PMID:24742990

  9. Differential eosinophil and mast cell regulation: Mast cell viability and accumulation in inflammatory tissue are independent of proton-sensing receptor GPR65

    PubMed Central

    Zhu, Xiang; Mose, Eucabeth; Hogan, Simon P.

    2014-01-01

    Extracellular acidification has been observed in allergic inflammatory diseases. Recently, we demonstrated that the proton-sensing receptor G protein-coupled receptor 65 (GPR65) regulates eosinophil survival in an acidic environment in vitro and eosinophil accumulation in an allergic lung inflammation model. For mast cells, another inflammatory cell type critical for allergic responses, it remains unknown whether GPR65 is expressed and/or regulates mast cell viability. Thus, in the present study, we employed in vitro experiments and an intestinal anaphylaxis model in which both mastocytosis and eosinophilia can be observed, particularly in the gastrointestinal tract, to enable us to directly compare the effect of GPR65 expression on these two cell types. We identified GPR65 expression on mast cells; however, unlike eosinophil viability, mast cell viability in vitro is not affected by acidification or GPR65 expression. Mechanistically, we determined that mast cells do not respond to extracellular acidification with increased cAMP levels. Furthermore, in the intestinal anaphylaxis model, we observed a significant reduction of eosinophils (59.1 ± 9.2% decrease) in the jejunum of allergen-challenged GPR65-deficient mice compared with allergen-challenged wild-type mice, despite the degree of antigen sensitization and the expression levels of Th2 cytokines (Il4, Il13) and eosinophil chemokines (Ccl11, Ccl24) in the jejunum being comparable. In contrast, the accumulation of mast cells in allergen-challenged mice was not affected by GPR65 deficiency. In conclusion, our study demonstrates differential regulation of eosinophils and mast cells in inflammatory tissue, with mast cell viability and accumulation being independent of GPR65. PMID:24742990

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  12. Effects of depsidones from Hypogymnia physodes on HeLa cell viability and growth.

    PubMed

    Stojanović, I Z; Najman, S; Jovanović, O; Petrović, G; Najdanović, J; Vasiljević, P; Smelcerović, A

    2014-01-01

    The anti-proliferative activitiy of Hypogymnia physodes methanol extracts (ME) and its main constituents, physodalic acid (P1), physodic acid (P2), and 3-hydroxy physodic acid (P3), was tested on human cancer HeLa cell lines. Three lichen depsidones, P1, P2 and P3, were isolated from H. physodes ME using column chromatography and their structures were determined by UV, ESI TOF MS, 1H and 13C NMR. The content of P1, P2 and P3 in ME was determined using reversed-phase highperformance liquid chromatography with photodiode array detection. P1-3 represented even 70 % of the studied extract. The HeLa cells were incubated during 24 and 72 h in the presence of ME and depsidones P1, P2 and P3, at concentrations of 10-1000 μg/ml. Compounds P2 and P3 showed higher activity than compound P1. Half maximal inhibitory concentrations (IC50, μg/ml) of P1, P2, P3 and ME for 24-h incubation were 964, 171, 97 and 254 μg/ml, respectively, while for 72-h incubation they were 283, 66, 63 and 68 μg/ml. As far as we know, this is the first report on the effect of H. physodes ME and their depsidones on HeLa cells. PMID:24785112

  13. Morphology, cell viability, karyotype, expression of surface markers and plasticity of three human primary cell line cultures before and after the cryostorage in LN2 and GN2.

    PubMed

    Del Pino, Alberto; Ligero, Gertrudis; López, María B; Navarro, Héctor; Carrillo, Jose A; Pantoll, Siobhan C; Díaz de la Guardia, Rafael

    2015-02-01

    Primary cell line cultures from human skin biopsies, adipose tissue and tumor tissue are valuable samples for research and therapy. In this regard, their derivation, culture, storage, transport and thawing are important steps to be studied. Towards this end, we wanted to establish the derivation, and identify the culture characteristics and the loss of viability of three human primary cell line cultures (human adult dermal fibroblasts (hADFs), human adult mesenchymal stem cells (hMSCs), and primary culture of tumor cells from lung adenocarcinoma (PCTCLA)). Compared to fresh hADFs, hMSCs and PCTCLA, thawed cells stored in a cryogenic Dewar tanks with liquid nitrogen (LN2), displayed 98.20% ± 0.99, 95.40% ± 1.41 and 93.31% ± 3.83 of cell viability, respectively. Thawed cells stored in a Dry Vapor Shipper container with gas phase (GN2), for 20 days, in addition displayed 4.61% ± 2.78, 3.70% ± 4.09 and 9.13% ± 3.51 of average loss of cells viability, respectively, showing strong correlation between the loss of viability in hADFs and the number of post-freezing days in the Dry Vapor Shipper. No significant changes in morphological characteristics or in the expression of surface markers (being hADFs, hMSCs and PCTCLA characterized by positive markers CD73+; CD90+; CD105+; and negative markers CD14-; CD20-; CD34-; and CD45-; n=2) were found. Chromosome abnormalities in the karyotype were not found. In addition, under the right conditions hMSCs were differentiated into adipogenic, osteogenic and chondrogenic lineages in vitro. In this paper, we have shown the characteristics of three human primary cell line cultures when they are stored in LN2 and GN2. PMID:25445570

  14. 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

  15. The stress responsive and morphologically regulated hsp90 gene from Paracoccidioides brasiliensis is essential to cell viability

    PubMed Central

    Nicola, André M; Andrade, Rosângela V; Dantas, Alessandra S; Andrade, Patrícia A; Arraes, Fabrício BM; Fernandes, Larissa; Silva-Pereira, Ildinete; Felipe, Maria Sueli S

    2008-01-01

    Background Paracoccidioides brasiliensis is a dimorphic fungus that causes the most prevalent systemic mycosis in Latin America. The response to heat shock is involved in pathogenesis, as this pathogen switches from mycelium to yeast forms in a temperature dependent fashion that is essential to establish infection. HSP90 is a molecular chaperone that helps in the folding and stabilization of selected polypeptides. HSP90 family members have been shown to present important roles in fungi, especially in the pathogenic species, as an immunodominant antigen and also as a potential antifungal therapeutic target. Results In this work, we decided to further study the Pbhsp90 gene, its expression and role in cell viability because it plays important roles in fungal physiology and pathogenesis. Thus, we have sequenced a Pbhsp90 cDNA and shown that this gene is present on the genome as a single copy. We have also confirmed its preferential expression in the yeast phase and its overexpression during dimorphic transition and oxidative stress. Treatment of the yeast with the specific HSP90 inhibitors geldanamycin and radicicol inhibited growth at 2 and 10 μM, respectively. Conclusion The data confirm that the Pbhsp90 gene encodes a morphologically regulated and stress-responsive protein whose function is essential to cell viability of this pathogen. This work also enforces the potential of HSP90 as a target for antifungal therapies, since the use of HSP90 inhibitors is lethal to the P. brasiliensis yeast cells in a dose-responsive manner. PMID:18808717

  16. Lipid emulsions differentially affect LPS-induced acute monocytes inflammation: in vitro effects on membrane remodeling and cell viability.

    PubMed

    Boisramé-Helms, Julie; Delabranche, Xavier; Klymchenko, Andrey; Drai, Jocelyne; Blond, Emilie; Zobairi, Fatiha; Mely, Yves; Hasselmann, Michel; Toti, Florence; Meziani, Ferhat

    2014-11-01

    The aim of this study was to assess how lipid emulsions for parenteral nutrition affect lipopolysaccharide (LPS)-induced acute monocyte inflammation in vitro. An 18 h long LPS induced human monocyte leukemia cell stimulation was performed and the cell-growth medium was supplemented with three different industrial lipid emulsions: Intralipid(®), containing long-chain triglycerides (LCT--soybean oil); Medialipid(®), containing LCT (soybean oil) and medium-chain triglycerides (MCT--coconut oil); and SMOFlipid(®), containing LCT, MCT, omega-9 and -3 (soybean, coconut, olive and fish oils). Cell viability and apoptosis were assessed by Trypan blue exclusion and flow cytometry respectively. Monocyte composition and membrane remodeling were studied using gas chromatography and NR12S staining. Microparticles released in supernatant were measured by prothrombinase assay. After LPS challenge, both cellular necrosis and apoptosis were increased (threefold and twofold respectively) and microparticle release was enhanced (sevenfold) after supplementation with Medialipid(®) compared to Intralipid(®), SMOFlipid(®) and monocytes in the standard medium. The monocytes differentially incorporated fatty acids after lipid emulsion challenge. Finally, lipid-treated cells displayed microparticles characterized by disrupted membrane lipid order, reflecting lipid remodeling of the parental cell plasma membrane. Our data suggest that lipid emulsions differentially alter cell viability, monocyte composition and thereby microparticle release. While MCT have deleterious effects, we have shown that parenteral nutrition emulsion containing LCT or LCT and MCT associated to n-3 and n-9 fatty acids have no effect on endotoxin-induced cell death and inflammation. PMID:25038627

  17. The influence of NIR femtosecond laser radiation on the viability of 3D stem cell clusters and tumor spheroids

    NASA Astrophysics Data System (ADS)

    Uchugonova, Aisada; Riemann, Iris; Stracke, Frank; Gorjup, Erwin; LeHarzic, Ronan; König, Karsten

    2007-02-01

    Adult human and rat pancreas stem cells as well as tumor spheroids were irradiated with femtosecond laser pulses in the near infrared (NIR) spectral range at high transient GW/cm2 and TW/cm2 intensities. The cellular response to the laser exposure was probed by the detection of modifications of NAD(P)H autofluorescence, the formation of reactive oxygen species (ROS) and DNA strand breaks (TUNEL-assay), and viability (live/dead test). The results confirm that long-term scanning of stem cells can be performed at appropriate laser exposure parameters without a measurable impact on the cellular metabolism and vitality. In addition, it was proven that a targeted inactivation of a particular single stem cells or a single tumour cell inside a 3D cell cluster using single point illumination at TW/cm2 laser intensities can be performed without affecting neighbouring cells. Therefore multiphoton microscopes can be considered as biosafe tools for long-term analysis of stem cells as well as highly precise optical knocking out of single cells within cell clusters and tissues.

  18. 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

  19. The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins.

    PubMed Central

    Hönlinger, A; Kübrich, M; Moczko, M; Gärtner, F; Mallet, L; Bussereau, F; Eckerskorn, C; Lottspeich, F; Dietmeier, K; Jacquet, M

    1995-01-01

    A multisubunit complex in the mitochondrial outer membrane is responsible for targeting and membrane translocation of nuclear-encoded preproteins. This receptor complex contains two import receptors, a general insertion pore and the protein Mom22. It was unknown if Mom22 directly interacts with preproteins, and two views existed about the possible functions of Mom22: a central role in transfer of preproteins from both receptors to the general insertion pore or a more limited function dependent on the presence of the receptor Mom19. For this report, we identified and cloned Saccharomyces cerevisiae MOM22 and investigated whether it plays a direct role in targeting of preproteins. A preprotein accumulated at the mitochondrial outer membrane was cross-linked to Mom22. The cross-linking depended on the import stage of the preprotein. Overexpression of Mom22 suppressed the respiratory defect of yeast cells lacking Mom19 and increased preprotein import into mom19 delta mitochondria, demonstrating that Mom22 can function independently of Mom19. Overexpression of Mom22 even suppressed the lethal phenotype of a double deletion of the two import receptors known so far (mom19 delta mom72 delta). Deletion of the MOM22 gene was lethal for yeast cells, identifying Mom22 as one of the few mitochondrial membrane proteins essential for fermentative growth. These results suggest that Mom22 plays an essential role in the mitochondrial receptor complex. It directly interacts with preproteins in transit and can perform receptor-like activities. PMID:7760834

  20. Activity and viability of polycyclic aromatic hydrocarbon‐degrading Sphingomonas sp. LB126 in a DC‐electrical field typical for electrobioremediation measures

    PubMed Central

    Shi, Lei; Müller, Susann; Loffhagen, Norbert; Harms, Hauke; Wick, Lukas Y.

    2008-01-01

    Summary There has been growing interest in employing electro‐bioremediation, a hybrid technology of bioremediation and electrokinetics for the treatment of contaminated soil. Knowledge however on the effect of weak electrokinetic conditions on the activity and viability of pollutant‐degrading microorganisms is scarce. Here we present data about the influence of direct current (DC) on the membrane integrity, adenosine triphosphate (ATP) pools, physico‐chemical cell surface properties, degradation kinetics and culturability of fluorene‐degrading Sphingomonas sp. LB126. Flow cytometry was applied to quantify the uptake of propidium iodide (PI) and the membrane potential‐related fluorescence intensities (MPRFI) of individual cells within a population. Adenosine tri‐phosphate contents and fluorene biodegradation rates of bulk cultures were determined and expressed on a per cell basis. The cells' surface hydrophobicity and electric charge were assessed by contact angle and zeta potential measurements respectively. Relative to the control, DC‐exposed cells exhibited up to 60% elevated intracellular ATP levels and yet remained unaffected on all other levels of cellular integrity and functionality tested. Our data suggest that direct current (X = 1 V cm−1; J = 10.2 mA cm−2) as typically used for electrobioremediation measures has no negative effect on the activity of the polycyclic aromatic hydrocarbon (PAH)‐degrading soil microorganism, thereby filling a serious gap of the current knowledge of the electrobioremediation methodology. PMID:21261821

  1. Mesenchymal stromal cells support the viability and differentiation of thymocytes through direct contact in autologous co-cultures.

    PubMed

    Azghadi, Seyed Mohammad Reza; Suciu, Maria; Gruia, Alexandra Teodora; Barbu-Tudoran, Lucian; Cristea, Mirabela Iustina; Mic, Ani Aurora; Muntean, Danina; Nica, Dragos Vasile; Mic, Felix Aurel

    2016-08-01

    The development of thymocytes and generation of mature T cells is a complex process that requires spatio-temporal interactions of thymocytes with the other cells of the thymus microenvironment. Recently, mesenchymal stromal cells were isolated from the neonatal human thymus and differentiated into chondrogenic, osteogenic, and adipogenic lineages, just like their bone marrow counterparts. However, their function in thymocyte homeostasis is unknown. In our autologous co-cultures of rat mesenchymal stromal cells and thymocytes, the stromal cells preserve the viability of cultured thymocytes and stimulate the development of CD4-CD8- double-negative and the maturation of mainly CD4+ single-positive thymocytes. Thymocytes also influence the stemness of bone marrow mesenchymal stromal cells, as their expression of CD44, a marker associated with cellular proliferation and migration, is reduced in co-cultures. Mesenchymal stromal cells' influence on thymocyte development requires direct physical contact between the two cells and is not mediated by a soluble factor. When the two types of cells were physically separated, the stimulative effects of mesenchymal stromal cells on thymocytes did not occur. Electron microscopy confirmed the close contact between the membranes of thymocytes and mesenchymal stromal cells. Our experiments suggest that membrane exchanges could occur between mesenchymal stromal cells and thymocytes, such as the transfer of CD44 from mesenchymal stromal cells to the thymocytes, but its functional significance for thymocytes development remains to be established. These results suggest that mesenchymal stromal cells could normally be a part of the in vivo thymic microenvironment and form a niche that could sustain and guide the development of thymocytes. PMID:27085705

  2. 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

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

    DOEpatents

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

    2011-12-13

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

  4. 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.

  5. 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.

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

    PubMed

    Khan, Sajjad; Trivedi, Vivek; Boateng, Joshua

    2016-03-16

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

  7. Knockdown of AKT3 (PKBγ) and PI3KCA Suppresses Cell Viability and Proliferation and Induces the Apoptosis of Glioblastoma Multiforme T98G Cells

    PubMed Central

    Paul-Samojedny, Monika; Suchanek, Renata; Borkowska, Paulina; Pudełko, Adam; Owczarek, Aleksander; Kowalczyk, Małgorzata; Machnik, Grzegorz; Fila-Daniłow, Anna; Kowalski, Jan

    2014-01-01

    Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor that is difficult to treat and has a very poor prognosis. Thus, new therapeutic strategies that target GBM are urgently needed. The PI3K/AKT/PTEN signaling pathway is frequently deregulated in a wide range of cancers. The present study was designed to examine the inhibitory effect of AKT3 or PI3KCA siRNAs on GBM cell growth, viability, and proliferation.T98G cells were transfected with AKT3 and/or PI3KCA siRNAs. AKT3 and PI3KCA protein-positive cells were identified using FC and Western blotting. The influence of specific siRNAs on T98G cell viability, proliferation, cell cycle, and apoptosis was evaluated as well using FC. Alterations in the mRNA expression of AKT3, PI3KCA, and apoptosis-related genes were analyzed using QRT-PCR. Knockdown of AKT3 and/or PI3KCA genes in T98G cells led to a significant reduction in cell viability, the accumulation of subG1-phase cells and, a reduced fraction of cells in the S and G2/M phases. Additionally, statistically significant differences in the BAX/BCL-2 ratio and an increased percentage of apoptotic cells were found. The siRNA-induced AKT3 and PI3KCA mRNA knockdown may offer a novel therapeutic strategy to control the growth of human GBM cells. PMID:24967401

  8. Extensive Reduction of Cell Viability and Enhanced Matrix Production in Pseudomonas aeruginosa PAO1 Flow Biofilms Treated with a d-Amino Acid Mixture

    PubMed Central

    Sanchez, Zoe; Tani, Akio

    2013-01-01

    Treatment of Pseudomonas aeruginosa PAO1 flow biofilms with a d-amino acid mixture caused significant reductions in cell biomass by 75% and cell viability by 71%. No biofilm disassembly occurred, and matrix production increased by 30%, thereby providing a thick protective cover for remaining viable or persister cells. PMID:23220960

  9. Effect of single-walled carbon nanotubes on tumor cells viability and formation of multicellular tumor spheroids

    NASA Astrophysics Data System (ADS)

    Yakymchuk, Olena M.; Perepelytsina, Olena M.; Dobrydnev, Alexey V.; Sydorenko, Mychailo V.

    2015-03-01

    This paper describes the impact of different concentrations of single-walled carbon nanotubes (SWCNTs) on cell viability of breast adenocarcinoma, MCF-7 line, and formation of multicellular tumor spheroids (MTS). Chemical composition and purity of nanotubes is controlled by Fourier transform infrared spectroscopy. The strength and direction of the influence of SWCNTs on the tumor cell population was assessed by cell counting and measurement of the volume of multicellular tumor spheroids. Effect of SWCNTs on the formation of multicellular spheroids was compared with the results obtained by culturing tumor cells with ultra dispersed diamonds (UDDs). Our results demonstrated that SWCNTs at concentrations ranging from 12.5 to 50 μg/ml did not have cytotoxic influence on tumor cells; instead, they had weak cytostatic effect. The increasing of SWCNTs concentration to 100 to 200 μg/ml stimulated proliferation of tumor cells, especially in suspension fractions. The result of this influence was in formation of more MTS in cell culture with SWCNTs compared with UDDs and control samples. In result, the median volume of MTS after cultivation with SWCNTs at 100 to 200 μg/ml concentrations is 3 to 5 times greater than that in samples which were incubated with the UDDs and is 2.5 times greater than that in control cultures. So, if SWCNTs reduced cell adhesion to substrate and stimulated formation of tumor cell aggregates volume near 7 · 10-3 mm3, at the same time, UDDs reduced adhesion and cohesive ability of cells and stimulated generation of cell spheroids volume no more than 4 · 10-3 mm3. Our results could be useful for the control of cell growth in three-dimensional culture.

  10. Inadequate Processing of Decellularized Dermal Matrix Reduces Cell Viability In Vitro and Increases Apoptosis and Acute Inflammation In Vivo

    PubMed Central

    Morris, Aaron H.; Chang, Julie; Kyriakides, Themis R.

    2016-01-01

    Abstract Decellularized tissue scaffolds are commonly used in the clinic because they can be used as substitutes for more traditional biomaterials, while imparting additional physiological effects. Nevertheless, reports of complications associated with their use are widespread and poorly understood. This study probes possible causes of these complications by examining cell viability and apoptosis in response to eluents from decellularized dermis. Using multiple sources of decellularized dermis, this study shows that typical decellularized scaffolds (prepared with commonly used laboratory techniques, as well as purchased from commercial sources) contain soluble components that are cytotoxic and that these components can be removed by extensive washes in cell culture media. In addition, this study demonstrates that these observed in vitro phenotypes correlate with increased apoptosis and acute inflammation when implanted subcutaneously in mice. PMID:27500014

  11. Effect of laser energy, substrate film thickness and bioink viscosity on viability of endothelial cells printed by Laser-Assisted Bioprinting

    NASA Astrophysics Data System (ADS)

    Catros, Sylvain; Guillotin, Bertrand; Bačáková, Markéta; Fricain, Jean-Christophe; Guillemot, Fabien

    2011-04-01

    Biofabrication of three dimensional tissues by Laser-Assisted Bioprinting (LAB) implies to develop specific strategies for assembling the extracellular matrix (ECM) and cells. Possible strategies consist in (i) printing cells onto or in the depth of ECM layer and/or (ii) printing bioinks containing both cells and ECM-like printable biomaterial. The aim of this article was to evaluate combinatorial effects of laser pulse energy, ECM thickness and viscosity of the bioink on cell viability. A LAB workstation was used to print Ea.hy926 endothelial cells onto a quartz substrate covered with a film of ECM mimicking Matrigel™. Hence, effect of laser energy, Matrigel™ film thickness and bioink viscosity was addressed for different experimental conditions (8-24 μJ, 20-100 μm and 40-110 mPa s, respectively). Cell viability was assessed by live/dead assay performed 24 h post-printing. Results show that increasing the laser energy tends to augment the cell mortality while increasing the thickness of the Matrigel™ film and the viscosity of the bioink support cell viability. Hence, critical printing parameters influencing high cell viability have been related to the cell landing conditions and more specifically to the intensity of the cell impacts occurring at the air-ECM interface and at the ECM-glass interface.

  12. 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

  13. The Effect of the Active Ingredient Thymoquinone on Flap Viability in Random Pattern Flaps in Rats.

    PubMed

    Kocak, Omer Faruk; Bozan, Nazim; Oksuz, Mustafa; Yuce, Serdar; Demir, Canser Yılmaz; Bulut, Gulay; Ragbetli, Murat Cetin

    2016-08-01

    Thymoquinone (TQ) is a plant extract that has been shown to have antioxidant, anti-inflammatory, angiogenic, antimicrobial, and anticarcinogenic effects. The aim of this study is to research how the use of TQ affects flap viability. 42 rats were placed into 6 groups, with 7 rats in each. A 3 × 10 cm McFarlane flap model was used on the test animals. The sham group had used neither surgical nor TQ treatment. The control group had surgery but no treatment afterwards. The preoperative TQ group was given oral doses of 2 mg/kg. TQ for 10 days preoperatively with no treatment after the surgical procedure. The postoperative TQ group received oral doses of 2 mg/kg TQ for 10 days after the surgical process. The preoperative + postoperative (pre + postoperative) TQ group was given oral doses of 2 mg/kg TQ for 10 days both preoperatively and postoperatively. Finally, the dimethylsulfoxide group received 10 mg/kg dimethylsulfoxide (DMSO) for 10 days both preoperatively and postoperatively. Ten days after surgery the findings were evaluated. The average rates of necrosis were found to be 29.7 % in the control group, 19.18 % in the preoperative TQ group, 13.05 % in the postoperative TQ group, 8.42 % in the pre + postoperative TQ group, and 29.03 % in the DMSO group. The experimental groups had better area measurement, histopathological, and electron microscopic results than the control group (All; p < 0.05). We believe that, because of its antioxidant, anti-inflammatory, and angiogenic properties, thymoquinone is an agent that can prevent ischemia-reperfusion damage and, therefore, prevent necrosis. PMID:27072137

  14. 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. PMID:26048174

  15. Bio-hybrid interfaces to study neuromorphic functionalities: New multidisciplinary evidences of cell viability on poly(anyline) (PANI), a semiconductor polymer with memristive properties.

    PubMed

    Juarez-Hernandez, Leon J; Cornella, Nicola; Pasquardini, Laura; Battistoni, Silvia; Vidalino, Laura; Vanzetti, Lia; Caponi, Silvia; Dalla Serra, Mauro; Iannotta, Salvatore; Pederzolli, Cecilia; Macchi, Paolo; Musio, Carlo

    2016-01-01

    The interfacing of artificial devices with biological systems is a challenging field that crosses several disciplines ranging from fundamental research (biophysical chemistry, neurobiology, material and surface science) to frontier technological application (nanotechnology, bioelectronics). The memristor is the fourth fundamental circuit element, whose electrical properties favor applications in signal processing, neural networks, and brain-computer interactions and it represents a new frontier for technological applications in many fields including the nanotechnologies, bioelectronics and the biosensors. Using multidisciplinary approaches, covering surface science, cell biology and electrophysiology, we successfully implemented a living bio-hybrid system constituted by cells adhering to films of poly(aniline) (PANI), a semiconductor polymer having memristive properties assembled with polyelectrolytes. Here we tested whether the PANI devices could support survivor, adhesion and differentiation of several cell lines, including the neuron-like SHSY5Y cells. Moreover, we performed electrophysiology on these cells showing that the biophysical properties are retained with differences occurring in the recorded ion currents. Taken together, the cell viability here reported is the key requirement to design and develop a reliable functional memristor-based bio-hybrid able to mimic neuronal activity and plasticity. PMID:26263829

  16. Cell viability modulation through changes of Ca(2+)-dependent signalling pathways.

    PubMed

    Wójcik-Piotrowicz, Karolina; Kaszuba-Zwoińska, Jolanta; Rokita, Eugeniusz; Thor, Piotr

    2016-05-01

    The aim of the study was to determine the correlations between intracellular calcium ion level and a cell's ability to survive. The intracellular concentration of Ca(2+) ions, maintained through different mechanisms, plays an important role in signalling in cells. The deregulation of these mechanisms by various cell stressors (e.g. cytotoxic agents) can disturb Ca(2+) homeostasis and influence Ca(2+)-dependent signalling pathways in the cell. Perturbations of intracellular electrochemical equilibrium may lead to changes in cell function or even to cell death. According to some experimental results, one of the cell stressors may be exposure to magnetic fields (MF). Because of the wide distribution of MF sources in our environment, magnetic fields have recently been intensively examined in relation to the occurrence of cancer. Nevertheless, two questions still remain unanswered: Is the influence of MF on cells positive or negative, and what mechanism(s) underlie the effects of MF action on cells? Most studies focus on the influence of MF on Ca(2+) ion fluxes as calcium ions play the role of intracellular second messengers, triggering many signalling cascades. Physical models assuming the mechanisms generating the disturbance of ionic transport and/or the dysfunction of ion-protein complexes in cells due to MF action have been widely discussed in the literature, but a detailed explanation of experimental results is still awaited. The dynamics of the concentration of intracellular calcium ions can be detected by various methods, including optical and non-optical techniques. This review combines an insight into basic intracellular Ca(2+) regulative mechanisms and common techniques used to detect changes in Ca(2+) concentration inside the cell. The emphasis here is on the determination of Ca(2+) regulative mechanisms developed in non-excitable cells (e.g. U937 cells, HeLa, etc.), which are probably mainly involved in cell responses to external stress (e.g. MF stimuli

  17. Solar cell activation system

    SciTech Connect

    Apelian, L.

    1983-07-05

    A system for activating solar cells involves the use of phosphorescent paint, the light from which is amplified by a thin magnifying lens and used to activate solar cells. In a typical system, a member painted with phosphorescent paint is mounted adjacent a thin magnifying lens which focuses the light on a predetermined array of sensitive cells such as selenium, cadmium or silicon, mounted on a plastic board. A one-sided mirror is mounted adjacent the cells to reflect the light back onto said cells for purposes of further intensification. The cells may be coupled to rechargeable batteries or used to directly power a small radio or watch.

  18. Differential concentration-specific effects of caffeine on cell viability, oxidative stress, and cell cycle in pulmonary oxygen toxicity in vitro

    SciTech Connect

    Tiwari, Kirti Kumar; Chu, Chun; Couroucli, Xanthi; Moorthy, Bhagavatula; Lingappan, Krithika

    2014-08-08

    Highlights: • Caffeine at 0.05 mM decreases oxidative stress in hyperoxia. • Caffeine at 1 mM decreases cell viability, increases oxidative stress in hyperoxia. • Caffeine at 1 but not 0.05 mM, abrogates hyperoxia-induced G2/M arrest. - Abstract: Caffeine is used to prevent bronchopulmonary dysplasia (BPD) in premature neonates. Hyperoxia contributes to the development of BPD, inhibits cell proliferation and decreases cell survival. The mechanisms responsible for the protective effect of caffeine in pulmonary oxygen toxicity remain largely unknown. A549 and MLE 12 pulmonary epithelial cells were exposed to hyperoxia or maintained in room air, in the presence of different concentrations (0, 0.05, 0.1 and 1 mM) of caffeine. Caffeine had a differential concentration-specific effect on cell cycle progression, oxidative stress and viability, with 1 mM concentration being deleterious and 0.05 mM being protective. Reactive oxygen species (ROS) generation during hyperoxia was modulated by caffeine in a similar concentration-specific manner. Caffeine at 1 mM, but not at the 0.05 mM concentration decreased the G2 arrest in these cells. Taken together this study shows the novel funding that caffeine has a concentration-specific effect on cell cycle regulation, ROS generation, and cell survival in hyperoxic conditions.

  19. Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies

    PubMed Central

    Lu, Yujie; Darne, Chinmay D.; Tan, I-Chih; Zhu, Banghe; Hall, Mary A.; Lazard, ZaWaunyka W.; Davis, Alan R.; Simpson, LaShan; Sevick-Muraca, Eva M.; Olmsted-Davis, Elizabeth A.

    2013-01-01

    Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models. PMID:24104323

  20. Correct Timing of Proliferation and Differentiation is Necessary for Normal Inner Ear Development and Auditory Hair Cell Viability

    PubMed Central

    Kopecky, Benjamin J.; Jahan, Israt; Fritzsch, Bernd

    2013-01-01

    Background Hearing restoration through hair cell regeneration will require revealing the dynamic interactions between proliferation and differentiation during development to avoid the limited viability of regenerated hair cells. Pax2-Cre N-Myc conditional knockout (CKO) mice highlighted the need of N-Myc for proper neurosensory development and possible redundancy with L-Myc. The late-onset hair cell death in the absence of early N-Myc expression could be due to mis-regulation of genes necessary for neurosensory formation and maintenance, such as Neurod1, Atoh1, Pou4f3, and Barhl1. Results Pax2-Cre N-Myc L-Myc double CKO mice show that proliferation and differentiation are linked together through Myc and in the absence of both Mycs, altered proliferation and differentiation results in morphologically abnormal ears. In particular, the organ of Corti apex is re-patterned into a vestibular-like organization and the base is truncated and fused with the saccule. Conclusions These data indicate that therapeutic approaches to restore hair cells must take into account a dynamic interaction of proliferation and differentiation regulation of basic Helix-Loop-Helix transcription factors in attempts to stably replace lost cochlear hair cells. In addition, our data indicate that Myc is an integral component of the evolutionary transformation process that resulted in the organ of Corti development. PMID:23193000

  1. Inhibition of ErbB2 by herceptin reduces viability and survival, induces apoptosis and oxidative stress in Calu-3 cell line.

    PubMed

    Dogan, Irem; Cumaoglu, Ahmet; Aricioglu, Aysel; Ekmekci, Abdullah

    2011-01-01

    Human epidermal growth factor receptor 2 (ErbB2) amplification and overexpression has been seen in many cancer types including non-small cell lung cancer (NSCLC). Thus, ErbB2 is an important target for cancer therapies. Increased ErbB2 expression has been associated with drug resistance in cancer cells. Herceptin is a humanized monoclonal antibody that targets the extracellular domain of ErbB2. In this study, we aimed to block ErbB2 signaling with Herceptin and assess cytotoxicity and effects on apoptosis, oxidative stress, nuclear factor kappa-B (NF-kB), and Survivin expression in Calu-3 cell line. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay were used to assess cell viability as a marker of proliferation. Acridine orange/ethidium bromide (AO/EB) staining and caspase 3/7 activity were measured as the markers of apoptosis. The relative expressions of NF-kB-p50 and Survivin mRNAs were evaluated. Activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), and the levels of glutathione (GSH) and reactive oxygen species (ROS) were determined in a time- and dose-dependent manner. Our results show that Herceptin treatment inhibits cell proliferation and activates apoptosis but without effects on Survivin and NF-kB expression in Calu-3 cell line. Intracellular glutathione levels and SOD and CAT activities were decreased in a time- and dose-dependent manner associated with oxidative stress. Also, ROS were increased at 24 h. These results provide evidence that Herceptin can be used as a cytotoxic and apoptotic agent in NSCLC. PMID:20936496

  2. Transcription Factor Stat3 Stimulates Metastatic Behavior of Human Prostate Cancer Cells in Vivo, whereas Stat5b Has a Preferential Role in the Promotion of Prostate Cancer Cell Viability and Tumor Growth

    PubMed Central

    Gu, Lei; Dagvadorj, Ayush; Lutz, Jacqueline; Leiby, Benjamin; Bonuccelli, Gloria; Lisanti, Michael P.; Addya, Sankar; Fortina, Paolo; Dasgupta, Abhijit; Hyslop, Terry; Bubendorf, Lukas; Nevalainen, Marja T.

    2010-01-01

    Identification of the molecular changes that promote viability and metastatic behavior of prostate cancer is critical for the development of improved therapeutic interventions. Stat5a/b and Stat3 are both constitutively active in locally-confined and advanced prostate cancer, and both transcription factors have been reported to be critical for the viability of prostate cancer cells. We recently showed that Stat3 promotes metastatic behavior of human prostate cancer cells not only in vitro but also in an in vivo experimental metastases model. In this work, we compare side-by-side Stat5a/b versus Stat3 in the promotion of prostate cancer cell viability, tumor growth, and induction of metastatic colonization in vivo. Inhibition of Stat5a/b induced massive death of prostate cancer cells in culture and reduced both subcutaneous and orthotopic prostate tumor growth, whereas Stat3 had a predominant role over Stat5a/b in promoting metastases formation of prostate cancer cells in vivo in nude mice. The molecular mechanisms underlying the differential biological effects induced by these two transcription factors involve largely different sets of genes regulated by Stat5a/b versus Stat3 in human prostate cancer model systems. Of the two Stat5 homologs, Stat5b was more important for supporting growth of prostate cancer cells than Stat5a. This work provides the first mechanistic comparison of the biological effects induced by transcription factors Stat5a/b versus Stat3 in prostate cancer. PMID:20167868

  3. Viability of adhered bacterial cells: tracking MinD protein oscillations

    NASA Astrophysics Data System (ADS)

    Barrett, Matt; Colville, Keegan; Schultz-Nielsen, Chris; Jericho, Manfred; Dutcher, John

    2010-03-01

    To study bacterial cells using atomic force microscopy, it is necessary to immobilize the cells on a substrate. Because bacterial cells and common substrates such as glass and mica have a net negative charge, positively charged polymers such as poly-L-lysine (PLL) and polyethyleneimine (PEI) are commonly used as adhesion layers. However, the use of adhesion polymers could stress the cell and even render it inviable. Viable E. coli cells use oscillations of Min proteins along the axis of the rod-shaped cells to ensure accurate cell division. By tagging MinD proteins with GFP, oscillations can be observed using fluorescence microscopy. For a healthy cell in an ideal environment, the oscillation period is measured to be ˜40 s. Prior experiments have shown that PLL increases the oscillation period significantly (up to 80%). In the present study, we have used epifluorescence and total internal reflection fluorescence (TIRF) to track MinD protein oscillations in E. coli bacteria adhered to a variety of positively charged polymers on mica as a function of polymer surface coverage.

  4. Inhibition of HDAC3- and HDAC6-Promoted Survivin Expression Plays an Important Role in SAHA-Induced Autophagy and Viability Reduction in Breast Cancer Cells

    PubMed Central

    Lee, Jane Ying-Chieh; Kuo, Ching-Wen; Tsai, Shing-Ling; Cheng, Siao Muk; Chen, Shang-Hung; Chan, Hsiu-Han; Lin, Chun-Hui; Lin, Kun-Yuan; Li, Chien-Feng; Kanwar, Jagat R.; Leung, Euphemia Y.; Cheung, Carlos Chun Ho; Huang, Wei-Jan; Wang, Yi-Ching; Cheung, Chun Hei Antonio

    2016-01-01

    SAHA is a class I HDAC/HDAC6 co-inhibitor and an autophagy inducer currently undergoing clinical investigations in breast cancer patients. However, the molecular mechanism of action of SAHA in breast cancer cells remains unclear. In this study, we found that SAHA is equally effective in targeting cells of different breast cancer subtypes and tamoxifen sensitivity. Importantly, we found that down-regulation of survivin plays an important role in SAHA-induced autophagy and cell viability reduction in human breast cancer cells. SAHA decreased survivin and XIAP gene transcription, induced survivin protein acetylation and early nuclear translocation in MCF7 and MDA-MB-231 breast cancer cells. It also reduced survivin and XIAP protein stability in part through modulating the expression and activation of the 26S proteasome and heat-shock protein 90. Interestingly, targeting HDAC3 and HDAC6, but not other HDAC isoforms, by siRNA/pharmacological inhibitors mimicked the effects of SAHA in modulating the acetylation, expression, and nuclear translocation of survivin and induced autophagy in MCF7 and MDA-MB-231 cancer cells. Targeting HDAC3 also mimicked the effect of SAHA in up-regulating the expression and activity of proteasome, which might lead to the reduced protein stability of survivin in breast cancer cells. In conclusion, this study provides new insights into SAHA's molecular mechanism of actions in breast cancer cells. Our findings emphasize the complexity of the regulatory roles in different HDAC isoforms and potentially assist in predicting the mechanism of novel HDAC inhibitors in targeted or combinational therapies in the future. PMID:27065869

  5. Tissue-specific mechanical and geometrical control of cell viability and actin cytoskeleton alignment

    NASA Astrophysics Data System (ADS)

    Wang, Dong; Zheng, Wenfu; Xie, Yunyan; Gong, Peiyuan; Zhao, Fang; Yuan, Bo; Ma, Wanshun; Cui, Yan; Liu, Wenwen; Sun, Yi; Piel, Matthieu; Zhang, Wei; Jiang, Xingyu

    2014-08-01

    Different tissues have specific mechanical properties and cells of different geometries, such as elongated muscle cells and polygonal endothelial cells, which are precisely regulated during embryo development. However, the mechanisms that underlie these processes are not clear. Here, we built an in vitro model to mimic the cellular microenvironment of muscle by combining both mechanical stretch and geometrical control. We found that mechanical stretch was a key factor that determined the optimal geometry of myoblast C2C12 cells under stretch, whereas vascular endothelial cells and fibroblasts had no such dependency. We presented the first experimental evidence that can explain why myoblasts are destined to take the elongated geometry so as to survive and maintain parallel actin filaments along the stretching direction. The study is not only meaningful for the research on myogenesis but also has potential application in regenerative medicine.

  6. Effects of Non-Collagenous Proteins, TGF-β1, and PDGF-BB on Viability and Proliferation of Dental Pulp Stem Cells

    PubMed Central

    Tabatabaei, Fahimeh Sadat

    2016-01-01

    ABSTRACT Objectives The dentin matrix servers as a reservoir of growth factors, sequestered during dentinogenesis. The aim of this study was to assess the viability and proliferation of dental pulp stem cells in the presence of dentin matrix-derived non-collagenous proteins and two growth factors; platelet-derived growth factor BB and transforming growth factor beta 1. Material and Methods The dental pulp cells were isolated and cultured. The dentin proteins were extracted and purified. The MTT assay was performed for assessment of cell viability and proliferation in the presence of different concentrations of dentin proteins and growth factors during 24 - 72 h post-treatment. Results The cells treated with 250 ng/mL dentin proteins had the best viability and proliferation ability in comparison with other concentrations (P < 0.05). The MTT assay demonstrated that cells cultured with 5 ng/mL platelet-derived growth factor BB had the highest viability at each time point as compared to other groups (P < 0.05). However, in presence of platelet-derived growth factor BB alone and in combination with transforming growth factor beta 1 and dentin proteins (10 ng/mL), significant higher viability was seen at all time points (P < 0.05). The least viability and proliferation at each growth factor concentration was seen in cells treated with combination of transforming growth factor beta 1 and dentin proteins at 72 h (P < 0.05). Conclusions The results indicated that the triple combination of growth factors and matrix-derived non-collagenous proteins (especially at 10 ng/mL concentration) has mitogenic effect on dental pulp stem cells. PMID:27099698

  7. Evaluation of the effects of cryopreservation on viability, proliferation and colony formation of human spermatogonial stem cells in vitro culture.

    PubMed

    Mirzapour, T; Movahedin, M; Tengku Ibrahim, T A; Haron, A W; Nowroozi, M R

    2013-02-01

    Proliferation of spermatogonial stem cells (SSCs) in vitro system is very important. It can enhance SSCs numbers for success of transplantation and treatment of infertility in cancer patients. In this study, testicular cells that obtained from azoospermia patients (n=8) by enzymatic digestion were cryopreserved at the beginning and after 2 weeks of culture. Then, frozen-thawed SSCs were co-cultured on fresh Sertoli cells (experimental group 1), and frozen-thawed Sertoli cells (experimental group 2) for another 3 weeks. In control group, fresh SSCs were co-cultured on fresh Sertoli cells. Viability rate after enzymatic digestion was 93.4%±5.0. Frozen-thawed testicular cells after 2 weeks of culture had a significantly (P<0.05) higher percentage of living cells compared to frozen-thawed testicular cells at the beginning of culture (59.2±7.05 and 46.3±8.40 respectively). The number of colonies in the experimental group 1 was significantly higher than experimental group 2 (19.6±2.8 and 8.33±1.5, respectively, P<0.05). The diameter of the colonies in the experimental group 1 was significantly higher than control and experimental group 2 (P<0.05) after 3 weeks of culture (269.7±52.1, 204.34±24.1 and 112.52±23.5 μm, respectively). Cryopreservation technique will raise the possibility of banking SSCs for men who have a cancer-related illness and waiting for radiotherapy and/or chemotherapy. PMID:22621173

  8. Treatment of Leptothrix Cells with Ultrapure Water Poses a Threat to Their Viability

    PubMed Central

    Kunoh, Tatsuki; Suzuki, Tomoko; Shiraishi, Tomonori; Kunoh, Hitoshi; Takada, Jun

    2015-01-01

    The genus Leptothrix, a type of Fe/Mn-oxidizing bacteria, is characterized by its formation of an extracellular and microtubular sheath. Although almost all sheaths harvested from natural aquatic environments are hollow, a few chained bacterial cells are occasionally seen within some sheaths of young stage. We previously reported that sheaths of Leptothrix sp. strain OUMS1 cultured in artificial media became hollow with aging due to spontaneous autolysis within the sheaths. In this study, we investigated environmental conditions that lead the OUMS1 cells to die. Treatment of the cells with ultrapure water or acidic buffers (pH 6.0) caused autolysis of the cells. Under these conditions, the plasma membrane and cytoplasm of cells were drastically damaged, resulting in leakage of intracellular electrolytes and relaxation of genomic DNA. The autolysis was suppressed by the presence of Ca2+. The hydrolysis of peptidoglycan by the lysozyme treatment similarly caused autolysis of the cells and was suppressed also by the presence of Ca2+. However, it remains unclear whether the acidic pH-dependent autolysis is attributable to damage of peptidoglycan. It was observed that L. discophora strain SP-6 cells also underwent autolysis when suspended in ultrapure water; it is however, uncertain whether this phenomenon is common among other members of the genus Leptothrix. PMID:25634812

  9. Treatment of leptothrix cells with ultrapure water poses a threat to their viability.

    PubMed

    Kunoh, Tatsuki; Suzuki, Tomoko; Shiraishi, Tomonori; Kunoh, Hitoshi; Takada, Jun

    2015-01-01

    The genus Leptothrix, a type of Fe/Mn-oxidizing bacteria, is characterized by its formation of an extracellular and microtubular sheath. Although almost all sheaths harvested from natural aquatic environments are hollow, a few chained bacterial cells are occasionally seen within some sheaths of young stage. We previously reported that sheaths of Leptothrix sp. strain OUMS1 cultured in artificial media became hollow with aging due to spontaneous autolysis within the sheaths. In this study, we investigated environmental conditions that lead the OUMS1 cells to die. Treatment of the cells with ultrapure water or acidic buffers (pH 6.0) caused autolysis of the cells. Under these conditions, the plasma membrane and cytoplasm of cells were drastically damaged, resulting in leakage of intracellular electrolytes and relaxation of genomic DNA. The autolysis was suppressed by the presence of Ca2+. The hydrolysis of peptidoglycan by the lysozyme treatment similarly caused autolysis of the cells and was suppressed also by the presence of Ca2+. However, it remains unclear whether the acidic pH-dependent autolysis is attributable to damage of peptidoglycan. It was observed that L. discophora strain SP-6 cells also underwent autolysis when suspended in ultrapure water; it is however, uncertain whether this phenomenon is common among other members of the genus Leptothrix. PMID:25634812

  10. A Viability Approach for Robustness Measurement, Organizational Autopoiesis, and Cell Turnover in a Multicellular System.

    PubMed

    Sarr, Abdoulaye; Désilles, Anya; Fronville, Alexandra; Rodin, Vincent

    2016-04-01

    In this article, we use the potential of computational biology to highlight the key role of cell apoptosis for studying some tissue's properties through in silico experiments of morphogenesis. Our morphogenesis model is a new approach focusing on the deterministic program within cells that controls their placement and their differentiation at the beginning of the embryogenesis. Indeed, when the tissue is made by just a few pair of cells, we consider that cellular mechanisms are related neither to the influence of mechanical forces nor to the spread of chemicals. Dynamics are based on spatial and logical choices, the other factors being involved when the tissue contains a large number of cells. We had established a mathematical formulation of such a model and had enlightened the link between phenotype (cell placement and cell differentiation) and genotype (cell program) at the early embryogenesis. Indeed, that work allowed for generating any early tissue and the associated program that designs it. We propose now to study and assess some properties of these tissues for further selection and classification purposes. More precisely, we present in this article novel methods to measure tissue robustness based on the backward morphogenesis of our model. We also show some implementations of their self-maintenance properties, on the one hand to deal with environment disturbances through autopoiesis and on the other hand to achieve a dynamical steady state which ensures tissue renewal. PMID:26958901

  11. Fuel Cell Cathode Contamination: Comparison of Prevention Strategies and their Viability

    NASA Astrophysics Data System (ADS)

    Tejaswi, Arjun

    Fuel cells are a major area of research in ongoing efforts to find alternate sources of energy. Today these efforts have become ever the more necessary in the face of spiraling costs of conventional sources of energy and concerns about global warming. Most fuel cells consume hydrogen to produce, for the most part, only water in their exhaust. They are also capable of achieving significantly higher efficiencies than conventional automobile internal combustion engines. Since cost still remains one of the most intractable challenges to the advent of fuel cells, it is imperative that every effort be made to lower the costs of fuel cell production, operation and maintenance as well as improving overall efficiency. The air circulation system of a fuel cell is designed to provide oxygen to the cathode of the fuel cell. Air taken from the surroundings, however, often contains pollutants including dust, SO2, NO 2 and various other gases. These gases may severely degrade various components of system, especially for polymer electrolyte membrane (PEM) type fuel cells, including the catalyst, membrane electrode assembly and other components. Moreover, these pollutants may lead to specific behavior based on ambient air composition at the test site thereby confusing researchers. In order to address these issues, this study seeks to identify these pollutants and examine the mitigation strategies to mitigate them. Also discussed is whether these pollutants have an effect debilitating enough to justify the extra cost and potential parasitic losses associated with these mitigation strategies. Adsorptive filtration is identified as the most appropriate cathode side air quality system for fuel cells. Performance of cathode side fuel cell filters are examined under varying relative humidity, temperature, air flow rate and pollutant concentration conditions. An estimated filter survival time under realistic conditions is also suggested.

  12. Viability and proliferation of L929, tumour and hybridoma cells in the culture media containing sericin protein as a supplement or serum substitute.

    PubMed

    Cao, Ting-Ting; Zhang, Yu-Qing

    2015-09-01

    Cell cultures often require the addition of animal serum and other supplements. In this study, silk sericin, a bioactive protein, recovered from the waste of silk floss production was hydrolysed into three pepsin-degraded sericin peptides with different ranges of molecular mass. Normal animal cells, tumour cells and hybridoma cells were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute. The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture. The cell culture results showed that with the degradation of sericin, for normal mouse fibroblast L929 cells, addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone. When all serum was replaced by sericin, cell viability in the sericin medium could reach about one half of that in FBS medium. When in a medium containing a mixture of FBS: sericin (6:4, v/v), the cell culture effect is about 80 %. For the cultures of four tumour and one hybridoma cells, regardless of the molecular weight range, these degraded sericin peptides could substitute all serum in FBS media. The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium. In other words, cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media. The mechanism of the sericin protein to promote cell growth and proliferation will be further investigated later. PMID:25895088

  13. Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields

    PubMed Central

    Wang, Jian; Xiang, Bo; Deng, Jixian; Freed, Darren H.; Arora, Rakesh C.; Tian, Ganghong

    2016-01-01

    After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF), Adipose-derived Stem Cells (ASCs) and those labeled by superparamagnetic iron oxide (SPIO) nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT) assay, proliferation by cell counting and bromodeoxyuridine (BrdU) incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor-1 (IGF-1), Transforming Growth Factor Beta 1 (TGF-β1), genetic markers comprising Stem Cell Antigen-1 (Sca1), Octamer-4 (Oct-4), ATP-binding Cassette Subfamily B Member 1 (ABCB1), adipogenic marker genes containing Lipoprotein Lipase (LPL), Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ), and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1) and Osterix (OSX). Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling. PMID:26880984

  14. Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields.

    PubMed

    Wang, Jian; Xiang, Bo; Deng, Jixian; Freed, Darren H; Arora, Rakesh C; Tian, Ganghong

    2016-01-01

    After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF), Adipose-derived Stem Cells (ASCs) and those labeled by superparamagnetic iron oxide (SPIO) nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT) assay, proliferation by cell counting and bromodeoxyuridine (BrdU) incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor-1 (IGF-1), Transforming Growth Factor Beta 1 (TGF-β1), genetic markers comprising Stem Cell Antigen-1 (Sca1), Octamer-4 (Oct-4), ATP-binding Cassette Subfamily B Member 1 (ABCB1), adipogenic marker genes containing Lipoprotein Lipase (LPL), Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ), and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1) and Osterix (OSX). Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling. PMID:26880984

  15. Addition of seminal plasma to post-thawing equine semen: what is the effect on sperm cell viability?

    PubMed

    de Andrade, A F C; Zaffalon, F G; Celeghini, E C C; Nascimento, J; Tarragó, O F B; Martins, S M M K; Alonso, M A; Arruda, R P

    2011-08-01

    Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autologous seminal plasma after semen thawing (ASP). The addition of 20% of seminal plasma led to an increase in the cell population that simultaneously show plasma and acrosomal membrane integrity (p < 0.05). The addition of seminal plasma did not alter the total motility, the amount of cells with mitochondrial membrane potential or the sperm velocities (average path velocity, straight-line velocity and curvilinear velocity). However, the beat/cross-frequency, straightness and linearity were reduced in ASP and HSP groups (p < 0.05). Unexpectedly, the addition of homologous seminal plasma reduced the proportion of cells with progressive motility (p < 0.05) and the addition of autologous seminal plasma reduced the amplitude of the lateral head displacement (p < 0.05). Based on the increase in the cell populations that had the plasma and acrosomal membrane integrity simultaneously identified in this study, we proposed that the addition of seminal plasma (autologous or homologous) into post-thawed semen before insemination could increase semen fertility. PMID:21121969

  16. Role of surface-electrical properties on the cell-viability of carbon thin films grown in nanodomain morphology

    NASA Astrophysics Data System (ADS)

    Javid, Amjed; Kumar, Manish; Yoon, Seokyoung; Lee, Jung Heon; Tajima, Satomi; Hori, Masaru; Geon Han, Jeon

    2016-07-01

    Carbon thin films, having a combination of unique physical and chemical properties, exhibit an interesting biocompatibility and biological response to living entities. Here, the carbon films are developed in the morphology form of nano-domains with nanoscale inter-domain separations, tuned by plasma conditions in the facing target magnetron sputtering process. The wettability and surface energy are found to have a close relation to the inter-domain separations. The chemical structure of carbon films exhibited the relative enhancement of sp3 in comparison to sp2 with the increase of domain separations. The cell-viability of these films shows promising results for L929 mouse fibroblast and Saos-2 bone cells, when inter-domain separation is increased. Electrical conductivity and surface energy are identified to play the key role in different time-scales during the cell-proliferation process. The contribution from electrical conductivity is dominant in the beginning of the cultivation, whereas with the passage of time (~3–5 d) the surface energy takes control over conductivity to enhance the cell proliferation.

  17. Hypoxia facilitates tumour cell detachment by reducing expression of surface adhesion molecules and adhesion to extracellular matrices without loss of cell viability.

    PubMed Central

    Hasan, N. M.; Adams, G. E.; Joiner, M. C.; Marshall, J. F.; Hart, I. R.

    1998-01-01

    The effects of acute hypoxia on integrin expression and adhesion to extracellular matrix proteins were investigated in two human melanoma cell lines, HMB-2 and DX3, and a human adenocarcinoma cell line, HT29. Exposure to hypoxia caused a significant down-regulation of cell surface integrins and an associated decrease in cell adhesion. Loss of cell adhesion and integrin expression were transient and levels returned to normal within 24 h of reoxygenation. Other cell adhesion molecules, such as CD44 and N-CAM, were also down-regulated after exposure of cells to hypoxia. Acute exposure to hypoxia of cells at confluence caused rapid cell detachment. Cell detachment preceded loss of viability. Detached HMB-2 and DX3 cells completely recovered upon reoxygenation, and floating cells re-attached and continued to grow irrespective of whether they were left in the original glass dishes or transferred to new culture vessels, while detached HT29 cells partly recovered upon reoxygenation. Cell detachment after decreased adhesion appears to be a stress response, which may be a factor enabling malignant cells to escape hypoxia in vivo, with the potential to form new foci of tumour growth. PMID:9667649

  18. Effect of low-dimensional alumina structures on viability of L 929 cells

    SciTech Connect

    Fomenko, Alla N. Korovin, Matvey S. Bakina, Olga V. Kazantsev, Sergey O. Glazkova, Elena A. Svarovskaya, Natalia V. Lozhkomoev, Aleksandr S.

    2015-10-27

    In the study, we estimated the cytotoxicity of alumina nanoparticles differing in shape (nanofibers, nanoplates, nanosheets, agglomerates of nanosheets) and close in physicochemical properties (particle size, specific surface area, phase composition, and zeta potential). The alumina structures were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD) data, low-temperature nitrogen adsorption, and dynamic light scattering (DLS). The cytotoxicity was estimated on fibroblast cells of the L929 line. It was found that a more adverse effect on the cells was exerted by alumina nanofibers and nanosheets. The action of nanosheets on the cells was inhibitory and was of about the same level, irrespective of the observation period. The effect of alumina nanosheet agglomerates and nanoplates on the cell proliferation was weak even at an exposure time of 72 h.

  19. Arctigenin Inhibits Lung Metastasis of Colorectal Cancer by Regulating Cell Viability and Metastatic Phenotypes.

    PubMed

    Han, Yo-Han; Kee, Ji-Ye; Kim, Dae-Seung; Mun, Jeong-Geon; Jeong, Mi-Young; Park, Sang-Hyun; Choi, Byung-Min; Park, Sung-Joo; Kim, Hyun-Jung; Um, Jae-Young; Hong, Seung-Heon

    2016-01-01

    Arctigenin (ARC) has been shown to have an anti-cancer effect in various cell types and tissues. However, there have been no studies concerning metastatic colorectal cancer (CRC). In this study, we investigated the anti-metastatic properties of ARC on colorectal metastasis and present a potential candidate drug. ARC induced cell cycle arrest and apoptosis in CT26 cells through the intrinsic apoptotic pathway via MAPKs signaling. In several metastatic phenotypes, ARC controlled epithelial-mesenchymal transition (EMT) through increasing the expression of epithelial marker E-cadherin and decreasing the expressions of mesenchymal markers; N-cadherin, vimentin, β-catenin, and Snail. Moreover, ARC inhibited migration and invasion through reducing of matrix metalloproteinase-2 (MMP-2) and MMP-9 expressions. In an experimental metastasis model, ARC significantly inhibited lung metastasis of CT26 cells. Taken together, our study demonstrates the inhibitory effects of ARC on colorectal metastasis. PMID:27618887