WorldWideScience

Sample records for viability cell cycle

  1. Effect of lycopene on cell viability and cell cycle progression in human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Teodoro Anderson

    2012-08-01

    Full Text Available Abstract Background Lycopene, a major carotenoid component of tomato, has a potential anticancer activity in many types of cancer. Epidemiological and clinical trials rarely provide evidence for mechanisms of the compound’s action, and studies on its effect on cancer of different cell origins are now being done. The aim of the present study was to determine the effect of lycopene on cell cycle and cell viability in eight human cancer cell lines. Methods Human cell lines were treated with lycopene (1–5 μM for 48 and 96 h. Cell viability was monitored using the method of MTT. The cell cycle was analyzed by flow cytometry, and apoptotic cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling (TUNEL and by DAPI. Results Our data showed a significant decrease in the number of viable cells in three cancer cells lines (HT-29, T84 and MCF-7 after 48 h treatment with lycopene, and changes in the fraction of cells retained in different cell cycle phases. Lycopene promoted also cell cycle arrest followed by decreased cell viability in majority of cell lines after 96 h, as compared to controls. Furthermore, an increase in apoptosis was observed in four cell lines (T-84, HT-29, MCF-7 and DU145 when cells were treated with lycopene. Conclusions Our findings show the capacity of lycopene to inhibit cell proliferation, arrest cell cycle in different phases and increase apoptosis, mainly in breast, colon and prostate lines after 96 h. These observations suggest that lycopene may alter cell cycle regulatory proteins depending on the type of cancer and the dose of lycopene administration. Taken together, these data indicated that the antiproliferative effect of lycopene was cellular type, time and dose-dependent.

  2. Function of trehalose and glycogen in cell cycle progression and cell viability in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Silljé, H H; Paalman, J W; ter Schure, E G; Olsthoorn, S Q; Verkleij, A J; Boonstra, Johannes; Verrips, C T

    Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation.

  3. High Nutrient Levels and TORC1 Activity Reduce Cell Viability following Prolonged Telomere Dysfunction and Cell Cycle Arrest

    Directory of Open Access Journals (Sweden)

    Julia Klermund

    2014-10-01

    Full Text Available Cells challenged with DNA damage activate checkpoints to arrest the cell cycle and allow time for repair. Successful repair coupled to subsequent checkpoint inactivation is referred to as recovery. When DNA damage cannot be repaired, a choice between permanent arrest and cycling in the presence of damage (checkpoint adaptation must be made. While permanent arrest jeopardizes future lineages, continued proliferation is associated with the risk of genome instability. We demonstrate that nutritional signaling through target of rapamycin complex 1 (TORC1 influences the outcome of this decision. Rapamycin-mediated TORC1 inhibition prevents checkpoint adaptation via both Cdc5 inactivation and autophagy induction. Preventing adaptation results in increased cell viability and hence proliferative potential. In accordance, the ability of rapamycin to increase longevity is dependent upon the DNA damage checkpoint. The crosstalk between TORC1 and the DNA damage checkpoint may have important implications in terms of therapeutic alternatives for diseases associated with genome instability.

  4. Acoustic perfusion processes for hybridoma cultures: viability, cell cycle and metabolic analysis

    NARCIS (Netherlands)

    Dalm, M.C.F.

    2007-01-01

    For the production of glycosylated proteins, such as monoclonal antibodies, hormones, and blood clothing factors, generally mammalian cells are used. Mammalian cells are preferred over other expression systems, such as bacteria or yeast, because they are able to glycosylate proteins in a human-like

  5. Differential concentration-specific effects of caffeine on cell viability, oxidative stress, and cell cycle in pulmonary oxygen toxicity in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Tiwari, Kirti Kumar; Chu, Chun; Couroucli, Xanthi; Moorthy, Bhagavatula; Lingappan, Krithika, E-mail: lingappa@bcm.edu

    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.

  6. Differential concentration-specific effects of caffeine on cell viability, oxidative stress, and cell cycle in pulmonary oxygen toxicity in vitro.

    Science.gov (United States)

    Tiwari, Kirti Kumar; Chu, Chun; Couroucli, Xanthi; Moorthy, Bhagavatula; Lingappan, Krithika

    2014-08-08

    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 1mM) of caffeine. Caffeine had a differential concentration-specific effect on cell cycle progression, oxidative stress and viability, with 1mM 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 1mM, 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Cell type and transfection reagent-dependent effects on viability, cell content, cell cycle and inflammation of RNAi in human primary mesenchymal cells

    DEFF Research Database (Denmark)

    Yang, Hsiao Yin; Vonk, Lucienne A.; Licht, Ruud

    2014-01-01

    % amidation), for siRNA delivery into primary mesenchymal cells including nucleus pulposus cells, articular chondrocytes and mesenchymal stem cells (MSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing by 20 nM or 200 nM siRNA at day...... 3 and day 6 post-transfection. In addition to silencing efficiency, non-specific effects such as cytotoxicity, change in DNA content and differentiation potential of cells were evaluated. Among the four transfection reagents, the commercial liposome-based agent was the most efficient reagent for siRNA...... delivery at 20 nM siRNA, followed by chitosan. Transfection using cationic liposomes, chitosan and PEI showed some decrease in viability and DNA content to varying degrees that was dependent on the siRNA dose and cell type evaluated, but independent of GAPDH knockdown. Some effects on DNA content were...

  8. Quantification of Intracellular Ice Formation and Recrystallization During Freeze-Thaw Cycles and Their Relationship with the Viability of Pig Iliac Endothelium Cells.

    Science.gov (United States)

    Liu, Xiaoli; Zhao, Gang; Shu, Zhiquan; Niu, Dan; Zhang, Zhiguo; Zhou, Ping; Cao, Yunxia; Gao, Dayong

    2016-12-01

    Quantitative evaluation of the inherent correlation between cell cryoinjuries and intracellular ice formation (IIF) together with recrystallization (IIR) is of primary importance for both optimization of biopreservation and cryotherapy. The objective of this study is to thoroughly explore the roles of IIF on cell viability by using pig iliac endothelium cells (PIECs) as model cells during freezing and thawing. The experimental results indicated that both the probabilities of IIF (PIF) and IIR (PIR) increased along with the increase of cooling rates (p evaluation with Hoechst 33342/propidium iodide double staining showed that most of the cells were killed (viability <20%) by the abovementioned freeze-thaw cycles, which indicated that the cooling rates investigated were all too rapid since large amounts of IIF and IIR were introduced. Another interesting phenomenon is that the presence of a low concentration of DMSO (1 M) tends to improve cell viability while increasing the PIF and PIR during freezing/thawing, contrary to the common belief that larger PIF corresponds to greater cryoinjury. This may be attributed to the intrinsic protection effect of DMSO by reduction of solution injury or other potential injuries. These findings may be of potential application value for both cryopreservation and cryosurgery by providing helpful additions to the existing studies on investigation of cryoinjuries of PIECs.

  9. The in vitro effects of caffeine on viability, cycle cycle profiles, proliferation, and apoptosis of glioblastomas.

    Science.gov (United States)

    Jiang, J; Lan, Y-Q; Zhang, T; Yu, M; Liu, X-Y; Li, L-H; Chen, X-P

    2015-09-01

    We studied the effects of caffeine on cell viability, cell cycle profiles, proliferation, and apoptosis in rat C6 and human U87MG glioblastoma cell lines. Cell viability was quantified by the methyl thiazolyl tetrazolium (MTT) assay. Flow cytometry was used to quantify the relative number of cells in different phases of the cell cycle, while cell proliferation was quantified using the Cell Counting Kit-8. The proportion of apoptotic cells was determined by flow cytometry, and expression of apoptosis-related proteins Caspase-3, Cyt-C, Bax and Bcl-2 by Western blot. Caffeine at doses of up to 0.5 mM did not affect cell viability in both rat C6 and human U87MG glioblastoma cells. Further studies were done using the dose of 0.5 mM. Percentage of cells in the G0/G1 phase was markedly increased, while percentage of cells in the S phase decreased, after cell treatment with caffeine. Cell proliferation was significantly inhibited by caffeine. Furthermore, caffeine induced cell apoptosis, decreased expression of Bcl-2, and increased expression of Cyt-C and Caspase-3. Caffeine inhibits proliferation and induces apoptosis in glioblastoma cells. Our results provide the experimental basis for further studies of potential role of caffeine in the treatment of glioblastomas.

  10. The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell-cycle progression in malignant bladder cancer cells in vitro and in vivo.

    Science.gov (United States)

    Zhou, Ning; Singh, Kamini; Mir, Maria C; Parker, Yvonne; Lindner, Daniel; Dreicer, Robert; Ecsedy, Jeffrey A; Zhang, Zhongfa; Teh, Bin T; Almasan, Alexandru; Hansel, Donna E

    2013-04-01

    Despite more than 70,000 new cases of bladder cancer in the United States annually, patients with advanced disease have a poor prognosis due to limited treatment modalities. We evaluated Aurora kinase A, identified as an upregulated candidate molecule in bladder cancer, as a potential therapeutic target. Gene expression in human bladder cancer samples was evaluated using RNA microarray and quantitative reverse transcriptase PCR. Effects of the Aurora kinase A inhibitor MLN8237 (Millennium) on cell dynamics in malignant T24 and UM-UC-3 and papilloma-derived RT4 bladder cells were evaluated in vitro and in vivo in a mouse xenograft model. A set of 13 genes involved in the mitotic spindle checkpoint, including Aurora kinases A and B, were upregulated in human urothelial carcinoma compared with normal urothelium. The Aurora kinase A inhibitor MLN8237 induced cell-cycle arrest, aneuploidy, mitotic spindle failure, and apoptosis in the human bladder cancer cell lines T24 and UM-UC-3. MLN8237 also arrested tumor growth when administered orally over 4 weeks in a mouse bladder cancer xenograft model. Finally, in vitro sequential administration of MLN8237 with either paclitaxel or gemcitabine resulted in synergistic cytotoxic effects in T24 cells. Mitotic spindle checkpoint dysfunction is a common characteristic of human urothelial carcinoma and can be exploited with pharmacologic Aurora A inhibition. Given our demonstration of the ability of the Aurora A inhibitor MLN8237 to inhibit growth of bladder cancer in vitro and in vivo, we conclude that Aurora kinase inhibitors warrant further therapeutic investigation in bladder cancer. ©2013 AACR.

  11. The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

    Science.gov (United States)

    Zhou, Ning; Singh, Kamini; Mir, Maria C.; Parker, Yvonne; Lindner, Daniel; Dreicer, Robert; Ecsedy, Jeffrey A; Zhang, Zhongfa; Teh, Bin T.; Almasan, Alexandru; Hansel, Donna E.

    2013-01-01

    PURPOSE Despite over 70,000 new cases of bladder cancer in the United States annually, patients with advanced disease have a poor prognosis due to limited treatment modalities. We evaluated Aurora kinase A, identified as an upregulated candidate molecule in bladder cancer, as a potential therapeutic target. EXPERIMENTAL DESIGN Gene expression in human bladder cancer samples was evaluated using RNA microarray and quantitative reverse-transcriptase PCR. Effects of the Aurora kinase A inhibitor MLN8237 (Millennium) on cell dynamics in malignant T24 and UM-UC-3 and papilloma-derived RT4 bladder cells were evaluated in vitro and in vivo in a mouse xenograft model. RESULTS A set of 13 genes involved in the mitotic spindle checkpoint, including Aurora kinases A and B, were upregulated in human urothelial carcinoma compared to normal urothelium. The Aurora kinase A inhibitor MLN8237 induced cell cycle arrest, aneuploidy, mitotic spindle failure, and apoptosis in the human bladder cancer cell lines T24 and UM-UC-3. MLN8237 also arrested tumor growth when administered orally over 4 weeks in a mouse bladder cancer xenograft model. Finally, in vitro sequential administration of MLN8237 with either paclitaxel or gemcitabine resulted in synergistic cytotoxic effects in T24 cells. CONCLUSIONS Mitotic spindle checkpoint dysfunction is a common characteristic of human urothelial carcinoma, and can be exploited with pharmacologic Aurora A inhibition. Given our demonstration of the ability of the Aurora A inhibitor MLN8237 to inhibit growth of bladder cancer in vitro and in vivo, we conclude that Aurora kinase inhibitors warrant further therapeutic investigation in bladder cancer. PMID:23403633

  12. Cell type and transfection reagent-dependent effects on viability, cell content, cell cycle and inflammation of RNAi in human primary mesenchymal cells

    NARCIS (Netherlands)

    Yang, Hsiao-yin; Vonk, Lucienne A; Licht, Ruud; van Boxtel, Antonetta M G; Bekkers, Joris E J; Kragten, Angela H M; Hein, San; Varghese, Oommen P; Howard, Kenneth A; Öner, F Cumhur; Dhert, Wouter J A; Creemers, Laura B

    2014-01-01

    The application of RNA interference (RNAi) has great therapeutic potential for degenerative diseases of cartilaginous tissues by means of fine tuning the phenotype of cells used for regeneration. However, possible non-specific effects of transfection per se might be relevant for future clinical

  13. Viability of mesenchymal stem cells during electrospinning

    Directory of Open Access Journals (Sweden)

    G. Zanatta

    2012-02-01

    Full Text Available 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.

  14. Effect of salt hyperosmotic stress on yeast cell viability

    Directory of Open Access Journals (Sweden)

    Logothetis Stelios

    2007-01-01

    Full Text Available During fermentation for ethanol production, yeasts are subjected to different kinds of physico-chemical stresses such as: initially high sugar concentration and low temperature; and later, increased ethanol concentrations. Such conditions trigger a series of biological responses in an effort to maintain cell cycle progress and yeast cell viability. Regarding osmostress, many studies have been focused on transcriptional activation and gene expression in laboratory strains of Saccharomyces cerevisiae. The overall aim of this present work was to further our understanding of wine yeast performance during fermentations under osmotic stress conditions. Specifically, the research work focused on the evaluation of NaCl-induced stress responses of an industrial wine yeast strain S. cerevisiae (VIN 13, particularly with regard to yeast cell growth and viability. The hypothesis was that osmostress conditions energized specific genes to enable yeast cells to survive under stressful conditions. Experiments were designed by pretreating cells with different sodium chloride concentrations (NaCl: 4%, 6% and 10% w/v growing in defined media containing D-glucose and evaluating the impact of this on yeast growth and viability. Subsequent fermentation cycles took place with increasing concentrations of D-glucose (20%, 30%, 40% w/v using salt-adapted cells as inocula. We present evidence that osmostress induced by mild salt pre-treatments resulted in beneficial influences on both cell viability and fermentation performance of an industrial wine yeast strain.

  15. Developmental kinetics of the first cell cycles of bovine in vitro PRODUCED EMBRYOS IN RELATION TO THEIR IN VITRO VIABILITY AND SEX

    DEFF Research Database (Denmark)

    Holm, P; Shukri, N.N; Vajta, Gabor

    1998-01-01

    The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3......) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period...... were included (n=392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38. The respective lengths of the first 4 cell cycles of viable embryos were 32.0 + 3.9, g...

  16. Monitoring cell growth, viability, and apoptosis.

    Science.gov (United States)

    Butler, Michael; Spearman, Maureen; Braasch, Katrin

    2014-01-01

    The accurate determination of cell growth and viability is pivotal to monitoring a bioprocess. Direct methods to determine the cell growth and/or viability in a bioprocess include microscopic counting, electronic particle counting, image analysis, in situ biomass monitoring, and dieletrophoretic cytometry. These methods work most simply when a fixed volume sample can be taken from a suspension culture. Manual microscopic counting is laborious but affords the advantage of allowing cell viability to be determined if a suitable dye is included. Electronic particle counting is a rapid total cell count method for replicate samples, but some data distortion may occur if the sample has significant cell debris or cell aggregates. Image analysis based on the use of digital camera images acquired through a microscope has advanced rapidly with the availability of several commercially available software packages replacing manual microscopic counting and viability determination. Biomass probes detect cells by their dielectric properties or their internal concentration of NADH and can be used as a continuous monitor of the progress of a culture. While the monitoring of cell growth and viability is an integral part of a bioprocess, the monitoring of apoptosis induction is also becoming more and more important in bioprocess control to increase volumetric productivity by extending bioprocess duration. Different fluorescent assays allow for the detection of apoptotic characteristics in a cell sample.Indirect methods of cell determination involve the chemical analysis of a culture component or a measure of metabolic activity. These methods are most useful when it is difficult to obtain intact cell samples. However, the relationship between these parameters and the cell number may not be linear through the phases of a cell culture. The determination of nucleic acid (DNA) or total protein can be used as an estimate of biomass, while the depletion of glucose from the media can be used

  17. Cell Viability in Arthroscopic Versus Open Autologous Chondrocyte Implantation.

    Science.gov (United States)

    Biant, Leela C; Simons, Michiel; Gillespie, Trudi; McNicholas, Michael J

    2017-01-01

    Autologous chondrocyte implantation (ACI) is an effective method of repair of articular cartilage defects. It is a 2-stage operation, with the second stage most commonly performed via mini-arthrotomy. Arthroscopic ACI is gaining popularity, as it is less invasive and may accelerate early rehabilitation. However, handling and manipulation of the implant have been shown to cause chondrocyte cell death. To assess the number and viability of cells delivered via an open versus arthroscopic approach in ACI surgery. Controlled laboratory study. Sixteen ACI surgeries were performed on young cadaveric knees by 2 experienced surgeons: 8 via mini-arthrotomy and 8 arthroscopically. Live and dead cells were stained and counted on implants after surgery. The cell number and viability were assessed using confocal laser scanning microscopy. Surgery was timed from knife to skin until the end of cycling the knee 10 times after implantation of the cell-membrane construct. On receipt of cell membranes after transportation from the laboratory, ≥92% of the cells were viable. There were significantly more remaining cells (8.47E+07 arthroscopic vs 1.41E+08 open; P arthroscopic vs 37.34% open; P arthroscopic technique. Open surgery was of a significantly shorter duration (6 vs 32 minutes; P arthroscopic technique. The viability of cells delivered for ACI via an arthroscopic approach was 16 times less than via an open approach. The mini-arthrotomy approach is recommended until long-term clinical comparative data are available.

  18. Optimizing cell viability in droplet-based cell deposition

    NARCIS (Netherlands)

    Hendriks, Jan; Willem Visser, Claas; Henke, Sieger; Leijten, Jeroen; Saris, Daniël B F|info:eu-repo/dai/nl/241604443; Sun, Chao; Lohse, Detlef; Karperien, Marcel

    2015-01-01

    Biofabrication commonly involves the use of liquid droplets to transport cells to the printed structure. However, the viability of the cells after impact is poorly controlled and understood, hampering applications including cell spraying, inkjet bioprinting, and laser-assisted cell transfer. Here,

  19. Exercise regulates breast cancer cell viability

    DEFF Research Database (Denmark)

    Dethlefsen, Christine; Lillelund, Christian; Midtgaard, Julie

    2016-01-01

    Purpose: Exercise decreases breast cancer risk and disease recurrence, but the underlying mechanisms are unknown. Training adaptations in systemic factors have been suggested as mediating causes. We aimed to examine if systemic adaptations to training over time, or acute exercise responses......, in breast cancer survivors could regulate breast cancer cell viability in vitro. Methods: Blood samples were collected from breast cancer survivors, partaking in either a 6-month training intervention or across a 2 h acute exercise session. Changes in training parameters and systemic factors were evaluated...... and pre/post exercise-conditioned sera from both studies were used to stimulate breast cancer cell lines (MCF-7, MDA-MB-231) in vitro. Results: Six months of training increased VO2peak (16.4 %, p

  20. Puget Sound steelhead life cycle model analyses - Population Viability Analysis

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This research was initiated by the Puget Sound Steelhead Technical Recovery Team to develop viability criteria for threatened Puget Sound steelhead and to support...

  1. Viability of dielectrophoretically trapped neuronal cortical cells in culture

    NARCIS (Netherlands)

    Heida, Tjitske; Vulto, P; Rutten, Wim; Marani, Enrico

    2001-01-01

    Negative dielectrophoretic trapping of neural cells is an efficient way to position neural cells on the electrode sites of planar micro-electrode arrays. The preservation of viability of the neural cells is essential for this approach. This study investigates the viability of postnatal cortical rat

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

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    2014-01-01

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

  3. Femtosecond optical transfection of cells:viability and efficiency

    National Research Council Canada - National Science Library

    D. Stevenson; B. Agate; X. Tsampoula; P. Fischer; C. T. A. Brown; W. Sibbett; A. Riches; F. Gunn-Moore; K. Dholakia

    2006-01-01

    .... However, there remains no study into the true efficiency of this procedure. Here, we present a detailed analysis of transfection efficiency and cell viability for femtosecond optical transfection using a titanium sapphire laser at 800 nm...

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

    Science.gov (United States)

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

    2010-04-01

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

  5. Inhibition of Geranylgeranyl Transferase-I Decreases Cell Viability of HTLV-1-Transformed Cells

    Directory of Open Access Journals (Sweden)

    Cynthia A. Pise-Masison

    2011-10-01

    Full Text Available Human T-cell leukemia virus type-1 (HTLV-1 is the etiological agent of adult T-cell leukemia (ATL, an aggressive and highly chemoresistant malignancy. Rho family GTPases regulate multiple signaling pathways in tumorigenesis: cytoskeletal organization, transcription, cell cycle progression, and cell proliferation. Geranylgeranylation of Rho family GTPases is essential for cell membrane localization and activation of these proteins. It is currently unknown whether HTLV-1-transformed cells are preferentially sensitive to geranylgeranylation inhibitors, such as GGTI-298. In this report, we demonstrate that GGTI-298 decreased cell viability and induced G2/M phase accumulation of HTLV-1-transformed cells, independent of p53 reactivation. HTLV-1-LTR transcriptional activity was inhibited and Tax protein levels decreased following treatment with GGTI-298. Furthermore, GGTI-298 decreased activation of NF-κB, a downstream target of Rho family GTPases. These studies suggest that protein geranylgeranylation contributes to dysregulation of cell survival pathways in HTLV-1-transformed cells.

  6. Inhibition of geranylgeranyl transferase-I decreases cell viability of HTLV-1-transformed cells.

    Science.gov (United States)

    Edwards, Dustin C; McKinnon, Katherine M; Fenizia, Claudio; Jung, Kyung-Jin; Brady, John N; Pise-Masison, Cynthia A

    2011-10-01

    Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL), an aggressive and highly chemoresistant malignancy. Rho family GTPases regulate multiple signaling pathways in tumorigenesis: cytoskeletal organization, transcription, cell cycle progression, and cell proliferation. Geranylgeranylation of Rho family GTPases is essential for cell membrane localization and activation of these proteins. It is currently unknown whether HTLV-1-transformed cells are preferentially sensitive to geranylgeranylation inhibitors, such as GGTI-298. In this report, we demonstrate that GGTI-298 decreased cell viability and induced G(2)/M phase accumulation of HTLV-1-transformed cells, independent of p53 reactivation. HTLV-1-LTR transcriptional activity was inhibited and Tax protein levels decreased following treatment with GGTI-298. Furthermore, GGTI-298 decreased activation of NF-κB, a downstream target of Rho family GTPases. These studies suggest that protein geranylgeranylation contributes to dysregulation of cell survival pathways in HTLV-1-transformed cells.

  7. Mitochondrial dynamics and the cell cycle

    Science.gov (United States)

    Nuclear-mitochondrial (NM) communication impacts many aspects of plant development including vigor, sterility and viability. Dynamic changes in mitochondrial number, shape, size, and cellular location takes place during the cell cycle possibly impacting the process itself and leading to distribution...

  8. Cell structure and percent viability by a slide centrifuge technique.

    Science.gov (United States)

    Fitzgerald, M G; Hosking, C S

    1982-01-01

    It was found that a slide centrifuge (Cytospin) preparation of a cell suspension allowed a reliable assessment of not only cell structure but also the percentage of non-viable cells. The non-viable cells appeared as "smear" cells and paralleled in number the cells taking up trypan blue. Direct experiment showed the unstained viable cells in a trypan blue cell suspension remained intact in a Cytospin preparation while the cells taking up trypan blue were the "smear" cells. The non-viability of the "smear" cells was confirmed by their inability to survive in culture. Images PMID:7040483

  9. Apoptosis and cell cycle

    Directory of Open Access Journals (Sweden)

    Petrović Marija

    2014-01-01

    Full Text Available Apoptosis, a form of programmed cell death, is used to eliminate individual cells surrounded by normal cell population. It is a controlled way of cell death in which the cell actively participates by conducting precise, gene-regulated program of self-destruction, that is, cell 'suicide.' Active synthesis of macromolecules is necessary during this process. Death of individual cells is necessary to maintain a balance in living systems, so the process of apoptosis is continuously present in the body, which allows normal development, tissue homeostasis, and many other physiological processes. The molecular mechanisms that regulate apoptosis are functionally linked to other cellular mechanisms, such as control of the cell cycle, cell proliferation and differentiation, genomic stability and cellular metabolism. Damage to the DNA molecule, caused both spontaneously and under the influence of various chemical and physical agents, leads to the cell cycle arrest and activation of mechanisms that repair the damage. Depending on the type and extent of the damage, the cell either continues progression through the cell cycle, or activates the mechanisms that lead to apoptosis. Disturbances in the regulation of apoptosis and cell cycle present the molecular and biological basis of many diseases. Because of the importance of these processes during the development and progression of tumors, their use as biological markers is one of the main strategies in the formation of therapeutic approaches for the treatment of cancer.

  10. The Use of Water Vapor as a Refrigerant: Impact of Cycle Modifications on Commercial Viability

    Energy Technology Data Exchange (ETDEWEB)

    Brandon F. Lachner, Jr.; Gregory F. Nellis; Douglas T. Reindl

    2004-08-30

    This project investigated the economic viability of using water as the refrigerant in a 1000-ton chiller application. The most attractive water cycle configuration was found to be a flash-intercooled, two-stage cycle using centrifugal compressors and direct contact heat exchangers. Component level models were developed that could be used to predict the size and performance of the compressors and heat exchangers in this cycle as well as in a baseline, R-134a refrigeration cycle consistent with chillers in use today. A survey of several chiller manufacturers provided information that was used to validate and refine these component models. The component models were integrated into cycle models that were subsequently used to investigate the life-cycle costs of both an R-134a and water refrigeration cycle. It was found that the first cost associated with the water as a refrigerant cycle greatly exceeded the savings in operating costs associated with its somewhat higher COP. Therefore, the water refrigeration cycle is not an economically attractive option to today's R-134a refrigeration system. There are a number of other issues, most notably the requirements associated with purging non-condensable gases that accumulate in a direct contact heat exchanger, which will further reduce the economic viability of the water cycle.

  11. Different effects of sonoporation on cell morphology and viability

    Directory of Open Access Journals (Sweden)

    Ji-Zhen Zhang

    2012-05-01

    Full Text Available The objective of our study was to investigate changes in cell morphology and viability after sonoporation. Sonoportion was achieved by ultrasound (21 kHz exposure on adherent human prostate cancer DU145 cells in the cell culture dishes with the presence of microbubble contrast agents and calcein (a cell impermeant dye. We investigated changes in cell morphology immediately after sonoporation under scanning electron microscope (SEM and changes in cell viability immediately and 6 h after sonoporation under fluorescence microscope. It was shown that various levels of intracellular calcein uptake and changes in cell morphology can be caused immediately after sonoporation: smooth cell surface, pores in the membrane and irregular cell surface. Immediately after sonoporation, both groups of cells with high levels of calcein uptake and low levels of calcein uptake were viable; 6 h after sonoporation, group of cells with low levels of calcein uptake still remained viable, while group of cells with high levels of calcein uptake died. Sonoporation induces different effects on cell morphology, intracellular calcein uptake and cell viability.

  12. Viability studies of optically trapped T-cells

    Science.gov (United States)

    McAlinden, Niall; Glass, David G.; Millington, Owain; Wright, Amanda J.

    2011-10-01

    We present a viability study of optically trapped live T cell hybridomas. T cells form an important part of the adaptive immune response system which is responsible for fighting particular pathogens or diseases. The cells of interest were directly trapped by a laser operating at a wavelength of 1064 nm and their viability measured as a function of time. Cell death was monitored using an inverted fluorescent microscope to observe the uptake by the cell of the fluorescent dye propidium iodide. Studies were undertaken at various laser powers and beam profiles. There is a growing interest in optically trapping immune cells and this is the first study that investigates the viability of a T cell when trapped using a conventional optical trapping system. In such experiments it is crucial that the T cell remains viable and trapping the cell directly means that any artefacts due to a cell-bead interface are removed. Our motivation behind this experiment is to use optical tweezers to gain a greater understanding of the interaction forces between T cells and antigen presenting cells. Measuring these interactions has become important due to recent theories which indicate that the strength of this interaction may underlie the activation of the T-cell and subsequent immune response.

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

    Science.gov (United States)

    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.

  14. Interactions between Plant Extracts and Cell Viability Indicators ...

    African Journals Online (AJOL)

    Interactions between Plant Extracts and Cell Viability. Indicators during Cytotoxicity Testing: Implications for. Ethnopharmacological Studies. Sze Mun Chan1, Kong Soo Khoo2 and Nam Weng Sit1*. 1Department of Biomedical Science, 2Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman,.

  15. Specific cell cycle synchronization with butyrate and cell cycle analysis

    Science.gov (United States)

    Synchronized cells have been invaluable for many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. To explore the possibility of using butyrate-blocked cells to obtain synchronized cells, we investigated the property of the cell cyc...

  16. Activin Receptor Signaling Regulates Prostatic Epithelial Cell Adhesion and Viability

    Directory of Open Access Journals (Sweden)

    Derek P. Simon

    2009-04-01

    Full Text Available Mutational changes coupled with endocrine, paracrine, and/or autocrine signals regulate cell division during carcinogenesis. The hormone signals remain undefined, although the absolute requirement in vitro for fetal serum indicates the necessity for a fetal serum factor(s in cell proliferation. Using prostatic cancer cell (PCC lines as a model of cancer cell proliferation, we have identified the fetal serum component activin A and its signaling through the activin receptor type II (ActRII, as necessary, although not sufficient, for PCC proliferation. Activin A induced Smad2 phosphorylation and PCC proliferation, but only in the presence of fetal bovine serum (FBS. Conversely, activin A antibodies and inhibin A suppressed FBS-induced PCC proliferation confirming activin A as one of multiple serum components required for PCC proliferation. Basic fibroblast growth factor was subsequently shown to synergize activin A-induced PCC proliferation. Inhibition of ActRII signaling using a blocking antibody or antisense-P decreased mature ActRII expression, Smad2 phosphorylation, and the apparent viability of PCCs and neuroblastoma cells grown in FBS. Suppression of ActRII signaling in PCC and neuroblastoma cells did not induce apoptosis as indicated by the ratio of active/inactive caspase 3 but did correlate with increased cell detachment and ADAM-15 expression, a disintegrin whose expression is strongly correlated with prostatic metastasis. These findings indicate that ActRII signaling is required for PCC and neuroblastoma cell viability, with ActRII mediating cell fate via the regulation of cell adhesion. That ActRII signaling governs both cell viability and cell adhesion has important implications for developing therapeutic strategies to regulate cancer growth and metastasis.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    M.B. Aires

    2015-08-01

    Full Text Available 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Aires, M.B. [Departamento de Morfologia, Universidade Federal de Sergipe, São Cristóvão, SE (Brazil); Santos, J.R.A. [Departamento de Enfermagem, Universidade Federal de Sergipe, São Cristóvão, SE (Brazil); Souza, K.S.; Farias, P.S. [Departamento de Morfologia, Universidade Federal de Sergipe, São Cristóvão, SE (Brazil); Santos, A.C.V. [Departamento de Enfermagem, Universidade Federal de Sergipe, São Cristóvão, SE (Brazil); Fioretto, E.T. [Departamento de Morfologia, Universidade Federal de Sergipe, São Cristóvão, SE (Brazil); Maria, D.A. [Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, SP (Brazil)

    2015-07-10

    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.

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

    Science.gov (United States)

    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. Copyright © 2012 John Wiley & Sons, Ltd.

  1. Effects of drinking desalinated seawater on cell viability and proliferation.

    Science.gov (United States)

    Macarrão, Camila Longhi; Bachi, André Luis Lacerda; Mariano, Mario; Abel, Lucia Jamli

    2017-06-01

    Desalination of seawater is becoming an important means to address the increasing scarcity of freshwater resources in the world. Seawater has been used as drinking water in the health, food, and medical fields and various beneficial effects have been suggested, although not confirmed. Given the presence of 63 minerals and trace elements in drinking desalinated seawater (63 DSW), we evaluated their effects on the behavior of tumorigenic and nontumorigenic cells through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and annexin-V-fluorescein isothiocyanate/propidium iodide staining. Our results showed that cell viability and proliferation in the presence of 63 DSW were significantly greater than in mineral water and in the presence of fetal bovine serum in a dose-dependent manner. Furthermore, 63 DSW showed no toxic effect on murine embryonic fibroblast (NIH-3T3) and murine melanoma (B16-F10) cells. In another assay, we also showed that pre-treatment of non-adherent THP-1 cells with 63 DSW reduces apoptosis incidence, suggesting a protective effect against cell death. We conclude that cell viability and proliferation were improved by the mineral components of 63 DSW and this effect can guide further studies on health effects associated with DSW consumption.

  2. Cytotoxicity and Effects on Cell Viability of Nickel Nanowires

    KAUST Repository

    Rodriguez, Jose E.

    2013-05-01

    Recently, magnetic nanoparticles are finding an increased use in biomedical applications and research. Nanobeads are widely used for cell separation, biosensing and cancer therapy, among others. Due to their properties, nanowires (NWs) are gaining ground for similar applications and, as with all biomaterials, their cytotoxicity is an important factor to be considered before conducting biological studies with them. In this work, the cytotoxic effects of nickel NWs (Ni NWs) were investigated in terms of cell viability and damage to the cellular membrane. Ni NWs with an average diameter of 30-34 nm were prepared by electrodeposition in nanoporous alumina templates. The templates were obtained by a two-step anodization process with oxalic acid on an aluminum substrate. Characterization of NWs was done using X-Ray diffraction (XRD) and energy dispersive X-Ray analysis (EDAX), whereas their morphology was observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cell viability studies were carried out on human colorectal carcinoma cells HCT 116 by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell proliferation colorimetric assay, whereas the lactate dehydrogenase (LDH) homogenous membrane fluorimetric assay was used to measure the degree of cell membrane rupture. The density of cell seeding was calculated to obtain a specific cell number and confluency before treatment with NWs. Optical readings of the cell-reduced MTT products were measured at 570 nm, whereas fluorescent LDH membrane leakage was recorded with an excitation wavelength of 525 nm and an emission wavelength of 580 - 640 nm. The effects of NW length, cell exposure time, as well as NW:cell ratio, were evaluated through both cytotoxic assays. The results show that cell viability due to Ni NWs is affected depending on both exposure time and NW number. On the other hand, membrane rupture and leakage was only significant at later exposure times. Both

  3. Endothelin receptor B antagonists decrease glioma cell viability independently of their cognate receptor

    Directory of Open Access Journals (Sweden)

    Patterson Paul H

    2008-11-01

    Full Text Available Abstract Background Endothelin receptor antagonists inhibit the progression of many cancers, but research into their influence on glioma has been limited. Methods We treated glioma cell lines, LN-229 and SW1088, and melanoma cell lines, A375 and WM35, with two endothelin receptor type B (ETRB-specific antagonists, A-192621 and BQ788, and quantified viable cells by the capacity of their intracellular esterases to convert non-fluorescent calcein AM into green-fluorescent calcein. We assessed cell proliferation by labeling cells with carboxyfluorescein diacetate succinimidyl ester and quantifying the fluorescence by FACS analysis. We also examined the cell cycle status using BrdU/propidium iodide double staining and FACS analysis. We evaluated changes in gene expression by microarray analysis following treatment with A-192621 in glioma cells. We examined the role of ETRB by reducing its expression level using small interfering RNA (siRNA. Results We report that two ETRB-specific antagonists, A-192621 and BQ788, reduce the number of viable cells in two glioma cell lines in a dose- and time-dependent manner. We describe similar results for two melanoma cell lines. The more potent of the two antagonists, A-192621, decreases the mean number of cell divisions at least in part by inducing a G2/M arrest and apoptosis. Microarray analysis of the effects of A-192621 treatment reveals up-regulation of several DNA damage-inducible genes. These results were confirmed by real-time RT-PCR. Importantly, reducing expression of ETRB with siRNAs does not abrogate the effects of either A-192621 or BQ788 in glioma or melanoma cells. Furthermore, BQ123, an endothelin receptor type A (ETRA-specific antagonist, has no effect on cell viability in any of these cell lines, indicating that the ETRB-independent effects on cell viability exhibited by A-192621 and BQ788 are not a result of ETRA inhibition. Conclusion While ETRB antagonists reduce the viability of glioma cells

  4. Ponatinib reduces viability, migration, and functionality of human endothelial cells.

    Science.gov (United States)

    Gover-Proaktor, Ayala; Granot, Galit; Shapira, Saar; Raz, Oshrat; Pasvolsky, Oren; Nagler, Arnon; Lev, Dorit L; Inbal, Aida; Lubin, Ido; Raanani, Pia; Leader, Avi

    2017-06-01

    Tyrosine kinase inhibitors (TKIs) have revolutionized the prognosis of chronic myeloid leukemia. With the advent of highly efficacious therapy, the focus has shifted toward managing TKI adverse effects, such as vascular adverse events (VAEs). We used an in vitro angiogenesis model to investigate the TKI-associated VAEs. Our data show that imatinib, nilotinib, and ponatinib reduce human umbilical vein endothelial cells (HUVECs) viability. Pharmacological concentrations of ponatinib induced apoptosis, reduced migration, inhibited tube formation of HUVECs, and had a negative effect on endothelial progenitor cell (EPC) function. Furthermore, in HUVECs transfected with VEGF receptor 2 (VEGFR2), the effect of ponatinib on tube formation and on all parameters representing normal endothelial cell function was less prominent than in control cells. This is the first report regarding the pathogenesis of ponatinib-associated VAEs. The antiangiogenic effect of ponatinib, possibly mediated by VEGFR2 inhibition, as shown in our study, is another piece in the intricate puzzle of TKI-associated VAEs.

  5. Microscale Electro-Hydrodynamic Cell Printing with High Viability.

    Science.gov (United States)

    He, Jiankang; Zhao, Xiang; Chang, Jinke; Li, Dichen

    2017-12-01

    Cell printing has gained extensive attentions for the controlled fabrication of living cellular constructs in vitro. Various cell printing techniques are now being explored and developed for improved cell viability and printing resolution. Here an electro-hydrodynamic cell printing strategy is developed with microscale resolution (95%). Unlike the existing electro-hydrodynamic cell jetting or printing explorations, insulating substrate is used to replace conventional semiconductive substrate as the collecting surface which significantly reduces the electrical current in the electro-hydrodynamic printing process from milliamperes (>0.5 mA) to microamperes (printed cells. The smallest width of the electro-hydrodynamically printed hydrogel filament is 82.4 ± 14.3 µm by optimizing process parameters. Multiple hydrogels or multilayer cell-laden constructs can be flexibly printed under cell-friendly conditions. The printed cells in multilayer hydrogels kept alive and gradually spread during 7-days culture in vitro. This exploration offers a novel and promising cell printing strategy which might benefit future biomedical innovations such as microscale tissue engineering, organ-on-a-chip systems, and nanomedicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Cell adhesion and viability of human endothelial cells on electrospun polymer scaffolds

    OpenAIRE

    Matschegewski Claudia; Matthies Jörn-Bo; Grabow Niels; Schmitz Klaus-Peter

    2016-01-01

    The usage of electrospun polymer scaffolds is a promising approach for artificial heart valve design. This study aims at the evaluation of biological performance of nanofibrous polymer scaffolds poly(L-lactide) PLLA L210, PLLA L214 and polyamide-6 fabricated by electrospinning via analyzing viability, adhesion and morphology of human umbilical vein endothelial cells (EA.hy926). Nanofibrous surface topography was shown to influence cell phenotype and cell viability according to the observation...

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

    Science.gov (United States)

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

    2013-12-31

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

  8. The viability of MCM-41 as separator in secondary alkaline cells

    Science.gov (United States)

    Meskon, S. R.; Othman, R.; Ani, M. H.

    2018-01-01

    The viability of MCM-41 membrane as a separator material in secondary alkaline cell is investigated. The inorganic membrane was employed in an alkaline nickel-zinc system. MCM-41 mesoporous material consists of arrays of hexagonal nano-pore channels. The membrane was synthesized using sol-gel route from parent solution comprising of quarternary ammonium surfactant, cethyltrimethylammonium bromide C16H33(CH3)3NBr (CTAB), hydrochloric acid (HCl), deionized water (H2O), ethanol (C2H5OH), and tetraethylortosilicate (TEOS). Both the anodic zinc/zinc oxide and cathodic nickel hydroxide electrodeposited film were coated with MCM-41 membrane. The Ni/MCM-41/Zn alkaline cell was then subjected to 100-cycle durability test and the structural stability of MCM-41 separator throughout the progression of the charge-discharge cycles is studied. X-ray diffraction (XRD) analysis on the dismantled cell shows that MCM-41 began to transform to lamellar MCM-50 on the 5th cycle and transformed almost completely on the 25th cycle. The phase transformation of MCM-41 hexagonal structure into gel-like MCM-50 prevents the mesoporous cell separator from diminished in the caustic alkaline surround. This work has hence demonstrated MCM-41 membrane is viable to be employed in secondary alkaline cells.

  9. Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest

    Directory of Open Access Journals (Sweden)

    Avdieiev S. S.

    2015-08-01

    Full Text Available Aim. To analyze the effect of the CHI3L2 protein on malignant and non-malignant cell viability, and determined the CHI3L2 impact on the cell cycle and signaling pathways involved in the cell cycle regulation. Methods. MTT-based cell proliferation assay, FACS, western blot analysis. Results. The CHI3L2 protein inhibits the glioma cells viability and potentiates the effect of anti-cancer cytotoxic agents. The CHI3L2 treatment results in the G1/S transition arrest. CHI3L2 provoked a dramatic reduction of pRB phosphorylation and a significant decrease in the cyclin D1 expression, whereas the p53 and p21 expression levels were substantially increased. Conclusions. The CHI3L2 protein, which is overexpressed in human gliomas, is a negative regulator of the glioma cells viability. The reduced cell viability after the CHI3L2 treatment could be due to the activation of pRB and p53 and the downregulation of cyclin D.

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

    Science.gov (United States)

    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.

  11. Hesperidin inhibits ovarian cancer cell viability through endoplasmic reticulum stress signaling pathways

    OpenAIRE

    Zhao, Jun; Li, Yali; Gao, Jinfang; De, Yinshan

    2017-01-01

    Hesperidin is a vitamin P flavonoid compound primarily present in citrus fruits. The aim of the present study was to investigate whether hesperidin inhibits ovarian cancer cell viability via endoplasmic reticulum stress signaling pathways. A2780 cells were treated with various doses of hesperidin for 6, 12 or 24 h, and the viability of A2780 cells was assessed using the MTT assay. Hesperidin decreased the viability of A2780 cells and increased cytotoxicity in a dose- and time-dependent manner...

  12. Effect of different carbon nanotubes on cell viability and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    De Nicola, Milena [Dipartimento di Biologia, Universita di Roma Tor Vergata (Italy); Gattia, Daniele Mirabile [Divisione nuovi materiali ENEA Casaccia (Italy); Bellucci, Stefano [INFN Laboratori Nazionali di Frascati (Italy); De Bellis, Giovanni [INFN Laboratori Nazionali di Frascati (Italy); Micciulla, Federico [INFN Laboratori Nazionali di Frascati (Italy); Pastore, Roberto [INFN Laboratori Nazionali di Frascati (Italy); Tiberia, Alessandra [INFN Laboratori Nazionali di Frascati (Italy); Cerella, Claudia [Dipartimento di Biologia, Universita di Roma Tor Vergata (Italy); D' Alessio, Maria [Dipartimento di Biologia, Universita di Roma Tor Vergata (Italy); Antisari, Marco Vittori [Divisione nuovi materiali ENEA Casaccia (Italy); Marazzi, Renzo [Divisione nuovi materiali ENEA Casaccia (Italy); Traversa, Enrico [Dipartimento di Chimica, Universita di Roma Tor Vergata (Italy); Magrini, Andrea [Cattedra Medicina del Lavoro, Universita di Roma Tor Vergata (Italy); Bergamaschi, Antonio [Cattedra di Medicina del Lavoro, Universita Cattolica del Sacro Cuore, Rome (Italy); Ghibelli, Lina [Dipartimento di Biologia, Universita di Roma Tor Vergata (Italy)

    2007-10-03

    Carbon nanotubes (CNTs) are a focus of intense research for their potential applications in multiple diverse applications, including innovative biomedical applications. Due to their very recent discovery, little information is available about the biocompatibility and toxicity of this new class of nanoparticle, and a systematic study on biological interference is lacking. Thus, we decided to explore the toxicity of three different types of carbon nanotube, differing in preparation (arc discharge versus catalysed chemical vapour deposition); size (10-50 versus 100-150 nm wide x 1-10 {mu}m long); contaminants (amorphous C, graphite, fullerenes or iron) and morphological type (multi-walled, MW, or single-walled, SW) on human leukemic U937 cells. We found that these carbon nanotubes exert a strong effect on the proliferation of the reporter U937 monocytic cell. However, these CNTs did not significantly affect the cell viability. These results show that CNTs, though not directly exerting a direct cytotoxic effect, are nonetheless able to deeply alter cell behaviour, and thus we recommend thorough analyses to limit health risk due to uncontrolled exposure.

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

    Directory of Open Access Journals (Sweden)

    Junxin Wang

    2016-01-01

    Full Text Available 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.

  14. The Abbreviated Pluripotent Cell Cycle

    Science.gov (United States)

    Kapinas, Kristina; Grandy, Rodrigo; Ghule, Prachi; Medina, Ricardo; Becker, Klaus; Pardee, Arthur; Zaidi, Sayyed K.; Lian, Jane; Stein, Janet; van Wijnen, Andre; Stein, Gary

    2013-01-01

    Human embryonic stem cells and induced pluripotent stem cells proliferate rapidly and divide symmetrically producing equivalent progeny cells. In contrast, lineage committed cells acquire an extended symmetrical cell cycle. Self-renewal of tissue-specific stem cells is sustained by asymmetric cell division where one progeny cell remains a progenitor while the partner progeny cell exits the cell cycle and differentiates. There are three principal contexts for considering the operation and regulation of the pluripotent cell cycle: temporal, regulatory andstructural. The primary temporal context that the pluripotent self-renewal cell cycle of human embryonic stem cells (hESCs) is a short G1 period without reducing periods of time allocated to S phase, G2, and mitosis. The rules that govern proliferation in hESCs remain to be comprehensively established. However, several lines of evidence suggest a key role for the naïve transcriptome of hESCs, which is competent to stringently regulate the ESC cell cycle. This supports the requirements of pluripotent cells to self propagate while suppressing expression of genes that confer lineage commitment and/or tissue specificity. However, for the first time, we consider unique dimensions to the architectural organization and assembly of regulatory machinery for gene expression in nuclear microenviornments that define parameters of pluripotency. From both fundamental biological and clinical perspectives, understanding control of the abbreviated embryonic stem cell cycle can provide options to coordinate control of proliferation versus differentiation. Wound healing, tissue engineering, and cell-based therapy to mitigate developmental aberrations illustrate applications that benefit from knowledge of the biology of the pluripotent cell cycle. PMID:22552993

  15. Retinoids, retinoid analogs, and lactoferrin interact and differentially affect cell viability of 2 bovine mammary cell types in vitro.

    Science.gov (United States)

    Wang, Y; Baumrucker, C R

    2010-07-01

    Two bovine mammary cell types (BME-UV1 and MeBo cells) were used to evaluate the effect of natural retinoids, retinoid analogs, and bovine lactoferrin (bLf) on cell viability in vitro. Experiments with Alamar Blue showed a linear relationship between fluorescence and cell viability index. The BME-UV1 cells exhibited twice the metabolic activity but required half the doubling time of the MeBo cells. The BME-UV1 cells were very sensitive to all-trans retinoic acid (atRA) inhibition of cell viability (Pretinoid-induced inhibition of cell viability, depending on the type of bovine mammary cell studied.

  16. Cell adhesion and viability of human endothelial cells on electrospun polymer scaffolds

    Directory of Open Access Journals (Sweden)

    Matschegewski Claudia

    2016-09-01

    Full Text Available The usage of electrospun polymer scaffolds is a promising approach for artificial heart valve design. This study aims at the evaluation of biological performance of nanofibrous polymer scaffolds poly(L-lactide PLLA L210, PLLA L214 and polyamide-6 fabricated by electrospinning via analyzing viability, adhesion and morphology of human umbilical vein endothelial cells (EA.hy926. Nanofibrous surface topography was shown to influence cell phenotype and cell viability according to the observation of diminished cell spreading accompanied with reduced cell viability on nonwovens. Among those, highest biocompatibility was assessed for PLLA L214, although being generally low when compared to the planar control surface. Electrospinning was demonstrated as an innovative technique for the fabrication of advanced biomaterials aiming at guided cellular behavior as well as the design of novel implant platforms. A better understanding of cell–biomaterial interactions is desired to further improve implant development.

  17. Evaluation of the viability of HL60 cells in contact with commonly used microchip materials

    NARCIS (Netherlands)

    Wolbers, F.; ter Braak, Paulus Martinus; le Gac, Severine; Lüttge, Regina; Andersson, Helene; Vermes, I.; van den Berg, Albert; Jensen, K.F; Han, J.; Harrison, D.J.; Voldman, J.

    2005-01-01

    This paper presents beneficial data when deciding to perform cell experiments in lab-on- a-chip devices. The choice of material can influence the viability of mammalian cells. PDMS, precoated with serum or not, suits well for HL60 cells, demonstrating the best results in the viability experiments,

  18. MiR-101-3p Regulates the Viability of Lung Squamous Carcinoma Cells via Targeting EZH2.

    Science.gov (United States)

    Hou, Yu; Li, Lan; Ju, Yunhe; Lu, Yulin; Chang, Li; Xiang, Xudong

    2017-10-01

    The aim of this study was to investigate the effects of miR-101-3p on the viability, migration, invasion, and mitosis of lung squamous carcinoma cells by inhibiting EZH2. In this study, RT-qPCR was used to detect the expression of miR-101-3p and EZH2 in both tissues and cells at RNA level. The dual luciferase reporter gene system was used to determine whether there was targeting relationship between miR-101-3p and EZH2-3'UTR. Western Blot was used to detect the expression of EZH2 as well as the proliferation and invasion related proteins. The CCK-8 assay, Transwell invasion assay, wound healing assay and flow cytometry were conducted to test the cell viability, invasion, migration and apoptosis. The results of RT-qPCR and Western blot showed that miR-101-3p was low-expressed and EZH2 was overexpressed in lung squamous cell carcinoma tissues and cells. Meanwhile the Western blot confirmed the effects of EZH2 expression on the proliferation and invasion of carcinoma cells. The results of luciferase assay and RT-qPCR showed that miR-101-3p had a negative regulation effect on EZH2. The CCK-8 assay, Transwell invasion assay, wound healing assay and flow cytometry results showed that the inhibition of EZH2 or the up-regulation of miR-101-3p inhibited the viability, migration, invasion and cell cycle but promoted cell apoptosis of lung squamous cell carcinoma. MiR-101-3p could inhibit the viability, migration, invasion, and cell cycle of lung squamous carcinoma cells by inhibiting the EZH2. J. Cell. Biochem. 118: 3142-3149, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. Improvement In Rabbit Corneal Cell Suspension Viability After Freezing With Gingko Biloba Extrakt

    Directory of Open Access Journals (Sweden)

    Murad Aktan

    2007-01-01

    Full Text Available We investigated whether the addition of Gingko Biloba extract (EGb 761 to rabbit corneal epithelial medium before cell freezing improved cell viability after freezing then thawing. After removal of corneas, they were treated with enzymes and the corneal epithelium was prepared as a single cell suspension in freezing media with or without EGb 761. After freezing for two weeks then thawing, a higher cell viability was found in the cornea cell suspensions which had been frozen pretreated with EGb 761 in the media. The improvement with corneal cell viability with EGb 761 pretreatment is postulated to be based on the antioxidant capacity of the plant extract.

  20. Maltose-binding protein isolated from Escherichia coli induces Toll-like receptor 2-mediated viability in U937 cells.

    Science.gov (United States)

    Xiaoxia, Zhao; Weihua, Ni; Qingyong, Zhang; Fengli, Wang; Yingying, Li; Xiaxia, Sun; Zhonghui, Liu; Guixiang, Tai

    2011-07-01

    Stimulation of Toll-like receptors (TLRs) by microbial products has been utilised to potentiate immune responses against haematologic malignancies. The maltose-binding protein (MBP) of Escherichia coli could induce the activation of immune cells via TLR4. The aim of the present study was to investigate whether TLRs mediated the biological effects of MBP on U937 and Jurkat cells in vitro. METHODS We observed the effect of MBP on U937 and Jurkat cells by using the WST, cell cycle analysis and morphological observation. Further, cells were stimulated with MBP for indicated times and doses, and detected by RT-PCR, western blotting, immunohistochemistry and immunofluorescence staining to investigate the mechanisms involved in cell viability. MBP enhanced the viability of U937 and Jurkat cells, and the effects were blocked by anti-TLR2, but not anti-TLR4 in U937 cells. Further studies confirmed that MBP was able to directly bind to U937 and Jurkat cells and modulate TLR expression. The effects of MBP depended on the activation of NF-κB and MAP kinase in U937 and Jurkat cells. Our results demonstrated that MBP could directly promote U937 cell viability via TLR2. It suggested that MBP may be used as an adjuvant for participating in the immunotherapy of haematologic malignancies.

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

    Energy Technology Data Exchange (ETDEWEB)

    Whitney, Jon R [ORNL; Rodgers, Amanda [Virginia Polytechnic Institute and State University; Harvie, Erica [Virginia Polytechnic Institute and State University; Carswell, William [Virginia Polytechnic Institute and State University; Torti, Suzy [Wake Forest University, Winston-Salem; Puretzky, Alexander A [ORNL; Rouleau, Christopher M [ORNL; Geohegan, David B [ORNL; Rylander, Christopher [Virginia Polytechnic Institute and State University; Rylander, Nichole M [Virginia Polytechnic Institute and State University

    2012-01-01

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

  2. Cell motility, morphology, viability and proliferation in response to nanotopography on silicon black

    DEFF Research Database (Denmark)

    Lopacinska, Joanna M.; Gradinaru, Cristian; Wierzbicki, Rafal

    2012-01-01

    standard measurements of cell viability, proliferation, and morphology on various surfaces. We also analyzed the motility of cells on the same surfaces, as recorded in time lapse movies of sparsely populated cell cultures. We find that motility and morphology vary strongly with nano-patterns, while...... viability and proliferation show little dependence on substrate type. We conclude that motility analysis can show a wide range of cell responses e. g. over a factor of two in cell speed to different nano-topographies, where standard assays, such as viability or proliferation, in the tested cases show much...

  3. Autoradiography and the Cell Cycle.

    Science.gov (United States)

    Jones, C. Weldon

    1992-01-01

    Outlines the stages of a cell biology "pulse-chase" experiment in which the students apply autoradiography techniques to learn about the concept of the cell cycle. Includes (1) seed germination and plant growth; (2) radioactive labeling and fixation of root tips; (3) feulgen staining of root tips; (4) preparation of autoradiograms; and…

  4. Stand-Alone Solar Organic Rankine Cycle Water Pumping System and Its Economic Viability in Nepal

    Directory of Open Access Journals (Sweden)

    Suresh Baral

    2015-12-01

    Full Text Available The current study presents the concept of a stand-alone solar organic Rankine cycle (ORC water pumping system for rural Nepalese areas. Experimental results for this technology are presented based on a prototype. The economic viability of the system was assessed based on solar radiation data of different Nepalese geographic locations. The mechanical power produced by the solar ORC is coupled with a water pumping system for various applications, such as drinking and irrigation. The thermal efficiency of the system was found to be 8% with an operating temperature of 120 °C. The hot water produced by the unit has a temperature of 40 °C. Economic assessment was done for 1-kW and 5-kW solar ORC water pumping systems. These systems use different types of solar collectors: a parabolic trough collector (PTC and an evacuated tube collector (ETC. The economic analysis showed that the costs of water are $2.47/m3 (highest and $1.86/m3 (lowest for the 1-kW system and a 150-m pumping head. In addition, the cost of water is reduced when the size of the system is increased and the pumping head is reduced. The minimum volumes of water pumped are 2190 m3 and 11,100 m3 yearly for 1 kW and 5 kW, respectively. The payback period is eight years with a profitability index of 1.6. The system is highly feasible and promising in the context of Nepal.

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

    Directory of Open Access Journals (Sweden)

    David G. Popovich

    2011-01-01

    Full Text Available 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.

  6. Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

    Science.gov (United States)

    Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V McNeil; Segarra, Verónica A

    2017-01-01

    Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

  7. Carrageenan delays cell cycle progression in human cancer cells in vitro demonstrated by FUCCI imaging.

    Science.gov (United States)

    Prasedya, Eka Sunarwidhi; Miyake, Masao; Kobayashi, Daisuke; Hazama, Akihiro

    2016-08-04

    Carrageenan is a sulfated polysaccharide that exists in red seaweeds recently shown to have anticancer properties. Previous findings show various effects of carrageenan suppressing tumor cell growth. One of the hallmarks of cancer is uncontrolled proliferation, a consequence of loss of normal cell-cycle control, that underlies tumor growth. Recently there is an increasing interest in potential anticancer agents that affect cell cycle in cancer cells. Thus, in this study we investigated the effects of carrageenan on the tumor cell cycle. Using human cervical carcinoma cells (HeLa) cells as and human umbilical vein endothelial cells (HUVEC), the cytotoxic effects of kappa carrageenan (k-CO) and lambda carrageenan (λ-CO) at the concentrations of 250-2500 μg/mL were observed. Cell viability was determined using the MTT assay while cell death rates were determined using staining with calcein-AM/propidium iodide. Cell-cycle profile and progression were demonstrated with HeLa cells expressing FUCCI (fluorescence ubiquitination-based cell-cycle indicator) probes (HeLa-FUCCI). Carrageenan had no significant effect on HUVEC (normal cells). In contrast both forms of carrageenan were cytotoxic towards HeLa cells (cancer cells). Furthermore, according to cell-cycle analysis with FUCCI cells, the cell cycle of HeLa cells was delayed in specific phases due to different carrageenan treatments. Considering these results, it could be suggested that carrageenan affects the cell-cycle of HeLa cells not only by arresting the cell cycle in specific phases but also by delaying the time needed for the cell to progress through the cell cycle. Additionally, different types of carrageenans have different effects on cell cycle progression. This effect of carrageenan towards cancer cells could possibly be developed into a tumor cell-specific anticancer agent.

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kuncharin, Yanin [Medical Microbiology Interdisciplinary Program, Graduate School, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Sangphech, Naunpun [Biotechnology Program, Faculty of Science, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Kueanjinda, Patipark [Medical Microbiology Interdisciplinary Program, Graduate School, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Bhattarakosol, Parvapan [Medical Microbiology Interdisciplinary Program, Graduate School, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Palaga, Tanapat, E-mail: tanapat.p@chula.ac.th [Department of Microbiology, Faculty of Science, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand)

    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. Microfluidic high viability neural cell separation using viscoelastically tuned hydrodynamic spreading

    DEFF Research Database (Denmark)

    Wu, Zhigang; Hjort, Klas; Wicher, Grzegorz

    2008-01-01

    A high viability microfluidic cell separation technique of high throughput was demonstrated based on size difference continuous mode hydrodynamic spreading with viscoelastic tuning. Using water with fluorescent dye as sample fluid and in parallel introducing as elution a viscoelastic biocompatible...

  11. The Effect of Disinfection on Viability and Function of Baboon Red Blood Cells and Platelets

    National Research Council Canada - National Science Library

    Valeri, C

    1997-01-01

    .... For the past 10 years our laboratory has used baboons to evaluate the effects of various disinfection treatments on autologous red blood cell and platelet viability and function in vitro and in vivo...

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

    Science.gov (United States)

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

    2011-06-01

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

  13. [Airborne fine particle decreases the cell viability and induces inflammation in human bronchial epithelial cells].

    Science.gov (United States)

    Hong, Zhicong; Luo, Xianyang; Cai, Chengfu; Xu, Jian; Zhuang, Guoshun

    2017-09-28

    To investigate the effects of airborne fine particle on cell viability and inflammation in human bronchial epithelial cells.
 Methods: Atmospheric PM2.5 samples were collected by PM2.5 sampler. PM2.5 morphology was observed by scanning electron microscope (SEM). Human bronchial epithelial cells (BEAS-2B) were treated with PM2.5 at different concentrations (0, 50, 100, 200, 400, 800 μg/mL) for 12, 24 or 48 hours, and the cell activity were evaluated by cell counting kit-8 (CCK-8). The mRNA expression levels of (granulocyte-macrophage colony stimulating factor,GM-CSF) and TNF-α were detected by quantitative real-time PCR (qRT-PCR). Western blot was used to detect the protein expressions of GM-CSF and TNF-α.
 Results: According to SEM, the shape of PM2.5 varied, and the diameter was different and mostly equal to or less than 2.5 μm. CCK-8 assay showed that different concentrations of PM2.5 exposure for 12 hours, 24 hours and 48 hours resulted in loss of cell viability of BEAS-2B cells (P<0.05). Different concentrations of PM2.5 increased the mRNA and protein expression of GM-CSF and TNF-α, and the higher concentration of PM2.5 induced higher expression, which have statistical significant difference between the groups (P<0.05).
 Conclusion: Atmospheric PM2.5 can cause inflammatory response in human bronchial epithelial cells. They can reduce cell viability, which may be related to the PM2.5 trigger and aggravation of bronchopulmonary inflammatory diseases.

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

    Science.gov (United States)

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

    2012-01-01

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

  15. Viability and Functionality of Cells Delivered from Peptide Conjugated Scaffolds

    OpenAIRE

    Vacharathit, Voranaddha; Silva, Eduardo A.; Mooney, David J.

    2011-01-01

    Many cell-based therapies aim to transplant functional cells to revascularize damaged tissues and ischemic areas. However, conventional cell therapy is not optimally efficient: massive cell death, damage, and non-localization of cells both spatially and temporally all likely contribute to poor tissue functionality. An alginate cell depot system has been proposed as an alternative means to deliver outgrowth endothelial cells (OECs) in a spatiotemporally controllable manner while protecting the...

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

    Directory of Open Access Journals (Sweden)

    Ming Liu

    2016-12-01

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

  17. Evaluation of resistance development and viability recovery by a non-enveloped virus after repeated cycles of aPDT.

    Science.gov (United States)

    Costa, Liliana; Tomé, João P C; Neves, Maria G P M S; Tomé, Augusto C; Cavaleiro, José A S; Faustino, Maria A F; Cunha, Ângela; Gomes, Newton C M; Almeida, Adelaide

    2011-09-01

    Nowadays, the emergence of drug resistant microorganisms is a public health concern. The antimicrobial photodynamic therapy (aPDT) has an efficient action against a wide range of microorganisms and can be viewed as an alternative approach for treating microbial infections. The aim of this study was to determine if a model target virus (T4-like bacteriophage), in the presence of the tricationic porphyrin 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide (Tri-Py(+)-Me-PF), can develop resistance to aPDT and recover its viability after photodynamic treatments. To assess the development of aPDT resistance after repeated treatments, a suspension of T4-like bacteriophage was irradiated with white light (40 Wm(-2)) for 120 min in the presence of 5.0 μM of Tri-Py(+)-Me-PF (99.99% of inactivation) and new phage suspensions were produced from the surviving phages, after each cycle of light exposure. The procedure was repeated ten times. To evaluate the recovery of viral viability after photoinactivation, a suspension of T4-like bacteriophage was irradiated with white light for 120 min in the presence of 5.0 μM of Tri-Py(+)-Me-PF on five consecutive days. In each day, an aliquot of the irradiated suspension was plated and the number of lysis plaques was counted after 24, 48, 72, 96 and 120 h of dark incubation at 37 °C. The profile of bacteriophage photoinactivation did not change after ten consecutive cycles and no recovery of viability was detected after five accumulated cycles of photodynamic treatment. The results suggest that aPDT represents a valuable and promising alternative therapy to treat viral infections, overcoming the problem of microbial resistance. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Functional interplay between cell cycle and cell phenotypes

    Science.gov (United States)

    Chen, Wei-Chiang; Wu, Pei-Hsun; Phillip, Jude M.; Khatau, Shyam B.; Choi, Jae Min; Dallas, Matthew R.; Konstantopoulos, Konstantinos; Sun, Sean X.; Lee, Jerry S.H.; Hodzic, Didier; Wirtz, Denis

    2013-01-01

    Cell cycle distribution of adherent cells is typically assessed using flow cytometry, which precludes the measurements of many cell properties and their cycle phase in the same environment. Here we develop and validate a microscopy system to quantitatively analyze the cell-cycle phase of thousands of adherent cells and their associated cell properties simultaneously. This assay demonstrates that population-averaged cell phenotypes can be written as a linear combination of cell-cycle fractions and phase-dependent phenotypes. By perturbing cell cycle through inhibition of cell-cycle regulators or changing nuclear morphology by depletion of structural proteins, our results reveal that cell cycle regulators and structural proteins can significantly interfere with each other’s prima facie functions. This study introduces a high-throughput method to simultaneously measure cell cycle and phenotypes at single-cell resolution, which reveals a complex functional interplay between cell cycle and cell phenotypes. PMID:23319145

  19. Fetal liver cell-containing hybrid organoids improve cell viability and albumin production upon transplantation.

    Science.gov (United States)

    Ye, Jingjia; Shirakigawa, Nana; Ijima, Hiroyuki

    2016-06-01

    Cell transplantation is a potential alternative for orthotopic liver transplantation because of the chronic donor shortage. Functional liver tissue is needed for cell transplantations. However, large functional liver tissue is difficult to construct because of the high oxygen consumption of hepatocytes. In our previous study, we developed a novel method to generate hybrid organoids. In this study, we used fetal liver cells (FLCs) to construct a hybrid organoid. Nucleus numbers, angiogenesis, and albumin production were measured in transplanted samples. Higher cell viability and larger liver tissue was found in FLC-containing samples than in hepatocyte-containing samples. Furthermore, the therapeutic efficiency of FLC-containing samples was evaluated by transplantation into Nagase analbuminemia rats. As a result, an increase in albumin concentration was found in rat blood. In summary, transplantation of a FLC-containing hybrid organoid is a potential approach for cell transplantation. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Loss of rachis cell viability is associated with ripening disorders in grapes

    OpenAIRE

    Hall, Geoffrey E.; Bondada, Bhaskar R.; Keller, Markus

    2010-01-01

    Rachises of grape (Vitis vinifera L.) clusters that appeared healthy or displayed symptoms of the ripening disorders berry shrivel (BS) or bunch-stem necrosis (BSN) were treated with the cellular viability stain fluorescein diacetate and examined by confocal microscopy. Clusters with BS and BSN symptoms experienced a decrease of cell viability throughout the rachis, and their berries contained 70–80% less sugar than healthy berries. The xylem-mobile dye basic fuchsin, infiltrated via the cut ...

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

    Science.gov (United States)

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

    2017-02-01

    Photocrosslinking hydrogel technologies are attractive for the biofabrication of cardiovascular soft tissues, but 3D printing success is dependent on multiple variables. In this study we systematically test variables associated with photocrosslinking hydrogels (photoinitiator type, photoinitiator concentration, and light intensity) for their effects on encapsulated cells in an extrusion 3D printable mixture of methacrylated gelatin/poly-ethylene glycol diacrylate/alginate (MEGEL/PEGDA3350/alginate). The fabrication conditions that produced desired hydrogel mechanical properties were compared against those that optimize aortic valve or mesenchymal stem cell viability. In the 3D hydrogel culture environment and fabrication setting studied, Irgacure can increase hydrogel stiffness with a lower proportional decrease in encapsulated cell viability compared to VA086. Human adipose derived mesenchymal stem cells (HADMSC) survived increasing photoinitiator concentrations in photo-encapsulation conditions better than aortic valve interstitial cells (HAVIC) and aortic valve sinus smooth muscle cells (HASSMC). Within the range of photo-encapsulation fabrication conditions tested with MEGEL/PEGDA/alginate (0.25-1.0% w/v VA086, 0.025-0.1% w/v Irgacure 2959, and 365 nm light intensity 2-136 mW/cm2), the highest viabilities achieved were 95, 93, and 93% live for HASSMC, HAVIC, and HADMSC respectively. These results identify parameter combinations that optimize cell viability during 3D printing for multiple cell types. These results also indicate that general oxidative stress is higher in photocrosslinking conditions that induce lower cell viability. However, suppressing this increase in intracellular oxidative stress did not improve cell viability, which suggests that other stress mechanisms also contribute.

  2. Assessment of a simple, non-toxic Alamar blue cell survival assay to monitor tomato cell viability.

    Science.gov (United States)

    Byth, H A; Mchunu, B I; Dubery, I A; Bornman, L

    2001-01-01

    The Alamar Blue (AB) assay, which incorporates a medox indicator that changes colour or fluorescence in response to metabolic activity, is commonly used to assess quantitatively the viability and/or proliferation of mammalian cells and micro-organisms. In this study the AB assay was adapted for the determination of the viability of plant cells. Cell suspension cultures of tomato, Lycopersicon esculentum, L., with differing viabilities, served as the experimental model for a comparison of the AB assay with the conventional 2,3,5-triphenyltetrazolium chloride (TTC) viability assay. The AB assay showed a sigmoidal relationship between cell viability and AB reduction (as quantified by spectrofluorometry or spectrophotometry), which was similar to that obtained using the TTC assay. Both assays detected a significant reduction in cell viability after 48 h exposure to virulent Ralstonia solanacearum (biovar III), while the TTC assay, in addition, revealed cell proliferation in control cells from 24 to 72 h. The TTC assay detected cell proliferation over a wider range of cell densities, while the AB assay was more rapid and versatile whilst being non-toxic and thus allowing subsequent cell analysis.

  3. Transcriptional regulation of the cell cycle

    NARCIS (Netherlands)

    Stahl, M.

    2006-01-01

    Transcriptional regulators play an important role during cell cycle progression. A subset of these even seems to have a critical function in regulating cell cycle transitions. In this thesis, I have addressed the importance of transcriptional control in the regulation of cell cycle progression, in

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

    Energy Technology Data Exchange (ETDEWEB)

    Matveeva, V. G., E-mail: matveeva-vg@mail.ru; Antonova, L. V., E-mail: antonova.la@mail.ru; Velikanova, E. A.; Sergeeva, E. A.; Krivkina, E. O.; Glushkova, T. V.; Kudryavtseva, Yu. A.; Barbarash, O. L.; Barbarash, L. S. [Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, 650002 (Russian Federation)

    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.

  5. Use of fluorescent redox indicators to evaluate cell proliferation and viability

    DEFF Research Database (Denmark)

    Rasmussen, E.S.

    1999-01-01

    The performance of two cell viability test kits based on the use of redox indicators yielding fluorescent products, the AlamarBlue assay and a resazurin-based in vitro toxicology assay kit from Sigma, was compared in the present study. Cultures of human neonatal foreskin fibroblasts were exposed...... to equal concentrations of the two dye solutions in the cell culture media. The fluorescence intensities of the cell culture media obtained in response to cell proliferation with the two dyes showed a pronounced similarity. Both dyes were noncytotoxic to cell cultures with high initial cell densities...... components were tentatively identified as resazurin and resorufin. The AlamarBlue assay has gained wide application as a cell viability indicator that allows continuous monitoring of cell proliferation or cytotoxicity in human and animal cells, bacteria, and fungi, but no studies with the deliberate use...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Salahinejad, E., E-mail: salahinejad@kntu.ac.ir [Faculty of Materials Science and Engineering, K.N. Toosi University of Technology, Tehran (Iran, Islamic Republic of); Ghaffari, M. [Bruker AXS Inc., 5465 East Cheryl Parkway, Madison, WI 53711 (United States); Vashaee, D. [Electrical and Computer Engineering Department, North Carolina State University, Raleigh, NC 27606 (United States); Tayebi, L. [Department of Developmental Sciences, Marquette University School of Dentistry, Milwaukee, WI 53201 (United States); Department of Engineering Science, University of Oxford, Oxford OX1 3PJ (United Kingdom)

    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. - Highlights: • Cell viability vs. corrosion resistance for medical-grade stainless steels • The stainless steel samples were prepared by powder metallurgy. • Unpenetrated additive played a critical role in the correlation.

  9. The effect of glucocorticoids on tendon cell viability in human tendon explants

    Science.gov (United States)

    Lui, Wai Ting; Chuen Fu, Sai; Man Lee, Kwong

    2009-01-01

    Background and purpose Previous studies on the culture of human tenocytes have shown that dexamethasone and triamcino-lone reduce cell viability, suppress cell proliferation, and reduce collagen synthesis. However, such cell cultures lack the extracellular matrix and three-dimensional structure of normal tendons, which affects their response to stimuli. We established a human tendon explant culture system and tested the effects of dexamethasone and triamcinolone on cell viability. Methods Primary human tendon explant cultures were prepared from healthy hamstring tendons. Tendon strips were harvested from hamstring tendons and cultured in 24-well plates in Dulbecco’s modification of Eagle’s Medium (DMEM) supplemented with 2% fetal calf serum. The tendon explants were treated with 0 μM (control), 10 μM, or 100 μM dexamethasone sodium phosphate or 0 μM (control), 10 μM, or 100 μM triamcinolone acetonide in DMEM for 96 h. Cell viability was measured by Alamar blue assay before and after glucocorticoid treatment. Results Incubation with 10 μM and 100 μM dexamethasone reduced cell viability in human tendon explants by 35% and 45%, respectively, as compared to a 6% increase in the controls (p = 0.01, mixed-effects ANOVA). Triamcinolone at 10 μM and 100 μM reduced cell viability by 33% and 36%, respectively, as compared to a 9% increase in the controls (p = 0.07, mixed-effects ANOVA). Interpretation Human tendon explant cultures can be used to study the effects of glucocorticoids on human tendon. Dexamethasone and triamcinolone suppress the cell viability of human tendon in its natural 3-dimensional environment with matrix anchorage. Human tendon explant cultures provide a species-specific model for further investigation of the effects of glucocorticoids on the metabolism of the extracellular matrix of human tendon, and on its mechanical properties. PMID:19421908

  10. Running title: Antioxidant and cell viability of Echinacea purpurea

    African Journals Online (AJOL)

    shieh cho han

    utilization and animal growth is an aim for safety and hygiene animal products. These additives are ... major part of feedstuff for livestock and poultry, the cell wall structure .... will replace traditional growth promoter antibiotics, and they can be ...

  11. Metabolic control of the cell cycle.

    Science.gov (United States)

    Kalucka, Joanna; Missiaen, Rindert; Georgiadou, Maria; Schoors, Sandra; Lange, Christian; De Bock, Katrien; Dewerchin, Mieke; Carmeliet, Peter

    2015-01-01

    Cell division is a metabolically demanding process, requiring the production of large amounts of energy and biomass. Not surprisingly therefore, a cell's decision to initiate division is co-determined by its metabolic status and the availability of nutrients. Emerging evidence reveals that metabolism is not only undergoing substantial changes during the cell cycle, but it is becoming equally clear that metabolism regulates cell cycle progression. Here, we overview the emerging role of those metabolic pathways that have been best characterized to change during or influence cell cycle progression. We then studied how Notch signaling, a key angiogenic pathway that inhibits endothelial cell (EC) proliferation, controls EC metabolism (glycolysis) during the cell cycle.

  12. Effect of methotrexate conjugated PAMAM dendrimers on the viability of MES-SA uterine cancer cells

    Directory of Open Access Journals (Sweden)

    Samreen Khatri

    2014-01-01

    Full Text Available The aim of this work was to synthesize methotrexate (MTX-polyamidoamine (PAMAM dendritic nanoconjugates and to study their effect on cell viability in uterine sarcoma cells. The amide-bonded PAMAM dendrimer-MTX conjugates were prepared by conjugation between the amine-terminated G5 dendrimer and the carboxylic groups of the MTX using a dicyclohexylcarbodiimide coupling reaction. The formation of conjugates was evaluated by ultraviolet (UV and 1 H nuclear magnetic resonance ( 1 H NMR spectroscopy studies. The cell survival of MES-SA cells, a uterine sarcoma cell line, was evaluated in the presence of the dendrimer-MTX nanoconjugate, using appropriate controls. The UV and 1 H NMR study confirmed the formation of covalent bonds between the drug and the dendrimer. The cell viability study indicated that the nanoconjugates had significantly improved cell killing compared to the free MTX.

  13. Bupivacaine decreases cell viability and matrix protein synthesis in an intervertebral disc organ model system.

    Science.gov (United States)

    Wang, Dong; Vo, Nam V; Sowa, Gwendolyn A; Hartman, Robert A; Ngo, Kevin; Choe, So Ra; Witt, William T; Dong, Qing; Lee, Joon Y; Niedernhofer, Laura J; Kang, James D

    2011-02-01

    Bupivacaine is a local anesthetic commonly used for back pain management in interventional procedures. Cytotoxic effects of bupivacaine have been reported in articular cartilage and, recently, in intervertebral disc cell culture. However, the relevance of these effects to discs in vivo remains unclear. This study examines the effect of bupivacaine on disc cell metabolism using an organotypic culture model system that mimics the in vivo environment. To assess the effect of bupivacaine on disc cell viability and matrix protein synthesis using an organotypic model system and to determine whether this anesthetic has toxic effects. Mouse intervertebral discs were isolated and maintained ex vivo in an organotypic culture then exposed to clinically relevant concentrations of bupivacaine, and the impact on disc cell viability and matrix proteoglycan (PG) and collagen syntheses were measured in the presence and absence of the drug. Mouse functional spine units (FSUs) were isolated from the lumbar spines of 10-week-old mice. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Total PG and collagen syntheses were determined by measuring the incorporation of radioactive (35)S-sulfate and (3)H-l-proline into PG and collagen, respectively. Organotypic cultures of mouse FSUs were exposed to different concentrations (0%-0.5%) of bupivacaine for variable amounts of time (0-2 hours). Cell viability within disc tissue was quantified by MTT staining and histologic assay. Matrix protein synthesis was measured by incorporation of radioactive (35)S-sulfate (for PG synthesis) and (3)H-l-proline (for collagen synthesis). Untreated mouse disc organs were maintained in culture for up to 1 month with minimal changes in tissue histology, cell viability, and matrix protein synthesis. Exposure to bupivacaine decreased cell viability in a dose- and time-dependent manner. Exposure to bupivacaine at concentrations less than or equal to 0.25% did

  14. Interactions between Plant Extracts and Cell Viability Indicators ...

    African Journals Online (AJOL)

    Methods: Hexane, chloroform, ethyl acetate, ethanol, methanol and water extracts were obtained for each plant by sequential solvent extraction. Cytotoxicity was evaluated in triplicate, from 640 to 5 μg/mL, two-fold, serially on monkey kidney epithelial (Vero) cells. Results: The hexane, chloroform and ethyl acetate extracts ...

  15. MAPK uncouples cell cycle progression from cell spreading and cytoskeletal organization in cycling cells.

    Science.gov (United States)

    Margadant, Coert; Cremers, Lobke; Sonnenberg, Arnoud; Boonstra, Johannes

    2013-01-01

    Integrin-mediated cytoskeletal tension supports growth-factor-induced proliferation, and disruption of the actin cytoskeleton in growth factor-stimulated cells prevents the re-expression of cyclin D and cell cycle re-entry from quiescence. In contrast to cells that enter the cell cycle from G0, cycling cells continuously express cyclin D, and are subject to major cell shape changes during the cell cycle. Here, we investigated the cell cycle requirements for cytoskeletal tension and cell spreading in cycling mammalian cells that enter G1-phase from mitosis. Disruption of the actin cytoskeleton at progressive time-points in G1-phase induced cell rounding, FA disassembly, and attenuated both integrin signaling and growth factor-induced p44/p42 mitogen-activated protein kinase activation. Although cyclin D expression was reduced, the expression of cyclin A and entry into S-phase were not affected. Moreover, expression of cyclin B1, progression through G2- and M-phase, and commitment to a new cell cycle occurred normally. In contrast, cell cycle progression was strongly prevented by inhibition of MAPK activity in G1-phase, whereas cell spreading, cytoskeletal organization, and integrin signaling were not impaired. MAPK inhibition also prevented cytoskeleton-independent cell cycle progression. Thus, these results uncouple the requirements for cell spreading and cytoskeletal organization from MAPK signaling, and show that cycling mammalian cells can proliferate independently of actin stress fibers, focal adhesions, or cell spreading, as long as a threshold level of MAPK activity is sustained.

  16. EPR, TEM and cell viability study of asbestiform zeolite fibers in cell media.

    Science.gov (United States)

    Cangiotti, Michela; Salucci, Sara; Battistelli, Michela; Falcieri, Elisabetta; Mattioli, Michele; Giordani, Matteo; Ottaviani, Maria Francesca

    2018-01-01

    Human monocyte U937 cell line was used as a model to verify the toxicity of erionite and offretite asbestiform zeolite fibers. As a presumed non-toxic reference, a fibrous scolecite zeolite was also used. To analyze the process of fiber ingestion into cells and the cells-fibers interactions, a spin-probe electron paramagnetic resonance (EPR) analysis was performed supported by transmission electron microscopy (TEM) and cell viability measurements as a function of the incubation time. Erionite fibers were fast internalized in the membrane mainly as aggregates with radical-solution drops trapped inside, and were found in the cytosol and at the nucleus. In 24h, first erionite fibers rich in sodium and potassium, and then calcium-rich erionite fibers, induced cell necrosis. The offretite fibers formed rounding electron-dense filaments which transformed in curved filaments, initially perturbing the cell structure and interacting at the external surface more than erionite fibers. Such interactions probably diminished the toxic effect of offretite on cells. Interestingly, the presumed non-toxic scolecite fibers were partially internalized, inducing formation of swollen mitochondria and squared cells. Overall, the toxic effect of the fibrous zeolites was related to fiber morphology, chemical distribution of sites, structural variations and formation of aggregates. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. "Constructing" the Cell Cycle in 3D

    Science.gov (United States)

    Koc, Isil; Turan, Merve

    2012-01-01

    The cycle of duplication and division, known as the "cell cycle," is the essential mechanism by which all living organisms reproduce. This activity allows students to develop an understanding of the main events that occur during the typical eukaryotic cell cycle mostly in the process of mitotic phase that divides the duplicated genetic material…

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

    Science.gov (United States)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2013-05-01

    The clinical outcome of transplantations of bioartificial tissues and organs depends on the presence of living cells. There are still no standard operative protocols that are simple, fast and reliable for confirming the presence of viable cells on bioartificial scaffolds prior to transplantation. By using mathematical modeling, we have developed a colorimetric-based system (colorimetric scale bar) to predict the cell viability and density for sufficient surface coverage. First, we refined a method which can provide information about cell viability and numbers in an in vitro setting: i) immunohistological staining by Phalloidin/DAPI and ii) a modified colorimetric cell viability assay. These laboratory-based methods and the developed colorimetric-based system were then validated in rat transplantation studies of unseeded and seeded tracheal grafts. This was done to provide critical information on whether the graft would be suitable for transplantation or if additional cell seeding was necessary. The potential clinical impact of the colorimetric scale bar was confirmed using patient samples. In conclusion, we have developed a robust, fast and reproducible colorimetric tool that can verify and warrant viability and integrity of an engineered tissue/organ prior to transplantation. This should facilitate a successful transplantation outcome and ensure patient safety. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Photothermal effects of multi-walled carbon nanotubes on the viability of BT-474 cancer cells.

    Science.gov (United States)

    Chou, Hung-Tao; Wang, Tsung-Pao; Lee, Chi-Young; Tai, Nyan-Hwa; Chang, Hwan-You

    2013-03-01

    Functionalized multi-walled carbon nanotubes (f-MWCNTs) were conjugated to an antibody of BT-474 cancer cells (f-MWCNTs-ab), and the photothermal effect of the f-MWCNTs-ab for BT-474 cancer cell destruction was demonstrated. After near-infrared irradiation, the f-MWCNTs-ab were more capable of killing cancer cells and possessed higher cell specificity than f-MWCNTs. Quantitative results showed that the viability of the cancer cells was affected by the concentration of the f-MWCNTs-ab solution, irradiation time, and settling time after irradiation. The membrane impermeable fluorescence dye ethidium bromide was used to detect cell viability after near-infrared irradiation, and the results agreed with those obtained from the Alamar Blue cell viability assay. The EtBr fluorescence results suggest that the cell membrane, attached to f-MWCNTs-ab, was damaged after irradiation, which led to cell death and necrosis. Using confocal microscopy, a few f-MWCNTs-ab were detected in the cell, indicating the endocytosis effect. The results not only explain the improved efficiency of thermotherapy but also indicate that necrosis may result from protein denaturation attributing to the heated f-MWCNTs-ab in the cell. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Host-Based Th2 Cell Therapy for Prolongation of Cardiac Allograft Viability

    Science.gov (United States)

    Foley, Jason E.; Costanzo, Carliann M.; Sennesh, Joel D.; Solomon, Michael A.; Fowler, Daniel H.

    2011-01-01

    Donor T cell transfusion, which is a long-standing approach to prevent allograft rejection, operates indirectly by alteration of host T cell immunity. We therefore hypothesized that adoptive transfer of immune regulatory host Th2 cells would represent a novel intervention to enhance cardiac allograft survival. Using a well-described rat cardiac transplant model, we first developed a method for ex vivo manufacture of rat host-type Th2 cells in rapamycin, with subsequent injection of such Th2.R cells prior to class I and class II disparate cardiac allografting. Second, we determined whether Th2.R cell transfer polarized host immunity towards a Th2 phenotype. And third, we evaluated whether Th2.R cell therapy prolonged allograft viability when used alone or in combination with a short-course of cyclosporine (CSA) therapy. We found that host-type Th2.R cell therapy prior to cardiac allografting: (1) reduced the frequency of activated T cells in secondary lymphoid organs; (2) shifted post-transplant cytokines towards a Th2 phenotype; and (3) prolonged allograft viability when used in combination with short-course CSA therapy. These results provide further support for the rationale to use “direct” host T cell therapy for prolongation of allograft viability as an alternative to “indirect” therapy mediated by donor T cell infusion. PMID:21559526

  2. Host-based Th2 cell therapy for prolongation of cardiac allograft viability.

    Directory of Open Access Journals (Sweden)

    Shoba Amarnath

    Full Text Available Donor T cell transfusion, which is a long-standing approach to prevent allograft rejection, operates indirectly by alteration of host T cell immunity. We therefore hypothesized that adoptive transfer of immune regulatory host Th2 cells would represent a novel intervention to enhance cardiac allograft survival. Using a well-described rat cardiac transplant model, we first developed a method for ex vivo manufacture of rat host-type Th2 cells in rapamycin, with subsequent injection of such Th2.R cells prior to class I and class II disparate cardiac allografting. Second, we determined whether Th2.R cell transfer polarized host immunity towards a Th2 phenotype. And third, we evaluated whether Th2.R cell therapy prolonged allograft viability when used alone or in combination with a short-course of cyclosporine (CSA therapy. We found that host-type Th2.R cell therapy prior to cardiac allografting: (1 reduced the frequency of activated T cells in secondary lymphoid organs; (2 shifted post-transplant cytokines towards a Th2 phenotype; and (3 prolonged allograft viability when used in combination with short-course CSA therapy. These results provide further support for the rationale to use "direct" host T cell therapy for prolongation of allograft viability as an alternative to "indirect" therapy mediated by donor T cell infusion.

  3. Epigenetic dynamics across the cell cycle

    DEFF Research Database (Denmark)

    Kheir, Tony Bou; Lund, Anders H.

    2010-01-01

    Progression of the mammalian cell cycle depends on correct timing and co-ordination of a series of events, which are managed by the cellular transcriptional machinery and epigenetic mechanisms governing genome accessibility. Epigenetic chromatin modifications are dynamic across the cell cycle...... a correct inheritance of epigenetic chromatin modifications to daughter cells. In this chapter, we summarize the current knowledge on the dynamics of epigenetic chromatin modifications during progression of the cell cycle....

  4. Fever-range hyperthermia vs. hypothermia effect on cancer cell viability, proliferation and HSP90 expression.

    Science.gov (United States)

    Kalamida, Dimitra; Karagounis, Ilias V; Mitrakas, Achilleas; Kalamida, Sofia; Giatromanolaki, Alexandra; Koukourakis, Michael I

    2015-01-01

    The current study examines the effect of fever-range hyperthermia and mild hypothermia on human cancer cells focusing on cell viability, proliferation and HSP90 expression. A549 and H1299 lung carcinoma, MCF7 breast adenocarcinoma, U87MG and T98G glioblastoma, DU145 and PC3 prostate carcinoma and MRC5 normal fetal lung fibroblasts cell lines were studied. After 3-day exposure to 34°C, 37°C and 40°C, cell viability was determined. Cell proliferation (ki67 index), apoptosis (Caspase 9) and HSP90 expression was studied by confocal microscopy. Viability/proliferation experiments demonstrated that MRC5 fibroblasts were extremely sensitive to hyperthermia, while they were the most resistant to hypothermia. T98G and A549 were thermo-tolerant, the remaining being thermo-sensitive to a varying degree. Nonetheless, as a universal effect, hypothermia reduced viability/proliferation in all cell lines. Hyperthermia sharply induced Caspase 9 in the U87MG most thermo-sensitive cell line. In T98G and A549 thermo-tolerant cell lines, the levels of Caspase 9 declined. Moreover, hyperthermia strongly induced the HSP90 levels in T98G, whilst a sharp decrease was recorded in the thermo-sensitive PC3 and U87MG cell lines. Hyperthermia sensitized thermo-sensitive cancer cell lines to cisplatin and temozolomide, whilst its sensitizing effect was diminished in thermo-tolerant cell lines. The existence of thermo-tolerant and thermo-sensitive cancer cell lines was confirmed, which further encourages research to classify human tumor thermic predilection for patient stratification in clinical trials. Of interest, mild hypothermia had a universal suppressing effect on cancer cell proliferation, further supporting the radio-sensitization hypothesis through reduction of oxygen and metabolic demands.

  5. Fever-range hyperthermia vs. hypothermia effect on cancer cell viability, proliferation and HSP90 expression.

    Directory of Open Access Journals (Sweden)

    Dimitra Kalamida

    Full Text Available The current study examines the effect of fever-range hyperthermia and mild hypothermia on human cancer cells focusing on cell viability, proliferation and HSP90 expression.A549 and H1299 lung carcinoma, MCF7 breast adenocarcinoma, U87MG and T98G glioblastoma, DU145 and PC3 prostate carcinoma and MRC5 normal fetal lung fibroblasts cell lines were studied. After 3-day exposure to 34°C, 37°C and 40°C, cell viability was determined. Cell proliferation (ki67 index, apoptosis (Caspase 9 and HSP90 expression was studied by confocal microscopy.Viability/proliferation experiments demonstrated that MRC5 fibroblasts were extremely sensitive to hyperthermia, while they were the most resistant to hypothermia. T98G and A549 were thermo-tolerant, the remaining being thermo-sensitive to a varying degree. Nonetheless, as a universal effect, hypothermia reduced viability/proliferation in all cell lines. Hyperthermia sharply induced Caspase 9 in the U87MG most thermo-sensitive cell line. In T98G and A549 thermo-tolerant cell lines, the levels of Caspase 9 declined. Moreover, hyperthermia strongly induced the HSP90 levels in T98G, whilst a sharp decrease was recorded in the thermo-sensitive PC3 and U87MG cell lines. Hyperthermia sensitized thermo-sensitive cancer cell lines to cisplatin and temozolomide, whilst its sensitizing effect was diminished in thermo-tolerant cell lines.The existence of thermo-tolerant and thermo-sensitive cancer cell lines was confirmed, which further encourages research to classify human tumor thermic predilection for patient stratification in clinical trials. Of interest, mild hypothermia had a universal suppressing effect on cancer cell proliferation, further supporting the radio-sensitization hypothesis through reduction of oxygen and metabolic demands.

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

    Science.gov (United States)

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

    2010-10-01

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

  7. Manual vs automated delivery of cells for transplantation: accuracy, reproducibility, and impact on viability.

    Science.gov (United States)

    Gobbel, Glenn T; Kondziolka, Douglas; Fellows-Mayle, Wendy; Uram, Martin

    2010-12-01

    Cellular transplantation holds promise for the management of a variety of neurological disorders. However, there is great variability in cell type, preparation methods, and implantation technique, which are crucial to clinical outcomes. We compared manual injection with automated injection using a prototype device to determine the possible value of a mechanized delivery system. Neural progenitor cells and bone marrow stromal cells were injected using manual or automated methods. Consistency of injection volumes and cell number and viability were evaluated immediately or 1 day after injection. When cells were delivered as a series of 3 manual injections from the same syringe, the variation in fluid volume was greater than for single manual injections. Automated delivery of a series of 3 injections resulted in a lower variability in the amount of delivery than manual injection for both cell lines (1.2%-2.6% coefficient of variability for automated delivery vs 4.3%-24.0% for manual delivery). The amount delivered from injection 1 to injection 3 increased significantly with manual injections, whereas the amount injected did not vary over the 3 injections for the automated unit. Cell viability 1 day after injection was typically 30% to 40% of the value immediately after injection for the bone marrow stromal cells and 30% to 70% for the neural progenitor cells. There were no significant differences in viability attributed to the method of injection. The automated delivery device led to enhanced consistency of volumetric cell delivery but did not improve cell viability in the methods tested. Automated techniques could be useful in standardizing reproducible procedures for cell transplantation and improve both preclinical and clinical research.

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

    Science.gov (United States)

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

    2017-05-01

    The increasing rate of cancer incidence has encouraged the search for novel natural sources of anticancer compounds. The presence of small quantities of taxol and taxanes in Corylus avellana L. has impelled new potential applications for this plant in the field of biomedicine. In the present work, the cell viability-reducing activity of stems and leaves from three different hazel trees was studiedagainst three human-derived cancer cell lines (HeLa, HepG2 and MCF-7). Both leaf and stem extracts significantly reduced viability of the three cell lines either after maceration with methanol or using taxane extraction methods. Since maceration reduced cell viability to a greater extent than taxane extraction methods, we scaled up the maceration extraction process using a method for solid/liquid extraction (Zippertex technology). Methanol leaf extracts promoted a higher reduction in viability of all cell lines assayed than stem extracts. Fractionation of methanol leaf extracts using silica gel chormatography led to the purification and identification of two compounds by HPLC-MS and NMR: (3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl) heptane 3-O-β-d-glucopyranoside and quercetin-3-O-rhamnoside. The isolated compounds decreased viability of HeLa and HepG2 cells to a greater extent than MCF-7 cells. Our results suggest a potential use of C. avellana extracts in the pharmacotherapy of cervical cancer and hepatocarcinoma and, to a lesser extent, breast cancer. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Cell cycle control in the kidney.

    Science.gov (United States)

    Thomasova, Dana; Anders, Hans-Joachim

    2015-10-01

    Proper control of the cell cycle is mandatory during homeostasis and disease. The balance of p53 and MDM2 integrates numerous signalling pathways to regulate the cell cycle, which is executed by multiple proteins including the cyclins, cyclin kinases and cyclin kinase inhibitors. Mutations or environmental factors that affect cell cycle control can lead to inappropriate hyperplasia or cancer as well as to cell loss and tissue atrophy. Normal kidney function is maintained largely by post-mitotic quiescent cells in the G0 phase with a low turnover. Early cell cycle activation during kidney injury contributes to cell death via mitotic catastrophe, i.e. death via mitosis, e.g. of cell with significant DNA damage. At later stages, cell cycle entry supports tissue regeneration and functional reconstitution via cell hypertrophy and/or cell proliferation. It is of note that so-called proliferation markers such as Ki67, PCNA or BrdU identify only cell cycle entry without telling whether this results in cell hypertrophy, cell division or mitotic catastrophe. With this in mind, some established concepts on kidney injury and regeneration are to be re-evaluated. Here, we discuss the components and functional roles of p53/MDM2-mediated cell cycle regulation in kidney homeostasis and disease. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

  11. Evaluation of Commercial Off-the-Shelf Solutions for Supporting Viability Retention of Yersinia Pestis Cells

    Science.gov (United States)

    2017-11-01

    swabs Thermo Fisher Scientific, Inc. R723140 13 Skim milk (filtered) Cloverland Farms Dairy (Baltimore, MD) – 14 Spent tryptic soy broth (sTSB) Thermo...9 5. Fate of Y. pestis A1122 cells sampled from surfaces and stored in half-strength skim milk ...11 7. Effect of AZTD on sustained viability of sampled Y. pestis in skim milk

  12. Short Peptides Enhance Single Cell Adhesion and Viability onMicroarrays

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-01-19

    Single cell patterning holds important implications forbiology, biochemistry, biotechnology, medicine, and bioinformatics. Thechallenge for single cell patterning is to produce small islands hostingonly single cells and retaining their viability for a prolonged period oftime. This study demonstrated a surface engineering approach that uses acovalently bound short peptide as a mediator to pattern cells withimproved single cell adhesion and prolonged cellular viabilityon goldpatterned SiO2 substrates. The underlying hypothesis is that celladhesion is regulated bythe type, availability, and stability ofeffective cell adhesion peptides, and thus covalently bound shortpeptides would promote cell spreading and, thus, single cell adhesion andviability. The effectiveness of this approach and the underlyingmechanism for the increased probability of single cell adhesion andprolonged cell viability by short peptides were studied by comparingcellular behavior of human umbilical cord vein endothelial cells on threemodelsurfaces whose gold electrodes were immobilized with fibronectin,physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently boundLys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and bindingproperties were characterized by reflectance Fourier transform infraredspectroscopy. Both short peptides were superior to fibronectin inproducing adhesion of only single cells, whereas the covalently boundpeptide also reduced apoptosis and necrosisof adhered cells. Controllingcell spreading by peptide binding domains to regulate apoptosis andviability represents a fundamental mechanism in cell-materialsinteraction and provides an effective strategy in engineering arrays ofsingle cells.

  13. The viability of a nonenzymatic reductive citric acid cycle - Kinetics and thermochemistry

    Science.gov (United States)

    Ross, D.S.

    2007-01-01

    The likelihood of a functioning nonenzymatic reductive citric acid cycle, recently proposed as the precursor to biosynthesis on early Earth, is examined on the basis of the kinetics and thermochemistry of the acetate ??? pyruvate ??? oxaloacetate ??? malate sequence. Using data derived from studies of the Pd-catalyzed phosphinate reduction of carbonyl functions it is shown that the rate of conversion of pyruvate to malate with that system would have been much too slow to have played a role in the early chemistry of life, while naturally occurring reduction systems such as the fayalite-magnetite-quartz and pyrrhotite-pyrite-magnetite mineral assemblages would have provided even slower conversions. It is also shown that the production of pyruvate from acetate is too highly endoergic to be driven by a naturally occurring energy source such as pyrophosphate. It is thus highly doubtful that the cycle can operate at suitable rates without enzymes, and most unlikely that it could have participated in the chemistry leading to life. ?? 2006 Springer Science + Business Media B.V.

  14. Microtissues Enhance Smooth Muscle Differentiation and Cell Viability of hADSCs for Three Dimensional Bioprinting

    Science.gov (United States)

    Yipeng, Jin; Yongde, Xu; Yuanyi, Wu; Jilei, Sun; Jiaxiang, Guo; Jiangping, Gao; Yong, Yang

    2017-01-01

    Smooth muscle differentiated human adipose derived stem cells (hADSCs) provide a crucial stem cell source for urinary tissue engineering, but the induction of hADSCs for smooth muscle differentiation still has several issues to overcome, including a relatively long induction time and equipment dependence, which limits access to abundant stem cells within a short period of time for further application. Three-dimensional (3D) bioprinting holds great promise in regenerative medicine due to its controllable construction of a designed 3D structure. When evenly mixed with bioink, stem cells can be spatially distributed within a bioprinted 3D structure, thus avoiding drawbacks such as, stem cell detachment in a conventional cell-scaffold strategy. Notwithstanding the advantages mentioned above, cell viability is often compromised during 3D bioprinting, which is often due to pressure during the bioprinting process. The objective of our study was to improve the efficiency of hADSC smooth muscle differentiation and cell viability of a 3D bioprinted structure. Here, we employed the hanging-drop method to generate hADSC microtissues in a smooth muscle inductive medium containing human transforming growth factor β1 and bioprinted the induced microtissues onto a 3D structure. After 3 days of smooth muscle induction, the expression of α-smooth muscle actin and smoothelin was higher in microtissues than in their counterpart monolayer cultured hADSCs, as confirmed by immunofluorescence and western blotting analysis. The semi-quantitative assay showed that the expression of α-smooth muscle actin (α-SMA) was 0.218 ± 0.077 in MTs and 0.082 ± 0.007 in Controls; smoothelin expression was 0.319 ± 0.02 in MTs and 0.178 ± 0.06 in Controls. Induced MTs maintained their phenotype after the bioprinting process. Live/dead and cell count kit 8 assays showed that cell viability and cell proliferation in the 3D structure printed with microtissues were higher at all time points compared to

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

    Directory of Open Access Journals (Sweden)

    Carla Marchetti

    2015-01-01

    Full Text Available 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.

  16. Resveratrol Inhibits Alpha-Melanocyte-Stimulating Hormone Signaling, Viability, and Invasiveness in Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Yu-Jen Chen

    2013-01-01

    Full Text Available Melanoma is a malignancy with high potential to invasion and treatment resistance. The α-melanocyte-stimulating hormone (α-MSH signal transduction involving Wnt/β-catenin, c-Kit, and microphthalmia-associated transcription factor (MITF, a known pathway to produce melanin, has been demonstrated as one of cancer stem cell characteristics. This study was aimed to examine the effect of resveratrol, an abundant ingredient of grape and medicinal plants, on α-MSH signaling, viability, and invasiveness in melanoma cells. By α-MSH treatment, the melanin production in B16 melanoma cells was augmented as a validation for activation of α-MSH signaling. The upregulated expression of α-MSH signaling-related molecules β-catenin, c-Kit, and MITF was suppressed by resveratrol and/or STI571 treatment. Nuclear translocation of MITF, a hallmark of α-MSH signaling activation, was inhibited by combined treatment of resveratrol and STI571. At effective concentration, resveratrol and/or STI571 inhibited cell viability and α-MSH-activated matrix metalloproteinase- (MMP-9 expression and invasion capacity of B16 melanoma cells. In conclusion, resveratrol enhances STI571 effect on suppressing the α-MSH signaling, viability, and invasiveness in melanoma cells. It implicates that resveratrol may have potential to modulate the cancer stem cell characteristics of melanoma.

  17. Polyphenolic Extracts of Edible Flowers Incorporated onto Atelocollagen Matrices and Their Effect on Cell Viability

    Directory of Open Access Journals (Sweden)

    Jorge López-García

    2013-10-01

    Full Text Available 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.

  18. Exercise regulates breast cancer cell viability: systemic training adaptations versus acute exercise responses.

    Science.gov (United States)

    Dethlefsen, Christine; Lillelund, Christian; Midtgaard, Julie; Andersen, Christina; Pedersen, Bente Klarlund; Christensen, Jesper Frank; Hojman, Pernille

    2016-10-01

    Exercise decreases breast cancer risk and disease recurrence, but the underlying mechanisms are unknown. Training adaptations in systemic factors have been suggested as mediating causes. We aimed to examine if systemic adaptations to training over time, or acute exercise responses, in breast cancer survivors could regulate breast cancer cell viability in vitro. Blood samples were collected from breast cancer survivors, partaking in either a 6-month training intervention or across a 2 h acute exercise session. Changes in training parameters and systemic factors were evaluated and pre/post exercise-conditioned sera from both studies were used to stimulate breast cancer cell lines (MCF-7, MDA-MB-231) in vitro. Six months of training increased VO2peak (16.4 %, p cancer cell viability in vitro. During 2 h of acute exercise, increases in serum lactate (6-fold, p exercise reduced viability by -9.2 % in MCF-7 (p = 0.04) and -9.4 % in MDA-MB-231 (p exercise session reduced breast cancer viability, while adaptations to 6 months of training had no impact. Our data question the prevailing dogma that training-dependent baseline reductions in risk factors mediate the protective effect of exercise on breast cancer. Instead, we propose that the cancer protection is driven by accumulative effects of repeated acute exercise responses.

  19. ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox.

    Science.gov (United States)

    Zhang, Di; Ren, Li; Chen, Guan-Qun; Zhang, Jie; Reed, Barbara M; Shen, Xiao-Hui

    2015-09-01

    Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability. We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O 2 (·-) , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA-GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.

  20. Effects of Trypsinization on Viability of Equine Chondrocytes in Cell Culture

    Directory of Open Access Journals (Sweden)

    B. C. Sutradhar, J. Park, G. Hong, S. H. Choi and G. Kim*

    2010-10-01

    Full Text Available Trypsin is an essential reagent for routine cell culture work. In the cultivation of mammalian cells, it has been extensively used for cell isolation from tissues or cell dislodging in subculturing. It may damage the cell membrane in contact of cells during long trypsinization. However, there is no specific report on time-dependent effect of trypsinization on cells. In the present study, we investigated the time dependent effects of trypsinization on equine chondrocytes. Cell viability after trypsinization with 0.25% trypsin-EDTA for 5 to 60 minutes was quantified by trypan blue exclusion assay, propidium iodide-Hoechst double staining, flow cytometry analysis and XTT assay. The results showed that trypsin-EDTA decreased the proliferation of equine chondrocytes depending on the exposure time of trypsinization. After 20 and 60 minutes of trypsinization, the cell membranes were strongly affected and the percentages of viable cells reduced to 91% and 85% respectively detected by trypan blue exclusion assay. Similar results were observed both in flow cytometric evaluation and propidium iodide-Hoechst double staining. The XTT assay result also showed decreased cell viability with the extended time of trypsinization. In order to minimize the time dependant cytotoxicity of trypsinization, as minimum as short time exposure is suggestive that maximizes live cell isolation from tissue as well as subculture of equine chondrocytes or other cells.

  1. Photothermal effects of multi-walled carbon nanotubes on the viability of BT-474 cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Chou, Hung-Tao [Department of Materials Science and Engineering, National Tsing Hua University, No. 101, Sec. 2 Kuang-Fu Rd., Hsin-chu 30013, Taiwan (China); Wang, Tsung-Pao [Department of Medical Science, National Tsing Hua University, No. 101, Sec. 2 Kuang-Fu Rd., Hsin-chu 30013, Taiwan (China); Lee, Chi-Young [Department of Materials Science and Engineering, National Tsing Hua University, No. 101, Sec. 2 Kuang-Fu Rd., Hsin-chu 30013, Taiwan (China); Tai, Nyan-Hwa, E-mail: nhtai@mx.nthu.edu.tw [Department of Materials Science and Engineering, National Tsing Hua University, No. 101, Sec. 2 Kuang-Fu Rd., Hsin-chu 30013, Taiwan (China); Chang, Hwan-You, E-mail: hychang@mx.nthu.edu.tw [Department of Medical Science, National Tsing Hua University, No. 101, Sec. 2 Kuang-Fu Rd., Hsin-chu 30013, Taiwan (China)

    2013-03-01

    Functionalized multi-walled carbon nanotubes (f-MWCNTs) were conjugated to an antibody of BT-474 cancer cells (f-MWCNTs-ab), and the photothermal effect of the f-MWCNTs-ab for BT-474 cancer cell destruction was demonstrated. After near-infrared irradiation, the f-MWCNTs-ab were more capable of killing cancer cells and possessed higher cell specificity than f-MWCNTs. Quantitative results showed that the viability of the cancer cells was affected by the concentration of the f-MWCNTs-ab solution, irradiation time, and settling time after irradiation. The membrane impermeable fluorescence dye ethidium bromide was used to detect cell viability after near-infrared irradiation, and the results agreed with those obtained from the Alamar Blue cell viability assay. The EtBr fluorescence results suggest that the cell membrane, attached to f-MWCNTs-ab, was damaged after irradiation, which led to cell death and necrosis. Using confocal microscopy, a few f-MWCNTs-ab were detected in the cell, indicating the endocytosis effect. The results not only explain the improved efficiency of thermotherapy but also indicate that necrosis may result from protein denaturation attributing to the heated f-MWCNTs-ab in the cell. Highlights: Black-Right-Pointing-Pointer f-MWCNTs conjugated with anti-HER2 antibody by chemical method. Black-Right-Pointing-Pointer Kill breast cancer cells by using low dose f-MWCNTs-ab due to photothermal effect. Black-Right-Pointing-Pointer Use EtBr fluorescent to prove that the cell membrane was broken by heated f-MWCNTs. Black-Right-Pointing-Pointer Few f-MWCNTs-ab were detected in the cell indicating the endocytosis effect. Black-Right-Pointing-Pointer Necrosis may result from protein denaturation due to contact with the heated CNTs.

  2. Effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell function and viability.

    Science.gov (United States)

    McDermott, Catherine; Chess-Williams, Russ; Grant, Gary D; Perkins, Anthony V; McFarland, Amelia J; Davey, Andrew K; Anoopkumar-Dukie, Shailendra

    2012-03-01

    We determined the effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell viability and function in vitro. RT4 urothelial cells were treated with pyocyanin (1 to 100 μM) for 24 hours. After exposure the treatment effects were measured according to certain end points, including changes in urothelial cell viability, reactive oxygen species formation, caspase-3 activity, basal and stimulated adenosine triphosphate release, SA-β-gal activity and detection of acidic vesicular organelles. The 24-hour pyocyanin treatment resulted in a concentration dependent decrease in cell viability at concentrations of 25 μM or greater, and increases in reactive oxygen species formation and caspase-3 activity at 25 μM or greater. Basal adenosine triphosphate release was significantly decreased at all tested pyocyanin concentrations while stimulated adenosine triphosphate release was significantly inhibited at pyocyanin concentrations of 12.5 μM or greater with no significant stimulated release at 100 μM. Pyocyanin treated RT4 cells showed morphological characteristics associated with cellular senescence, including SA-β-gal expression. This effect was not evident at 100 μM pyocyanin and may have been due to apoptotic cell death, as indicated by increased caspase-3 activity. An increase in acridine orange stained vesicular-like organelles was observed in RT4 urothelial cells after pyocyanin treatment. Exposure to pyocyanin alters urothelial cell viability, reactive oxygen species production and caspase-3 activity. Treatment also results in cellular senescence, which may affect the ability of urothelium to repair during infection. The virulence factor depressed stimulated adenosine triphosphate release, which to our knowledge is a novel finding with implications for awareness of bladder filling in patients with P. aeruginosa urinary tract infection. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier

  3. Lowering the concentration affects the migration and viability of intracerebroventricular-delivered human mesenchymal stem cells.

    Science.gov (United States)

    Kim, Hyeong Seop; Lee, Na Kyung; Yoo, Dongkyeom; Lee, Jeongmin; Choi, Soo Jin; Oh, Wonil; Chang, Jong Wook; Na, Duk L

    2017-11-04

    Due to their widely known therapeutic benefits, mesenchymal stem cells have been proposed as a novel treatment option for a wide range of diseases including Alzheimer's disease. To maximize these benefits, critical factors such as delivery route, cell viability, and cell migration must be accounted for. Out of the various delivery routes to the brain, the intracerebroventricular (ICV) route stands out due to the widespread distribution that can occur via cerebrospinal fluid flow. The major objective of this present study was to observe how altering cell concentration influences the migration and viability of human umbilical cord blood derived-mesenchymal stem cells (hUCB-MSCs), delivered via ICV injection, in the brains of wild-type (WT) mice. C3H/C57 WT mice were divided into three groups and were injected with 1 × 10(5) hUCB-MSCs suspended in varying volumes: high (3 μl), middle (5 μl), and low (7 μl) concentrations, respectively. Lowering the concentration increased the migratory capabilities and elevated the viability of hUCB-MSCs. These results suggest that cell concentration can affect the physiological state of hUCB-MSCs, and thus the extent of therapeutic efficacy that can be achieved. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    T. D. Jones

    2016-01-01

    Full Text Available 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.

  5. Effects of cannabinoids and related fatty acids upon the viability of P19 embryonal carcinoma cells.

    Science.gov (United States)

    Gustafsson, Sofia B; Wallenius, Anders; Zackrisson, Hanna; Popova, Dina; Plym Forshell, Linus; Jacobsson, Stig O P

    2013-11-01

    Compounds acting on the cannabinoid (CB) receptors are involved in the control of cell fate, and there is an emerging consensus that CBs have anticancer effects. However, the CB-mediated effects are contradictory since some studies suggest stimulatory effects on cancer cell proliferation, and CBs have been shown to stimulate both proliferation and differentiation of other mitotic cells such as stem and progenitor cells. In this study, the concentration-dependent effects of synthetic and endogenous CBs on the viability of mouse P19 embryonal carcinoma (EC) cells have been examined by using fluorescence assays of cell membrane integrity, cell proliferation, oxidative stress, and detection of apoptosis and necrosis. All compounds examined produced a concentration-dependent decrease in cell viability in the micromolar range, with the potent CB receptor agonist HU 210 and the enantiomer HU 211 (with no CB receptor activity) being the most potent compounds examined with apparent IC50 values of 1 and 0.6 μM, respectively. The endogenous CB anandamide showed similar potency and efficacy as structurally related polyunsaturated fatty acids with no reported activity at the CB receptors. The rapid (within hours) decrease in cell viability induced by the examined CBs suggests cytocidal rather than antiproliferative effects and is dependent on the plating cell population density with the highest toxicity around 100 cells/mm(2). The CB-induced cytotoxicity, which appears to involve CB receptors and the sphingomyelin-ceramide pathway, is a mixture of both apoptosis and necrosis that can be blocked by the antioxidants α-tocopherol and N-acetylcysteine. In conclusion, both synthetic and endogenous CBs produce seemingly unspecific cytotoxic effects in the P19 EC cells.

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

    Science.gov (United States)

    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. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  7. The effect of spiritual healing on in vitro tumour cell proliferation and viability - an experimental study

    DEFF Research Database (Denmark)

    Zachariae, R.; Hojgaard, L.; Zachariae, C.

    2005-01-01

    of cells to five different doses of healing or control. Researchers conducting the assays and statistical analyses were blinded to the experimental conditions. Main outcome measures were MTT viability, 3H-thymidine incorporation and counts of an adherent human breast cancer cell line (MCF-7...... three experimental days, doses, assays, and cells, 34 (51.6%) of 66 independent comparisons showed differences in the hypothesised direction (P = 0.90). The average effect size across cell lines, days, assays, and doses approached zero (Cohen's d = -0.01). The results do not support previous reports...

  8. Ribosome modulation factor, an important protein for cell viability encoded by the polyamine modulon.

    Science.gov (United States)

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

    2010-09-10

    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.

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

    Science.gov (United States)

    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

  10. A key inactivation factor of HeLa cell viability by a plasma flow

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Takehiko; Yokoyama, Mayo [Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan); Johkura, Kohei, E-mail: sato@ifs.tohoku.ac.jp [Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan)

    2011-09-21

    Recently, a plasma flow has been applied to medical treatment using effects of various kinds of stimuli such as chemical species, charged particles, heat, light, shock wave and electric fields. Among them, the chemical species are known to cause an inactivation of cell viability. However, the mechanisms and key factors of this event are not yet clear. In this study, we focused on the effect of H{sub 2}O{sub 2} in plasma-treated culture medium because it is generated in the culture medium and it is also chemically stable compared with free radicals generated by the plasma flow. To elucidate the significance of H{sub 2}O{sub 2}, we assessed the differences in the effects of plasma-treated medium and H{sub 2}O{sub 2}-added medium against inactivation of HeLa cell viability. These two media showed comparable effects on HeLa cells in terms of the survival ratios, morphological features of damage processes, permeations of H{sub 2}O{sub 2} into the cells, response to H{sub 2}O{sub 2} decomposition by catalase and comprehensive gene expression. The results supported that among chemical species generated in a plasma-treated culture medium, H{sub 2}O{sub 2} is one of the main factors responsible for inactivation of HeLa cell viability. (fast track communication)

  11. DMPD: Nitric oxide and cell viability in inflammatory cells: a role for NO inmacrophage function and fate. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15691589 Nitric oxide and cell viability in inflammatory cells: a role for NO inmacrophage function...(.png) (.svg) (.html) (.csml) Show Nitric oxide and cell viability in inflammatory cells: a role for NO inmacrophage function...ty in inflammatory cells: a role for NO inmacrophage function and fate. Authors Bosca L, Zeini M, Traves PG,

  12. [Induction of cell cycle arrest in bladder cancer RT4 cells by capsaicin].

    Science.gov (United States)

    Li, Qin; Wang, Xing-huan; Yang, Zhong-hua; Wang, Huai-peng; Yang, Zhi-wei; Li, Shi-wen; Zheng, Xin-min

    2010-05-11

    To study the effects of capsaicin on the growth of bladder cancer RT4 cell and its potential mechanism. Cell counting kit-8 (CCK-8) assay and flow cytometry were employed to observe the effects of capsaicin (50, 100, 150, 200, 250 micromol/L) on cell growth, cell cycle and apoptosis. Capsaicin (0 micromol/L) was used as a control. The effects of mRNA and protein of transient receptor potential cation channel subfamily V 1 (TRPV1) on RT4 cells were tested by RT-PCR and immunofluorescence respectively. And the expressions of cell cycle protein P53, P21, CDK2 were detected by Western blot after the treatment of capsaicin. 100 micromol/L capsaicin significantly decreased the viability of RT4 cell [82.0% +/- 6.2% vs 100.0% +/- 12.4% (control), P = 0.036] while the cell viability was 7.8% +/- 2.9% at 250 micromol/L (P = 0.000). It was in a dose-dependent manner. On the other hand, capsaicin induced the cell cycle arrest of bladder cancer RT4 cells G(0)/G(1) phase in a dose-dependent way. The cell proportion of G(0)/G(1) phase in the control was 37.4% +/- 5.6%, however, it was 72.4% +/- 5.3% at 250 micromol/L (P = 0.000). It was showed that TRPV1 mRNA and protein were expressed in RT4 cells. After a 48-hour treatment with capsaicin, the expressions of P53 and P21 were up-regulated in contrary to the expression of CDK2. Capsaicin induces the cell cycle arrest of bladder cancer RT4 cells G(0)/G(1) phase and growth inhibition via TRPV1 receptor by modulating the expression of P53, P21 and CDK2.

  13. Direct evaluation of myocardial viability and stem cell engraftment demonstrates salvage of the injured myocardium.

    Science.gov (United States)

    Kim, Paul J; Mahmoudi, Morteza; Ge, Xiaohu; Matsuura, Yuka; Toma, Ildiko; Metzler, Scott; Kooreman, Nigel G; Ramunas, John; Holbrook, Colin; McConnell, Michael V; Blau, Helen; Harnish, Phillip; Rulifson, Eric; Yang, Phillip C

    2015-03-27

    The mechanism of functional restoration by stem cell therapy remains poorly understood. Novel manganese-enhanced MRI and bioluminescence reporter gene imaging were applied to follow myocardial viability and cell engraftment, respectively. Human-placenta-derived amniotic mesenchymal stem cells (AMCs) demonstrate unique immunoregulatory and precardiac properties. In this study, the restorative effects of 3 AMC-derived subpopulations were examined in a murine myocardial injury model: (1) unselected AMCs, (2) ckit(+)AMCs, and (3) AMC-derived induced pluripotent stem cells (MiPSCs). To determine the differential restorative effects of the AMC-derived subpopulations in the murine myocardial injury model using multimodality imaging. SCID (severe combined immunodeficiency) mice underwent left anterior descending artery ligation and were divided into 4 treatment arms: (1) normal saline control (n=14), (2) unselected AMCs (n=10), (3) ckit(+)AMCs (n=13), and (4) MiPSCs (n=11). Cardiac MRI assessed myocardial viability and left ventricular function, whereas bioluminescence imaging assessed stem cell engraftment during a 4-week period. Immunohistological labeling and reverse transcriptase polymerase chain reaction of the explanted myocardium were performed. The unselected AMC and ckit(+)AMC-treated mice demonstrated transient left ventricular functional improvement. However, the MiPSCs exhibited a significantly greater increase in left ventricular function compared with all the other groups during the entire 4-week period. Left ventricular functional improvement correlated with increased myocardial viability and sustained stem cell engraftment. The MiPSC-treated animals lacked any evidence of de novo cardiac differentiation. The functional restoration seen in MiPSCs was characterized by increased myocardial viability and sustained engraftment without de novo cardiac differentiation, indicating salvage of the injured myocardium. © 2015 American Heart Association, Inc.

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

    Science.gov (United States)

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

    2015-10-01

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

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

    Science.gov (United States)

    Krasnow, Mark; Matthews, Mark; Shackel, Ken

    2008-01-01

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

  16. Polyvinyl Alcohol/Lithospermum Erythrorhizon Nanofibrous Membrane: Characterizations, In Vitro Drug Release, and Cell Viability

    Directory of Open Access Journals (Sweden)

    Ching-Wen Lou

    2017-11-01

    Full Text Available This study proposes an optimization process of the Lithospermum erythrorhizon (LE extraction with a higher purity of shikonin (SK. The influence of extraction temperature on the concentration of SK is examined, and an in vitro cell viability assay is used to examine the optimal concentration of SK. Afterwards, polyvinyl alcohol (PVA/LE solutions at ratios of 90/10, 80/20, and 70/30 w/w are electrospun into LE electrospun nanofibrous membranes (LENMs. The optimal manufacture parameters of LENMs are evaluated based on the test results of in vitro drug release test and cell viability assay. The optimal concentration occurs when the extraction temperature is −10 °C. The purity of the LE extract reaches 53.8% and the concentration of SK is 1.07 mg/mL. Moreover, the cell viability of nanofibrous membranes significantly increases to 136.8% when 0.7 μM SK is used. The diameter of nanofibers of LENM is decreased by 43.9% when the ratio of PVA solution to LE extract is 70/30 (w/w. 80/20 (w/w LENM has the maximum amount of drug release of 79% for a continuous period of 48 h. In particular, 90/10 (w/w LENM can create the maximum cell proliferation of 157.5% in a 24-h in vitro cell viability assay. This suggests that LENM has great potential to be used in facilitating tissue regeneration and wound healing.

  17. Toona Sinensis Extracts Induced Cell Cycle Arrest and Apoptosis in the Human Lung Large Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Cheng-Yuan Wang

    2010-02-01

    Full Text Available Toona sinensis extracts have been shown to exhibit anti-cancer effects in human ovarian cancer cell lines, human promyelocytic leukemia cells and human lung adenocarcinoma. Its safety has also been confirmed in animal studies. However, its anti-cancer properties in human lung large cell carcinoma have not been studied. Here, we used a powder obtained by freeze-drying the super-natant of centrifuged crude extract from Toona sinensis leaves (TSL-1 to treat the human lung carcinoma cell line H661. Cell viability was evaluated by the 3-(4-,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay. Flow cytometry analysis revealed that TSL-1 blocked H661 cell cycle progression. Western blot analysis showed decreased expression of cell cycle proteins that promote cell cycle progression, including cyclin-dependent kinase 4 and cyclin D1, and increased the expression of proteins that inhibit cell cycle progression, including p27. Furthermore, flow cytometry analysis showed that TSL-1 induced H661 cell apoptosis. Western blot analysis showed that TSL-1 reduced the expression of the anti-apoptotic protein B-cell lymphoma 2, and degraded the DNA repair protein, poly(ADP-ribose polymerase. TSL-1 shows potential as a novel therapeutic agent or for use as an adjuvant for treating human lung large cell carcinoma.

  18. Evaluation of tissue reaction, cell viability and cytokine production induced by Sealapex Plus

    Directory of Open Access Journals (Sweden)

    João Eduardo Gomes-Filho

    2011-08-01

    Full Text Available OBJECTIVE: The aim of this study was to investigate the effects of mineral trioxide aggregate (MTA, Sealapex, and a combination of Sealapex and MTA (Sealapex Plus on the reaction of subcutaneous connective tissue of rats, and on cell viability and cytokine production in mouse fibroblasts. MATERIAL AND METHODS: The tissue reaction was carried out with dentin tubes containing the materials implanted in the dorsal connective tissue of rats. The histological analysis was performed after 7 and 30 days. Millipore culture plate inserts with polyethylene tubes filled with materials were placed into 24-well cell culture plates with mouse fibroblasts to evaluate the cell viability by MTT assay. ELISA assays were also performed after 24 h of exposure of the mouse fibroblasts to set material disks. RESULTS: Histopathologic examination showed Von Kossa-positive granules that were birefringent to polarized light for all the studied materials at the tube openings. No material inhibited the cell viability in the in vitro test. It was detected IL-6 production in all root-end filling materials. MTA and Sealapex Plus induced a slight raise of mean levels of IL-1β. CONCLUSIONS: The results suggest that Sealapex Plus is biocompatible and stimulates the mineralization of the tissue.

  19. Clotrimazole decreases glycolysis and the viability of lung carcinoma and colon adenocarcinoma cells.

    Science.gov (United States)

    Penso, Julia; Beitner, Rivka

    2002-09-20

    Glycolysis is known to be the primary energy source in most cancer cells. We investigated here the effect of clotrimazole (1-(alpha-2-chlorotrityl)imidazole), the antifungal azole derivative, which was recently recognized as calmodulin antagonist, on the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis, and on ATP content and cell viability in LL/2 Lewis lung carcinoma cells and CT-26 colon adenocarcinoma cells. We found that clotrimazole induced a significant, dose- and time-dependent reduction in the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, ATP, and cell viability. These findings suggest that clotrimazole causes a reduction in glycolysis and ATP levels, which eventually leads to cell destruction after 3 h of treatment. Since cell proliferation was also reported to be inhibited by calmodulin antagonists, this substance is most promising agent in treatment of cancer by inhibiting both cell proliferation and the glycolytic supply of ATP required for cancer cell growth. Copyright 2002 Elsevier Science B.V.

  20. Lactobacillus decelerates cervical epithelial cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Katarina Vielfort

    Full Text Available We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells.

  1. Cell cycle control factors and skeletal development

    Directory of Open Access Journals (Sweden)

    Toru Ogasawara

    2013-05-01

    Full Text Available In the oral and maxillofacial region, conditions such as delayed bone healing after tooth extraction, bone fracture, trauma-induced bone or cartilage defects, and tumors or birth defects are common, and it is necessary to identify the molecular mechanisms that control skeletogenesis or the differentiation of cells, in order to establish new treatment strategies for these conditions. Multiple studies have been conducted to investigate the involvement of factors that may be crucial for skeletogenesis or the differentiation of cells, including transcription factors, growth factors and cell cycle factors. Several genetically engineered mouse models of cell cycle factors have been generated in research seeking to identify cell cycle factor(s involved in the differentiation of cells, carcinogenesis, etc. Many groups have also reported the importance of cell cycle factors in the differentiation of osteoblasts, osteoclasts, chondrocytes and other cell types. Herein, we review the phenotypes of the genetically engineered mouse models of cell cycle factors with a particular focus on the size, body weight and skeletal abnormalities of the mice, and we discuss the potential of cell cycle factors as targets of clinical applications.

  2. Microfluidic Cell Cycle Analysis of Spread Cells by DAPI Staining

    Directory of Open Access Journals (Sweden)

    Jing Sun

    2017-01-01

    Full Text Available Single-cell cell cycle analysis is an emerging technique that requires detailed exploration of the image analysis process. In this study, we established a microfluidic single-cell cell cycle analysis method that can analyze cells in small numbers and in situ on a microfluidic chip. In addition, factors that influenced the analysis were carefully investigated. U87 or HeLa cells were seeded and attached to microfluidic channels before measurement. Cell nucleic DNA was imaged by 4′-6-diamidino-2-phenylindole (DAPI staining under a fluorescent microscope and subsequently fluorescent intensities of the cell nuclei DNA were converted to depict histograms for cell cycle phases. DAPI concentration, microscopic magnification, exposure time and cell number were examined for optimal cell cycle analysis conditions. The results showed that as few as a few hundred cells could be measured by DAPI staining in the range of 0.4–0.6 μg/mL to depict histograms with typical cell cycle phase distribution. Microscopic magnification during image acquisition, however, could distort the phase distribution. Exposure time did not significantly affect the cell cycle analysis. Furthermore, cell cycle inhibitor rapamycin treatment changed the cell cycle phase distribution as expected. In conclusion, a method for microfluidic single-cell cell cycle analysis of spread cells in situ was developed. Factors such as dye concentration and microscopic magnification had more influence on cell cycle phase distribution. Further studies will focus on detail differentiation of cell cycle phases and the application of such a method for biological meanings.

  3. Viability and membrane potential analysis of Bacillus megaterium cells by impedance flow cytometry.

    Science.gov (United States)

    David, F; Hebeisen, M; Schade, G; Franco-Lara, E; Di Berardino, M

    2012-02-01

    Single cell analysis is an important tool to gain deeper insights into microbial physiology for the characterization and optimization of bioprocesses. In this study a novel single cell analysis technique was applied for estimating viability and membrane potential (MP) of Bacillus megaterium cells cultured in minimal medium. Its measurement principle is based on the analysis of the electrical cell properties and is called impedance flow cytometry (IFC). Comparatively, state-of-the-art fluorescence-based flow cytometry (FCM) was used to verify the results obtained by IFC. Viability and MP analyses were performed with cells at different well-defined growth stages, focusing mainly on exponential and stationary phase cells, as well as on dead cells. This was done by PI and DiOC(2)(3) staining assays in FCM and by impedance measurements at 0.5 and 10 MHz in IFC. In addition, transition growth stages of long-term cultures and agar plate colonies were characterized with both methods. FCM and IFC analyses of all experiments gave comparable results, quantitatively and qualitatively, indicating that IFC is an equivalent technique to FCM for the study of physiological cell states of bacteria. Copyright © 2011 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Ana Lúcia Vargas Arigony

    2013-01-01

    Full Text Available 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.

  7. Do Multiwell Plate High Throughput Assays Measure Loss of Cell Viability Following Exposure to Genotoxic Agents?

    Directory of Open Access Journals (Sweden)

    Razmik Mirzayans

    2017-08-01

    Full Text Available Cell-based assays in multiwell plates are widely used for radiosensitivity and chemosensitivity assessment with different mammalian cell types. Despite their relative ease of performance, such assays lack specificity as they do not distinguish between the cytostatic (reversible/sustained growth arrest and cytotoxic (loss of viability effects of genotoxic agents. We recently reported studies with solid tumor-derived cell lines demonstrating that radiosensitivity as measured by multiwell plate colorimetric (e.g., XTT and fluorimetric (e.g., CellTiter-Blue assays reflects growth arrest but not loss of viability. Herein we report similar observations with cancer cell lines expressing wild-type p53 (A549 lung carcinoma or mutant p53 (MDA–MB-231 breast carcinoma after treatment with the chemotherapeutic drug cisplatin. Importantly, we show that treatment of cancer cells with concentrations of cisplatin that result in 50% effect (i.e., IC50 in multiwell plate assays trigger the emergence of growth arrested cells that exhibit highly enlarged morphology, remain viable and adherent to the culture dish, and metabolize the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT to its formazan derivative. The emergence of markedly enlarged viable cells complicates the interpretation of chemosensitivity data obtained with multiwell plate high throughput assays. Relying solely on IC50 values could be misleading.

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

    Science.gov (United States)

    Zhou, Y; Guan, X X; Zhu, Z L; Guo, J; Huang, Y C; Hou, W W; Yu, H Y

    2010-12-01

    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). 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. After treating BMSCs with high caffeine concentrations (0.1-1mM), 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. 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.

  9. Analysis of the Interactions of Botanical Extract Combinations Against the Viability of Prostate Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Lynn S. Adams

    2006-01-01

    Full Text Available Herbal medicines are often combinations of botanical extracts that are assumed to have additive or synergistic effects. The purpose of this investigation was to compare the effect of individual botanical extracts with combinations of extracts on prostate cell viability. We then modeled the interactions between botanical extracts in combination isobolographically. Scutellaria baicalensis, Rabdosia rubescens, Panax-pseudo ginseng, Dendranthema morifolium, Glycyrrhiza uralensis and Serenoa repens were collected, taxonomically identified and extracts prepared. Effects of the extracts on cell viability were quantitated in prostate cell lines using a luminescent ATP cell viability assay. Combinations of two botanical extracts of the four most active extracts were tested in the 22Rv1 cell line and their interactions assessed using isobolographic analysis. Each extract significantly inhibited the proliferation of prostate cell lines in a time- and dose-dependent manner except repens. The most active extracts, baicalensis, D. morifolium, G. uralensis and R. rubescens were tested as two-extract combinations. baicalensis and D. morifolium when combined were additive with a trend toward synergy, whereas D. morifolium and R. rubescens together were additive. The remaining two-extract combinations showed antagonism. The four extracts together were significantly more effective than the two-by-two combinations and the individual extracts alone. Combining the four herbal extracts significantly enhanced their activity in the cell lines tested compared with extracts alone. The less predictable nature of the two-way combinations suggests a need for careful characterization of the effects of each individual herb based on their intended use.

  10. Effect of surface organic coatings of cellulose nanocrystals on the viability of mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Jimenez AS

    2017-09-01

    Full Text Available Ambar S Jimenez,1 Francesca Jaramillo,1 Usha D Hemraz,2 Yaman Boluk,3 Karina Ckless,1 Rajesh Sunasee1 1Department of Chemistry, State University of New York at Plattsburgh, Plattsburgh, NY, USA; 2National Research Council, Montreal, QC, Canada, 3Department of Civil & Environmental Engineering, University of Alberta and National Institute for Nanotechnology, National Research Council, Edmonton, AB, Canada Abstract: Cellulose nanocrystals (CNCs have emerged as promising candidates for a number of bio-applications. Surface modification of CNCs continues to gain significant research interest as it imparts new properties to the surface of the nanocrystals for the design of multifunctional CNCs-based materials. A small chemical surface modification can potentially lead to drastic behavioral changes of cell-material interactions thereby affecting the intended bio-application. In this work, unmodified CNCs were covalently decorated with four different organic moieties such as a diaminobutane fragment, a cyclic oligosaccharide (β-cyclodextrin, a thermoresponsive polymer (poly[N-isopropylacrylamide], and a cationic aminomethacrylamide-based polymer using different synthetic covalent methods. The effect of surface coatings of CNCs and the respective dose-response of the above organic moieties on the cell viability were evaluated on mammalian cell cultures (J774A.1 and MFC-7, using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays. Overall, the results indicated that cells exposed to surface-coated CNCs for 24 h did not display major changes in cell viability, membrane permeability as well as cell morphology. However, with longer exposure, all these parameters were somewhat affected, which appears not to be correlated with either anionic or cationic surface coatings of CNCs used in this study. Keywords: cellulose nanocrystals, surface coating, cell viability, MTT, LDH

  11. Analysis of the Interactions of Botanical Extract Combinations Against the Viability of Prostate Cancer Cell Lines

    Science.gov (United States)

    Adams, Lynn S.; Seeram, Navindra P.; Hardy, Mary L.; Carpenter, Catherine; Heber, David

    2006-01-01

    Herbal medicines are often combinations of botanical extracts that are assumed to have additive or synergistic effects. The purpose of this investigation was to compare the effect of individual botanical extracts with combinations of extracts on prostate cell viability. We then modeled the interactions between botanical extracts in combination isobolographically. Scutellaria baicalensis, Rabdosia rubescens, Panax-pseudo ginseng, Dendranthema morifolium, Glycyrrhiza uralensis and Serenoa repens were collected, taxonomically identified and extracts prepared. Effects of the extracts on cell viability were quantitated in prostate cell lines using a luminescent ATP cell viability assay. Combinations of two botanical extracts of the four most active extracts were tested in the 22Rv1 cell line and their interactions assessed using isobolographic analysis. Each extract significantly inhibited the proliferation of prostate cell lines in a time- and dose-dependent manner except S. repens. The most active extracts, S. baicalensis, D. morifolium, G. uralensis and R. rubescens were tested as two-extract combinations. S. baicalensis and D. morifolium when combined were additive with a trend toward synergy, whereas D. morifolium and R. rubescens together were additive. The remaining two-extract combinations showed antagonism. The four extracts together were significantly more effective than the two-by-two combinations and the individual extracts alone. Combining the four herbal extracts significantly enhanced their activity in the cell lines tested compared with extracts alone. The less predictable nature of the two-way combinations suggests a need for careful characterization of the effects of each individual herb based on their intended use. PMID:16550232

  12. Decrease in cell viability in an RMF, sigma(38), and OmpC triple mutant of Escherichia coli.

    Science.gov (United States)

    Samuel Raj, V; Füll, Christine; Yoshida, Madoka; Sakata, Kaori; Kashiwagi, Keiko; Ishihama, Akira; Igarashi, Kazuei

    2002-11-29

    In a speG-disrupted Escherichia coli mutant, which cannot metabolize spermidine to acetylspermidine, addition of spermidine to the medium caused a decrease in cell viability at the late stationary phase of growth. There were parallel decreases in the levels of ribosome modulation factor (RMF), the sigma(38) subunit of RNA polymerase, and the outer membrane protein C (OmpC). To clarify that these three proteins are strongly involved in cell viability, the rmf, rpoS (encoding sigma(38)), and ompC genes were disrupted. Viability of the triple mutant decreased to less than 1% of normal cells. The triple mutant had a reduced cell viability compared to any combination of double mutants, which also had a reduced cell viability. The single rmf and rpoS, but not ompC, mutant only slightly reduced cell viability. The results indicate that cooperative functions of these three proteins are necessary for cell viability at the late stationary phase. The triple mutant had a reduced level of ribosomes and of intracellular cations.

  13. The effect of Aloe vera gel on viability of dental pulp stem cells.

    Science.gov (United States)

    Sholehvar, Fatemeh; Mehrabani, Davood; Yaghmaei, Parichehr; Vahdati, Akbar

    2016-10-01

    Dental pulp stem cells (DPSCs) can play a prominent role in tissue regeneration. Aloe vera L. (Liliaceae) contains the polysaccharide of acemannan that was shown to be a trigger factor for cell proliferation, differentiation, mineralization, and dentin formation. This study sought to determine the viability of DPSCs in Aloe vera in comparison with Hank's balanced salt solution (HBSS). Twelve rabbits underwent anesthesia, and their incisor teeth were extracted; the pulp tissue was removed, chopped, treated with collagenase and plated in culture flasks. DPSCs from passage 3 were cultured in 24-well plates, and after 3 days, the culture media changed to 10, 25, 50, and 100% concentrations of Aloe vera at intervals of 45 and 90 min and 3 and 6 h. Distilled water was used as negative and HBSS as positive control for comparison. The cell morphology, viability, population doubling time (PDT), and growth kinetics were evaluated. RT-PCR was carried out for characterization and karyotyping for chromosomal stability. Aloe vera showed a significant higher viability than HBSS (74.74%). The 50% Aloe vera showed higher viability (97.73%) than other concentrations. PDT in 50% concentration was 35.1 h and for HBSS was 49.5 h. DPSCs were spindle shaped and were positive for CD73 and negative for CD34 and CD45. Karyotyping was normal. Aloe vera as an inexpensive and available herb can improve survival of avulsed or broken teeth in emergency cases as a transfer media. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    2016-05-04

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

  15. Trehalose preincubation increases mesenchymal (CD271+ stem cells post-cryopreservation viability

    Directory of Open Access Journals (Sweden)

    Indra Kusuma

    2016-10-01

    Full Text Available Background: Dimethyl sulfoxide (Me2SO is a common cryoprotective agent widely used in cell preservation system. Me2SO is currently known to cause epigenetic changes which are  critical in stem cells development and cellular differentiation. Therefore, it is imperative to develop cryopreservation techniques that protect cellular functions and avert Me2SO adverse effect. Trehalose was able to protect organism in extreme condition such as dehydration and cold. This study aimed to verify the protective effect of trehalose preincubation procedure in cryopreservation.Methods: The study was conducted using experimental design. Thawed mesenchymal (CD271+ stem cells from YARSI biorepository were used for the experiment. Trehalose preincubation was performed for 1 hour, internalized trehalose was confirmed by FTIR-ATR measurement. Three groups consisted of (1 cryopreserved without trehalose preincubation, (2 cryopreserved with trehalose preincubation, and (3 did not undergo cryopreservation were evaluated after 24 hours in LN2 for viability in culture. The absorbance from each group was measured at 450 nm. The analysis performed using paired student t test.Results: Viability of thawed mesenchymal (CD271+ stem cells that undergo trehalose preincubation prior cryopreservation was significantly higher (p<0.05 compared to group without trehalose preincubation. Higher viability observed between group with trehalose preincubation compared with controlled group suggests protection to trypsinization. Mesenchymal (CD271+ stem cells incubated for 1 hour in 100 mM trehalose supplemented medium  results in 15%  trehalose loading efficiency.Conclusion: These findings confirm the protective effect of trehalose preincubation in cryopreservation. Future research should be directed to elucidate the trehalose internalization mechanism and eventually the protective mechanism of trehalose in mammalian cell cryopreservation.

  16. The Effect of Thymoquinone on BEAS-2B Cell Viability and TGF-β1 Release

    Directory of Open Access Journals (Sweden)

    Hasret Ecevit

    2017-02-01

    Full Text Available Thymoquinone, one of the essential oil in the structure of cumin, is used for alternative therapy for many diseases from past to present. It was shown to have anti-carcinogenic and anti-inflammatory effects, as well as positive effects on fibrosis. However, there is no study on the effect of thymoquinone on cancer and fibrosis mechanism in bronchial epithelium cell line BEAS-2B. In our study, the effect of thymoquinone on cell viability and transforming growth factor-beta 1 (TGF-β1 level, which has an important role in the regulation of many biological processes including cancer and fibrosis-associated signal transduction, was evaluated. BEAS-2B cells were exposed to thymoquinone at 0–80 μmol/L concentrations for 24-, 48- and 72-hour durations. Cell viability was evaluated with 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT test. TGF-β1 level was determined with enzyme-linked immunosorbent assay (ELISA method from the collected supernatant. Cell viability was found to be increased at all concentrations and durations (10–80 μmol/L; 24, 48 and 72 h according to the control group (0 μmol/L; thymoquinone in ethanol (p < 0.0001. Moreover, thymoquinone was found to increase the level of TGF-β1 only at 80 μmol/L concentration and 24-hour exposure period (0 μmol/L, 53.41 ± 18.44 pgr/ml TGF-β1; 80 μmol/L, 174.5 ± 80.03 pgr/ml TGF-β1. As a result, thymoquinone was found to increase cell proliferation and encourage TGF-β1 release.

  17. Effects of cytokinins, cytokinin ribosides and their analogs on the viability of normal and neoplastic human cells.

    Science.gov (United States)

    Casati, Silvana; Ottria, Roberta; Baldoli, Erika; Lopez, Elisa; Maier, Jeanette A M; Ciuffreda, Pierangela

    2011-10-01

    We examined the effects of some cytokinins and cytokinin ribosides including a series of adenosine analogs differently substituted in the N(6) position, along with some hypoxanthine derivatives on the viability of normal and neoplastic human cells. Cytokinins such as trans-zeatin, isopentenyladenine and benzyladenine do not show any effect, while cytokinin ribosides such as trans-zeatin riboside, isopentenyladenosine, and benzylaminopurine riboside impair the viability of normal and neoplastic cells, apart from colon carcinoma LoVo cells.

  18. Influence of shear stress and size on viability of endothelial cells exposed to gold nanoparticles

    Science.gov (United States)

    Fede, C.; Albertin, Giovanna; Petrelli, L.; De Caro, R.; Fortunati, I.; Weber, V.; Ferrante, Camilla

    2017-09-01

    Screening nanoparticle toxicity directly on cell culture can be a fast and cheap technique. Nevertheless, to obtain results in accordance with those observed in live animals, the conditions in which cells are cultivated should resemble the one encountered in live systems. Microfluidic devices offer the possibility to satisfy this requirement, in particular with endothelial cell lines, because they are capable to reproduce the flowing media and shear stress experienced by these cell lines in vivo. In this work, we exploit a microfluidic device to observe how human umbilical vein endothelial cells (HUVEC) viability changes when subject to a continuous flow of culture medium, in which spherical citrate-stabilized gold nanoparticles of different sizes and at varying doses are investigated. For comparison, the same experiments are also run in multiwells where the cells do not experience the shear stress induced by the flowing medium. We discuss the results considering the influence of mode of exposure and nanoparticle size (24 and 13 nm). We observed that gold nanoparticles show a lower toxicity under flow conditions with respect to static and the HUVEC viability decreases as the nanoparticle surface area per unit volume increases, regardless of size.

  19. Hibiscus sabdariffa extractivities on cadmium-mediated alterations of human U937 cell viability and activation.

    Science.gov (United States)

    Okoko, Tebekeme; Ere, Diepreye

    2012-01-01

    To investigate the effect of the anthocyanin-rich extract of Hibiscus sabdariffa (H. sabdariffa) calyx on the viability of cadmium-treated U937 cells and cadmium-mediated activation of U937-derived macrophages. The macrophage cell line U937 was treated with cadmium (0.1 μ mol/L) and later incubated with the anthocyanin-rich extract and cell viability was assessed via trypan blue staining. In the other experiment, the U937 cells were transformed to the macrophage form by treatment with phorbol 12, myristate 13, and acetate and incubated with cadmium (10 μ mol/L). The anthocyanin-rich extract was added to the cells later and subsequently, the supernatant of each cell culture was analysed for the production of tumour necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), nitric oxide, and catalase activity as indices for the activation of macrophages. It revealed that the anthocynanin-rich extract significantly (P sabdariffa possesses significant immunoprotective effect. These corroborate the immense reported antioxidant and medicinal potential of the calyces of the plant which could be exploited for pharmacological and neutraceutical advantages. Copyright © 2012 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  20. [EFFECT OF TEMPERATURE ON THE VIABILITY OF PLANKTON CELLS AND MODEL LEGIONELLA PNEUMOPHILA BIOFILMS IN WATER].

    Science.gov (United States)

    Tartakovsky, I S; Karpoval, T I; Gruzdeva, O A; Marinenko, O V; Dronina, Yu E

    2015-01-01

    Study the effect of water temperature from 40 to 70 degrees C on viability of plankton forms and model Legionella pneumophila under experimental conditions. Monospecies legionella biofilms, obtained in plates for enzyme immunoassay during 96 hours at 28 degrees C, and water suspension of BCYE agar cultivated cells of L. pneumophila at a concentration of 10(3) - 10(5) CFU per liter were used in the study for evaluation of bactericidal effect of temperature on various legionella forms. Analysis of effects of various temperature regimens on plankton forms and model legionella biofilms has shown that at a temperature range from 50 to 60 degrees C a significant reduction of quantity of viable legionella cells occurs. Model legionella biofilms have partially conserved viability at a temperature of 60 degrees C and only exposition to a temperature of 70 degrees C resulted in death of legionella biofilms and plankton forms of bacteria. A dependence of viability conservation of legionella from the initial concentration of the causative agent in water and duration of exposition at varying temperature was shown. Short-term heating at a temperature of at least 70 degrees C has the most pronounced bactericidal effect on plankton forms and model L. pneumophila biofilms under experimental conditions. Such temperature regimen could be used as one of the prophylaxis approaches during maintenance of especially dangerous water system and, fist of all, systems of hot water supply.

  1. Cell cycle activation by plant parasitic nematodes

    NARCIS (Netherlands)

    Goverse, A.; Almeida Engler, de J.; Verhees, J.; Krol, van der S.; Helder, J.; Gheysen, G.

    2000-01-01

    Sedentary nematodes are important pests of crop plants. They are biotrophic parasites that can induce the (re)differentiation of either differentiated or undifferentiated plant cells into specialized feeding cells. This (re)differentiation includes the reactivation of the cell cycle in specific

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

    Science.gov (United States)

    Sullivan, M L; Bradford, B J

    2011-01-01

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

  3. Kinetic analysis of hybridoma cells viability under mechanical shear stress with and without serum protection.

    Science.gov (United States)

    Legazpi, Lorea; Laca, Adriana; Díaz, Mario

    2009-10-01

    The effect of a well-defined mild shear stress on hybridoma cell viability (HB-8852) in a serum-free culture medium has been analysed, and the role as shear protector of different concentrations of fetal bovine serum have been studied. Samples harvested from cultures in their late exponential growth phase, were subjected in a rheometer to a constant shear stress of 0.41 +/- 0.02 Pa, and the evolution of viable and total cell concentrations was determined and compared with static controls. A simple segregated kinetic model for the viable and dead cells was used to know the effect of serum concentration on the specific cell growth and death rate of the cells.

  4. All-trans retinoic acid influences viability, migration and adhesion of U251 glioblastoma cells

    Directory of Open Access Journals (Sweden)

    Marjanović-Vićentić Jelena

    2017-01-01

    Full Text Available Glioblastoma (GBM is one of the most aggressive and deadly forms of cancer. Literature data reveals that all-trans retinoic acid (ATRA has anticancer effects on different types of tumor cells. However, data about the effects of ATRA on glioblastoma cells are contradictory. In this study, we examined whether ATRA treatment affects features of human glioblastoma U251 cells. To that end, the cells were treated with different concentrations of ATRA. Results obtained by MTT and the crystal violet assays imply that ATRA affected the viability of U251 glioblastoma cells in a dose- and time-dependent manner. Fluorescence staining of microtubule cytoskeleton protein α-tubulin revealed that ATRA induced changes in cell morphology. Using semi-quantitative RT-PCR we found that the expression of SOX3 and GFAP genes, as markers of neural differentiation, was not changed upon ATRA treatment. Thus, the observed changes in cell morphology after ATRA treatment are not associated with neural differentiation of U251 glioblastoma cells. The scratch-wound healing assay revealed that ATRA changed the mode of U251 cell migration from collective to single cell motility. The cell-matrix adhesion assay demonstrated that the pharmacologically relevant concentration of ATRA lowered the cell-matrix adhesion capability of U251 cells. In conclusion, our results imply that further studies are needed before ATRA could be considered for the treatment of glioblastoma. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173051

  5. Effect of vital dyes on retinal pigmented epithelial cell viability and apoptosis: implications for chromovitrectomy

    Science.gov (United States)

    Penha, Fernando M; Pons, Marianne; Costa, Elaine Fiod; Rodrigues, Eduardo B.; Maia, Mauricio; Marin-Castaño, Maria E; Farah, Michel Eid

    2013-01-01

    Purpose To investigate in vitro effect of vital dyes on toxicity and apoptosis in a human retinal pigment epithelial (RPE) cell line. Methods ARPE-19 cells were exposed to brilliant blue-BriB, evans blue-EB, bromophenol blue-BroB, indocyanine green-ICG, infracyanine green-IfCG, light green-LG, fast green-FG, indigo carmine-IC and congo red-CR. BSS was used as the control. Five different concentrations and two times were tested. Cell viability was determined by MTS assay and apoptosis by Bax expression on western blot. Results All dyes significantly reduced cell viability after 3 minutes of exposure at all concentrations (pdyes exposure, except BriB; ICG had the highest Bax expression (pdye was BriB followed by LG, IfCG, FG, CR, IC, BroB, RB and ICG. ICG was toxic at all concentrations and exposure times tested. Moreover, BriB was the only dye that did not induce apoptosis in ARPE-19 cells. PMID:24022718

  6. Impact of bone harvesting techniques on cell viability and the release of growth factors of autografts.

    Science.gov (United States)

    Miron, Richard J; Gruber, Reinhard; Hedbom, Erik; Saulacic, Nikola; Zhang, Yufeng; Sculean, Anton; Bosshardt, Dieter D; Buser, Daniel

    2013-08-01

    Autogenous bone grafts obtained by different harvesting techniques behave differently during the process of graft consolidation; the underlying reasons are however not fully understood. One theory is that harvesting techniques have an impact on the number and activity of the transplanted cells which contribute to the process of graft consolidation. To test this assumption, porcine bone grafts were harvested with four different surgical procedures: bone mill, piezosurgery, bone drilling (bone slurry), and bone scraper. After determining cell viability, the release of molecules affecting bone formation and resorption was assessed by reverse transcription polymerase chain reaction and immunoassay. The mitogenic and osteogenic activity of the conditioned media was evaluated in a bioassay with isolated bone cells. Cell viability and the release of molecules affecting bone formation were higher in samples harvested by bone mill and bone scraper when compared with samples prepared by bone drilling and piezosurgery. The harvesting procedure also affected gene expression, for example, bone mill and bone scraper samples revealed significantly higher expression of growth factors such as bone morphogenetic protein-2 and vascular endothelial growth factor compared with the two other modalities. Receptor activator of nuclear factor kappa B ligand expression was lowest in bone scraper samples. These data can provide a scientific basis to better understand the impact of harvesting techniques on the number and activity of transplanted cells, which might contribute to the therapeutic outcome of the augmentation procedure. © 2012 Wiley Periodicals, Inc.

  7. A functional screen identifies specific microRNAs capable of inhibiting human melanoma cell viability.

    Directory of Open Access Journals (Sweden)

    Jos B Poell

    Full Text Available Malignant melanoma is an aggressive form of skin cancer with poor prognosis. Despite improvements in awareness and prevention of this disease, its incidence is rapidly increasing. MicroRNAs (miRNAs are a class of small RNA molecules that regulate cellular processes by repressing messenger RNAs (mRNAs with partially complementary target sites. Several miRNAs have already been shown to attenuate cancer phenotypes, by limiting proliferation, invasiveness, tumor angiogenesis, and stemness. Here, we employed a genome-scale lentiviral human miRNA expression library to systematically survey which miRNAs are able to decrease A375 melanoma cell viability. We highlight the strongest inhibitors of melanoma cell proliferation, including the miR-15/16, miR-141/200a and miR-96/182 families of miRNAs and miR-203. Ectopic expression of these miRNAs resulted in long-term inhibition of melanoma cell expansion, both in vitro and in vivo. We show specifically miR-16, miR-497, miR-96 and miR-182 are efficient effectors when introduced as synthetic miRNAs in several melanoma cell lines. Our study provides a comprehensive interrogation of miRNAs that interfere with melanoma cell proliferation and viability, and offers a selection of miRNAs that are especially promising candidates for application in melanoma therapy.

  8. A role for LRP4 in neuronal cell viability is related to apoE-binding.

    Science.gov (United States)

    Lu, Yonghao; Tian, Qing Bao; Endo, Shogo; Suzuki, Tatsuo

    2007-10-26

    The distribution pattern of apolipoprotein E (apoE) in cortical neurons in culture resembles that of low-density lipoprotein receptor-related protein 4 (LRP4). Both proteins are distributed in a punctate manner on the cell surface throughout neurons, including somas and dendrites. This finding prompted us to examine whether apoE is a ligand for LRP4 in the rat brain. ApoE and LRP4 from both Cos7 cells heterologous expressing LRP4 and brain homogenate were co-immunoprecipitated. We then examined the effect of antibody against the ligand-binding domain of LRP4 (anti-LB). Anti-LB applied to neuronal cells in culture down-regulated MAP2-immunoreactive neurons, reduced the viability of neurons and impaired synaptic structure. This effect was possibly due to a blockade of the binding of extraneuronal ligands, such as apoE/cholesterol, to LRP4 protein, since anti-LB suppressed binding of apoE to the LRP4 heterologously expressed in Cos7 cells. These results suggest that apoE is an endogenous ligand for LRP4 and may play a role as a receptor for extracellular signals, including those from glial cells, in the maintenance of the viability of neurons.

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

    Directory of Open Access Journals (Sweden)

    Meenakshi Sharma

    2016-01-01

    Full Text Available 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.

  10. Berberine promotes antiproliferative effects of epirubicin in T24 bladder cancer cells by enhancing apoptosis and cell cycle arrest
.

    Science.gov (United States)

    Zhuo, Yumin; Chen, Qibiao; Chen, Bo; Zhan, Xiongyu; Qin, Xiaoping; Huang, Jun; Lv, Xiuxiu

    2017-01-01

    The present study was aimed to observe the effect of berberine (Ber) on epirubicin (EPI)-induced growth inhibition, apoptosis, and cell cycle arrest in T24 bladder cancer cells. The cancer cells were exposed to EPI, with or without different concentrations of Ber. The viability of the cancer cells was measured by cell counting Kit-8, the apoptosis was determined by Hoechst 33258 staining and the expression of cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, and P53 proteins were detected by Western blot assay. In addition, cell cycle arrest and the production of reactive oxygen species (ROS) were also measured. We found that Ber enhanced the inhibitory effect of EPI on the viability of T24 cells and promoted EPI-induced cell cycle arrest at G0/G1 and apoptosis in T24 cells. EPI increased the expression of cleaved caspase-3, cleaved caspase-9, Bax, P53, and P21 proteins, all of which were enhanced by treatment with Ber. In contrast, Ber exposure further decreased the expression of Bcl-2 in EPI-treated T24 cells. Furthermore, we also demonstrated that Ber significantly increased ROS production in EPI-treated T24 cells. These data indicate that Ber enhances the antiproliferative effects of EPI in bladder cancer cells by promoting apoptosis and cell cycle arrest.
.

  11. Mitochondrial regulation of cell cycle progression through SLC25A43

    Energy Technology Data Exchange (ETDEWEB)

    Gabrielson, Marike; Reizer, Edwin [School of Health and Medical Sciences, Faculty of Medicine and Health, Örebro University, SE 70182 Örebro (Sweden); Stål, Olle [Department of Clinical and Experimental Medicine, Linköping University, SE 58185 Linköping (Sweden); Department of Oncology, Linköping University, SE 58185 Linköping (Sweden); Tina, Elisabet, E-mail: elisabet.tina@regionorebrolan.se [Department of Clinical Research Laboratory, Faculty of Medicine and Health, Örebro University, SE 70182 Örebro (Sweden)

    2016-01-22

    An increasing body of evidence is pointing towards mitochondrial regulation of the cell cycle. In a previous study of HER2-positive tumours we could demonstrate a common loss in the gene encoding for the mitochondrial transporter SLC25A43 and also a significant relation between SLC25A43 protein expression and S-phase fraction. Here, we investigated the consequence of suppressed SLC25A43 expression on cell cycle progression and proliferation in breast epithelial cells. In the present study, we suppressed SLC25A43 using siRNA in immortalised non-cancerous breast epithelial MCF10A cells and HER2-positive breast cancer cells BT-474. Viability, apoptosis, cell proliferation rate, cell cycle phase distribution, and nuclear Ki-67 and p21, were assessed by flow cytometry. Cell cycle related gene expressions were analysed using real-time PCR. We found that SLC25A43 knockdown in MCF10A cells significantly inhibited cell cycle progression during G{sub 1}-to-S transition, thus significantly reducing the proliferation rate and fraction of Ki-67 positive MCF10A cells. In contrast, suppressed SLC25A43 expression in BT-474 cells resulted in a significantly increased proliferation rate together with an enhanced G{sub 1}-to-S transition. This was reflected by an increased fraction of Ki-67 positive cells and reduced level of nuclear p21. In line with our previous results, we show a role for SLC25A43 as a regulator of cell cycle progression and proliferation through a putative mitochondrial checkpoint. These novel data further strengthen the connection between mitochondrial function and the cell cycle, both in non-malignant and in cancer cells. - Highlights: • Proposed cell cycle regulation through the mitochondrial transporter SLC25A43. • SLC25A43 alters cell proliferation rate and cell cycle progression. • Suppressed SLC25A43 influences transcription of cell cycle regulatory genes.

  12. Ciliary neurotrophic factor (CNTF) promotes skeletal muscle progenitor cell (MPC) viability via the phosphatidylinositol 3-kinase-Akt pathway.

    Science.gov (United States)

    Hiatt, Kelly; Lewis, Davina; Shew, Mathew; Bijangi-Vishehsaraei, Khadijeh; Halum, Stacey

    2014-12-01

    Muscle progenitor cells (MPCs) are currently being investigated as cellular vectors to deliver neurotrophic factor (NF) for the promotion of re-innervation after axonal injury. Ideally NF delivery in such a model would enhance axonal regeneration while simultaneously promoting MPC viability. To date, insulin-like growth factor 1 (IGF-1) is one of the few NFs known to promote both re-innervation and MPC viability. We herein identify ciliary neurotrophic factor (CNTF) as a factor that promotes MPC viability in culture, and demonstrate CNTF to impart greater viability effects on MPCs than IGF-1. We demonstrate that pharmacological inhibition via LY294002 results in abrogation of CNTF-mediated viability, suggesting that the CNTF-mediated MPC viability benefit occurs via the PI3-Akt pathway. Finally, we employ a genetic model, establishing MPC cultures from mice deficient in class IA PI-3 K (p85α(-/-) ) mice, and demonstrate that the viability benefit imparted by CNTF is completely abrogated in PI-3 K-deficient MPCs compared to wild-type controls. In summary, our investigations define CNTF as a promoter of MPC viability beyond IGF-1, and reveal that the CNTF-mediated MPC viability effects occur via the PI3-Akt pathway. Copyright © 2012 John Wiley & Sons, Ltd.

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

    Directory of Open Access Journals (Sweden)

    Nathalia R. Lestard

    2016-01-01

    Full Text Available 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.

  14. Extracellular vesicle associated long non-coding RNAs functionally enhance cell viability

    Directory of Open Access Journals (Sweden)

    Chris Hewson

    2016-10-01

    Full Text Available Cells communicate with one another to create microenvironments and share resources. One avenue by which cells communicate is through the action of exosomes. Exosomes are extracellular vesicles that are released by one cell and taken up by neighbouring cells. But how exosomes instigate communication between cells has remained largely unknown. We present evidence here that particular long non-coding RNA molecules are preferentially packaged into exosomes. We also find that a specific class of these exosome associated non-coding RNAs functionally modulate cell viability by direct interactions with l-lactate dehydrogenase B (LDHB, high-mobility group protein 17 (HMG-17, and CSF2RB, proteins involved in metabolism, nucleosomal architecture and cell signalling respectively. Knowledge of this endogenous cell to cell pathway, those proteins interacting with exosome associated non-coding transcripts and their interacting domains, could lead to a better understanding of not only cell to cell interactions but also the development of exosome targeted approaches in patient specific cell-based therapies. Keywords: Non-coding RNA, Extracellular RNA, Exosomes, Retroelement, Pseudogene

  15. Adherence and viability of intestinal bacteria to differentiated Caco-2 cells quantified by flow cytometry.

    Science.gov (United States)

    Grootaert, Charlotte; Boon, Nico; Zeka, Fjoralba; Vanhoecke, Barbara; Bracke, Marc; Verstraete, Willy; Van de Wiele, Tom

    2011-07-01

    Recent developments in host-microbe research give rise to a growing demand for rapid and accurate methods to quantify bacterial adhesion to epithelial cells. Here, we describe a new flow cytometric method to determine the amount and viability of gut bacteria, adhered to a monolayer of differentiated cells. The latter is a more relevant epithelium model than the suspended eukaryotic cells currently used in flow cytometric protocols. During the development of the method, we monitored the adhesion potential of six bacterial species and an intestinal microbial community to Caco-2 cells. The combination of SYBR Green I/propidium iodide was more efficient than carboxyfluorescein diacetate to stain the bacterial cells. In addition, a better separation between the Caco-2 background signal and viable and dead bacteria was obtained. A precise amount of Triton X-100 was used to detach adhered bacteria from Caco-2 cells and cell debris. Yet, a limited decrease in viability was observed for the intestinal microbial community treated with Triton X-100. The flow cytometric lower detection limit for pure bacterial cultures was 3.0-4.0log/mL, whereas a 5.0-5.5log/mL detection limit was obtained in the presence of Caco-2 cell background. The latter was sufficient to quantify adhered bacteria. To the best of our knowledge, this is the first description of a flow cytometric protocol that quantifies adhesion of both pure and mixed gut microbial cultures to a differentiated monolayer of Caco-2 cells and that allows to distinguish between viable and dead adhered bacteria. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Jarel K. Gandhi

    2015-09-01

    Full Text Available 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.

  17. Assessment of viability of sperm cells of Litopenaeus vannamei on cryopreservation

    Directory of Open Access Journals (Sweden)

    Marcela Fornari Uberti

    2014-06-01

    Full Text Available Aiming at assessing the cryopreservation potential of Litopenaeus vannamei sperm cells, 74 spermatophores were manually extracted from the sexually mature individuals. After the toxicity test to define the cryoprotectant concentration, suspensions of spermatic cells were cryopreserved in the groups in freezing solutions comprising different cryoprotectants such as dimethyl sulfoxide (DMSO and ethylene glycol (EG at 10% concentration. Each treatment was divided in subgroups for storage in liquid nitrogen during 0, 30, 60 and 90 days, in triplicate. After thawing at 25ºC for 40 seconds, cell viability in the suspensions was analyzed under the microscope in eosin-nigrosin stain and flow cytometry. There were no significant differences between the cryoprotectants used. For all the treatments, lower and higher mortalities occurred in the 0 and 90 days, respectively. By applying the eosin-nigrosin technique, lower and higher mortalities were 23.17 and 82.11% for DMSO and 29.94 and 83.72% for EG, while the flow cytometry registered mortalities of 2.42 and 55.13% for DMSO and 0.90 and 55.56% for EG. The Spearman correlation coefficient indicated a positive correlation (R=0.91 between the two techniques used. It was concluded that there was a decrease in cell viability within a longer cryopreservation time.

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

    Science.gov (United States)

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

    2013-05-01

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

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

    Science.gov (United States)

    Batiuskaite, Danute; Grinceviciute, Nora; Snitka, Valentinas

    2015-08-01

    The evaluation of the cyto- and bio-compatibility is a critical step in the development of graphene oxide (GO) as a new promising material for in vivo biomedical applications. In this study, we report the impact of GO, with and without the addition of bovine serum albumin, on healthy (Chinese hamster ovary) and a cancer (mouse hepatoma MH-22A) cells viability and the estimation of the intracellular distribution of GO inside the cells in vitro. The viability tests were performed using a colony formation assay. The intracellular distribution of GO was estimated using Raman spectroscopy and imaging. The viability of both cell lines decreased with increasing concentration of graphene oxide (12.5-50.0 μg/ml): in the case of Chinese hamster ovary cells viability decreased from 44% to 11%, in the case of mouse hepatoma MH-22A cells--from 22% to 3%. These cell lines significantly differed in their response to GO and GO-BSA formulations. The results of viability tests correlate with results of atomic force microscopy and Raman spectroscopy and imaging findings. The GO influence on cell morphology changes, cell structure, cells colony growth dynamics and GO accumulation inside the cells was higher in the case of mouse hepatoma MH-22A cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2003-01-01

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

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

    OpenAIRE

    Ngwenya, Bryne

    2015-01-01

    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 na...

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

    Science.gov (United States)

    Ivanov, Vladimir N; Hei, Tom K

    2014-12-01

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

  3. Single-cell model of prokaryotic cell cycle.

    Science.gov (United States)

    Abner, Kristo; Aaviksaar, Tõnis; Adamberg, Kaarel; Vilu, Raivo

    2014-01-21

    One of the recognized prokaryotic cell cycle theories is Cooper-Helmstetter (CH) theory which relates start of DNA replication to particular (initiation) cell mass, cell growth and division. Different aspects of this theory have been extensively studied in the past. In the present study CH theory was applied at single cell level. Universal equations were derived for different cell parameters (cell mass and volume, surface area, DNA amount and content) depending on constructivist cell cycle parameters (unit mass, replication and division times, cell age, cell cycle duration) based on selected growth laws of cell mass (linear, exponential). The equations derived can be integrated into single-cell models for the analysis and design of bacterial cells. © 2013 Published by Elsevier Ltd.

  4. DNA Damage and Cell Cycle Arrest Induced by Protoporphyrin IX in Sarcoma 180 Cells

    Directory of Open Access Journals (Sweden)

    Qing Li

    2013-09-01

    Full Text Available Background: Porphyrin derivatives have been widely used in photodynamic therapy as effective sensitizers. Protoporphyrin IX (PpIX, a well-known hematoporphyrin derivative component, shows great potential to enhance light induced tumor cell damage. However, PpIX alone could also exert anti-tumor effects. The mechanisms underlying those direct effects are incompletely understood. This study thus investigated the putative mechanisms underlying the anti-tumor effects of PpIX on sarcoma 180 (S180 cells. Methods: S180 cells were treated with different concentrations of PpIX. Following the treatment, cell viability was evaluated by the 3-(4, 5- dimethylthiazol-2-yl-2, 5-diphenyltetrazoliumbromide (MTT assay; Disruption of mitochondrial membrane potential was measured by flow cytometry; The trans-location of apoptosis inducer factor (AIF from mitochondria to nucleus was visualized by confocal laser scanning microscopy; DNA damage was detected by single cell gel electrophoresis; Cell cycle distribution was analyzed by DNA content with flow cytometry; Cell cycle associated proteins were detected by western blotting. Results: PpIX (≥ 1 µg/ml significantly inhibited proliferation and reduced viability of S180 cells in a dose-dependent manner. PpIX rapidly and significantly triggered mitochondrial membrane depolarization, AIF (apoptosis inducer factor translocation from mitochondria to nucleus and DNA damage, effects partially relieved by the specific inhibitor of MPTP (mitochondrial permeability transition pore. Furthermore, S phase arrest and upregulation of the related proteins of P53 and P21 were observed following 12 and 24 h PpIX exposure. Conclusion: PpIX could inhibit tumor cell proliferation by induction of DNA damage and cell cycle arrest in the S phase.

  5. A map of protein dynamics during cell-cycle progression and cell-cycle exit

    Science.gov (United States)

    Gookin, Sara; Min, Mingwei; Phadke, Harsha; Chung, Mingyu; Moser, Justin; Miller, Iain; Carter, Dylan

    2017-01-01

    The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the temporal dynamics of key cell-cycle proteins in asynchronously cycling human cells. We identify several unexpected patterns for core cell-cycle proteins in actively proliferating (CDK2-increasing) versus spontaneously quiescent (CDK2-low) cells, including Cyclin D1, the levels of which we find to be higher in spontaneously quiescent versus proliferating cells. We also identify proteins with concentrations that steadily increase or decrease the longer cells are in quiescence, suggesting the existence of a continuum of quiescence depths. Our single-cell measurements thus provide a rich resource for the field by characterizing protein dynamics during proliferation versus quiescence. PMID:28892491

  6. Fuel cell hybrid taxi life cycle analysis

    Energy Technology Data Exchange (ETDEWEB)

    Baptista, Patricia, E-mail: patricia.baptista@ist.utl.pt [IDMEC-Instituto Superior Tecnico, Universidade Tecnica de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa (Portugal); Ribau, Joao; Bravo, Joao; Silva, Carla [IDMEC-Instituto Superior Tecnico, Universidade Tecnica de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa (Portugal); Adcock, Paul; Kells, Ashley [Intelligent Energy, Charnwood Building, HolywellPark, Ashby Road, Loughborough, LE11 3GR (United Kingdom)

    2011-09-15

    A small fleet of classic London Taxis (Black cabs) equipped with hydrogen fuel cell power systems is being prepared for demonstration during the 2012 London Olympics. This paper presents a Life Cycle Analysis for these vehicles in terms of energy consumption and CO{sub 2} emissions, focusing on the impacts of alternative vehicle technologies for the Taxi, combining the fuel life cycle (Tank-to-Wheel and Well-to-Tank) and vehicle materials Cradle-to-Grave. An internal combustion engine diesel taxi was used as the reference vehicle for the currently available technology. This is compared to battery and fuel cell vehicle configurations. Accordingly, the following energy pathways are compared: diesel, electricity and hydrogen (derived from natural gas steam reforming). Full Life Cycle Analysis, using the PCO-CENEX drive cycle, (derived from actual London Taxi drive cycles) shows that the fuel cell powered vehicle configurations have lower energy consumption (4.34 MJ/km) and CO{sub 2} emissions (235 g/km) than both the ICE Diesel (9.54 MJ/km and 738 g/km) and the battery electric vehicle (5.81 MJ/km and 269 g/km). - Highlights: > A Life Cycle Analysis of alternative vehicle technologies for the London Taxi was performed. > The hydrogen powered vehicles have the lowest energy consumption and CO{sub 2} emissions results. > A hydrogen powered solution can be a sustainable alternative in a full life cycle framework.

  7. FEM-based oxygen consumption and cell viability models for avascular pancreatic islets

    Directory of Open Access Journals (Sweden)

    Buchwald Peter

    2009-04-01

    Full Text Available Abstract 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

  8. Monitoring of the Viability of Cells Immobilized by Sol-Gel Process

    Czech Academy of Sciences Publication Activity Database

    Kuncová, Gabriela; Podrazký, Ondřej; Ripp, S.; Trögl, Josef; Sayler, G. S.; Demnerová, K.; Vaňková, Radomíra

    2004-01-01

    Roč. 31, 1-3 (2004), s. 335-342 ISSN 0928-0707. [International Workshop on Sol-Gel and Technology-Part I (Sol-Gel'03) /12./. Sydney, 25.08.2003-29.08.2003] R&D Projects: GA ČR GA104/01/0461; GA MŠk OC 840.20; GA MŠk OC 840.10 Institutional research plan: CEZ:AV0Z4072921 Keywords : sol-gel process * cell entrapment * viability Subject RIV: CE - Biochemistry Impact factor: 1.150, year: 2004

  9. Changes in oscillatory dynamics in the cell cycle of early Xenopus laevis embryos.

    Directory of Open Access Journals (Sweden)

    Tony Y-C Tsai

    2014-02-01

    Full Text Available During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min and the subsequent 11 cycles are short (∼30 min and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development.

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

    Science.gov (United States)

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

    2005-07-01

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

  11. Different methods to quantify Listeria monocytogenesbiofilms cells showed different profile in their viability

    Directory of Open Access Journals (Sweden)

    Lizziane Kretli Winkelströter

    2015-03-01

    Full Text Available 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.

  12. American cranberry (Vaccinium macrocarpon) extract affects human prostate cancer cell growth via cell cycle arrest by modulating expression of cell cycle regulators.

    Science.gov (United States)

    Déziel, Bob; MacPhee, James; Patel, Kunal; Catalli, Adriana; Kulka, Marianna; Neto, Catherine; Gottschall-Pass, Katherine; Hurta, Robert

    2012-05-01

    Prostate cancer is one of the most common cancers in the world, and its prevalence is expected to increase appreciably in the coming decades. As such, more research is necessary to understand the etiology, progression and possible preventative measures to delay or to stop the development of this disease. Recently, there has been interest in examining the effects of whole extracts from commonly harvested crops on the behaviour and progression of cancer. Here, we describe the effects of whole cranberry extract (WCE) on the behaviour of DU145 human prostate cancer cells in vitro. Following treatment of DU145 human prostate cancer cells with 10, 25 and 50 μg ml⁻¹ of WCE, respectively for 6 h, WCE significantly decreased the cellular viability of DU145 cells. WCE also decreased the proportion of cells in the G2-M phase of the cell cycle and increased the proportion of cells in the G1 phase of the cell cycle following treatment of cells with 25 and 50 μg ml⁻¹ treatment of WCE for 6 h. These alterations in cell cycle were associated with changes in cell cycle regulatory proteins and other cell cycle associated proteins. WCE decreased the expression of CDK4, cyclin A, cyclin B1, cyclin D1 and cyclin E, and increased the expression of p27. Changes in p16(INK4a) and pRBp107 protein expression levels also were evident, however, the changes noted in p16(INK4a) and pRBp107 protein expression levels were not statistically significant. These findings demonstrate that phytochemical extracts from the American cranberry (Vaccinium macrocarpon) can affect the behaviour of human prostate cancer cells in vitro and further support the potential health benefits associated with cranberries.

  13. Verification of cell viability at progressively higher scanning forces using a hybrid atomic force and fluorescence microscope.

    Science.gov (United States)

    Barnes, C A; O'Hagan, B M G; Howard, C V; McKerr, G

    2007-11-01

    The prudent use of the atomic force microscope as a supra-vital live cell imaging tool requires that cell viability must be determined before and after scanning. Complementary optical techniques in conjunction with the fluorescent dyes rhodamine-123 and ethidium homodimer have been used within this study to determine cell viability after increasing loads are applied in contact mode. Guideline force ranges for five commonly cultured cell lines, human squamous carcinoma (A431), fibroblast, HeLa, Potorous tridactylis (PtK2) and rat intestinal epithelial (RIE) cells are given.

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

    NARCIS (Netherlands)

    Kort, R.; Keijser, B.J.; Caspers, M.P.M.; Schuren, F.H.; Montijn, R.

    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

  15. Arctigenin Inhibits Lung Metastasis of Colorectal Cancer by Regulating Cell Viability and Metastatic Phenotypes

    OpenAIRE

    Yo-Han Han; Ji-Ye Kee; Dae-Seung Kim; Jeong-geon Mun; Mi-Young Jeong; Sang-Hyun Park; Byung-Min Choi; Sung-Joo Park; Hyun-Jung Kim; Jae-Young Um; Seung-Heon Hong

    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-mese...

  16. T-Type Calcium Channels Are Required to Maintain Viability of Neural Progenitor Cells.

    Science.gov (United States)

    Kim, Ji-Woon; Oh, Hyun Ah; Lee, Sung Hoon; Kim, Ki Chan; Eun, Pyung Hwa; Ko, Mee Jung; Gonzales, Edson Luck T; Seung, Hana; Kim, Seonmin; Bahn, Geon Ho; Shin, Chan Young

    2018-02-21

    T-type calcium channels are low voltage-activated calcium channels that evoke small and transient calcium currents. Recently, T-type calcium channels have been implicated in neurodevelopmental disorders such as autism spectrum disorder and neural tube defects. However, their function during embryonic development is largely unknown. Here, we investigated the function and expression of T-type calcium channels in embryonic neural progenitor cells (NPCs). First, we compared the expression of T-type calcium channel subtypes (CaV3.1, 3.2, and 3.3) in NPCs and differentiated neural cells (neurons and astrocytes). We detected all subtypes in neurons but not in astrocytes. In NPCs, CaV3.1 was the dominant subtype, whereas CaV3.2 was weakly expressed, and CaV3.3 was not detected. Next, we determined CaV3.1 expression levels in the cortex during early brain development. Expression levels of CaV3.1 in the embryonic period were transiently decreased during the perinatal period and increased at postnatal day 11. We then pharmacologically blocked T-type calcium channels to determine the effects in neuronal cells. The blockade of T-type calcium channels reduced cell viability, and induced apoptotic cell death in NPCs but not in differentiated astrocytes. Furthermore, blocking T-type calcium channels rapidly reduced AKT-phosphorylation (Ser473) and GSK3β-phosphorylation (Ser9). Our results suggest that T-type calcium channels play essential roles in maintaining NPC viability, and T-type calcium channel blockers are toxic to embryonic neural cells, and may potentially be responsible for neurodevelopmental disorders.

  17. Purified Brominated Indole Derivatives from Dicathais orbita Induce Apoptosis and Cell Cycle Arrest in Colorectal Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Babak Esmaeelian

    2013-10-01

    Full Text Available Dicathais orbita is a large Australian marine gastropod known to produce bioactive compounds with anticancer properties. In this research, we used bioassay guided fractionation from the egg mass extract of D. orbita using flash column chromatography and identified fractions containing tyrindoleninone and 6-bromoisatin as the most active against colon cancer cells HT29 and Caco-2. Liquid chromatography coupled with mass spectrometry (LCMS and 1H NMR were used to characterize the purity and chemical composition of the isolated compounds. An MTT assay was used to determine effects on cell viability. Necrosis and apoptosis induction using caspase/LDH assay and flow cytometry (PI/Annexin-V and cell cycle analysis were also investigated. Our results show that semi-purified 6-bromoisatin had the highest anti-cancer activity by inhibiting cell viability (IC50 = ~100 µM and increasing caspase 3/7 activity in both of the cell lines at low concentration. The fraction containing 6-bromoisatin induced 77.6% apoptosis and arrested 25.7% of the cells in G2/M phase of cell cycle in HT29 cells. Tyrindoleninone was less potent but significantly decreased the viability of HT29 cells at IC50 = 390 µM and induced apoptosis at 195 µM by increasing caspase 3/7 activity in these cells. This research will facilitate the development of these molluscan natural products as novel complementary medicines for colorectal cancer.

  18. Effects of short-chain chlorinated paraffins exposure on the viability and metabolism of human hepatoma HepG2 cells.

    Science.gov (United States)

    Geng, Ningbo; Zhang, Haijun; Zhang, Baoqin; Wu, Ping; Wang, Feidi; Yu, Zhengkun; Chen, Jiping

    2015-03-03

    Short-chain chlorinated paraffins (SCCPs) have attracted considerable attention for their characteristic of persistent organic pollutants. However, very limited information is available for their toxic effects at environmentally relevant doses, limiting the evaluation of their health risks. In this study, cell viability assay and targeted metabolomic approach was used to evaluate the environmental dose (<100 μg/L) effect of SCCPs on HepG2 cells. Cell viability was found to be decreased with increases in exposure dose of SCCPs. Exposure for 48 h to C10-CPs resulted in a significant reduction in cell viability compared with 24 h, even at 1 μg/L. SCCPs exposure altered the intracellular redox status and caused significant metabolic disruptions. As a kind of peroxisome proliferator, SCCPs specifically stimulated the β-oxidation of unsaturated fatty acids and long-chain fatty acids. Meanwhile, SCCPs exposure disturbed glycolysis and amino acid metabolism, and led to the up-regulation of glutamate metabolism and urea cycle. The toxic effects of SCCPs might mainly involve the perturbation of energy production, protein biosynthesis, fatty acid metabolism, and ammonia recycling.

  19. A 'fragile cell' sub-population revealed during cytometric assessment of Saccharomyces cerevisiae viability in lipid-limited alcoholic fermentation.

    Science.gov (United States)

    Delobel, P; Pradal, M; Blondin, B; Tesniere, C

    2012-11-01

    To show that in anaerobic fermentation with limiting lipid nutrients, cell preparation impacts the viability assessment of yeast cells, and to identify the factors involved. Saccharomyces cerevisiae viability was determined using propidium iodide staining and the flow cytometry. Analyses identified intact cells, dead cells and, under certain conditions, the presence of a third subpopulation of apparently damaged cells. This intermediate population could account for up to 40% of the entire cell population. We describe, analyse and discuss the effects of different solutions for cell resuspension on the respective proportion of these three populations, in particular that of the intermediate population. We show that this intermediate cell population forms in the absence of Ca(2+)/Mg(2+). Cell preparation significantly impacts population viability assessment by FCM. The intermediate population, revealed under certain conditions, could be renamed as 'fragile cells'. For these cells, Ca(2+) and Mg(2+) reduce cell membrane permeability to PI. This is the first study that analyses and discusses the factors influencing the formation of an intermediate population when studying viability in yeast alcoholic fermentation. With a wider application in biological research, this study provides important support to the relatively new questioning of propidium iodide staining as a universal cell death indicator. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

  20. Dihydroartemisinin (DHA) induces ferroptosis and causes cell cycle arrest in head and neck carcinoma cells.

    Science.gov (United States)

    Lin, Renyu; Zhang, Ziheng; Chen, Lingfeng; Zhou, Yunfang; Zou, Peng; Feng, Chen; Wang, Li; Liang, Guang

    2016-10-10

    Head and neck cancer is the sixth most common cancer worldwide. Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, exhibits a wide range of biological roles including a highly efficient and specific anti-tumor activity. Here, we aimed to examine the effect of DHA on head and neck carcinoma cells and elucidate the potential mechanisms. We used five head and neck carcinoma cell lines and two non-tumorigenic normal epithelial cell lines to achieve our goals. Cells were exposed to DHA and subjected to cellular activity assays including viability, cell cycle analysis, cell death, and angiogenic phenotype. Our results show that DHA causes cell cycle arrest which is mediated through Forkhead box protein M1 (FOXM1). We also demonstrate that DHA induces ferroptosis and apoptosis in head and neck carcinoma cells. Lastly, our results show that DHA alters the angiogenic phenotype of cancer cells by reducing the expression of angiogenic factors and the ability of cancer cells to support endothelial cell tubule formation. Our study suggests that DHA specifically causes head and neck cancer cell death through contribution from both ferroptosis and apoptosis. DHA may represent an effective strategy in head and neck cancer treatment. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Cell-Cycle Position of Single MYC-Driven Cancer Cells Dictates Their Susceptibility to a Chemotherapeutic Drug.

    Science.gov (United States)

    Ryl, Tatsiana; Kuchen, Erika E; Bell, Emma; Shao, Chunxuan; Flórez, Andrés F; Mönke, Gregor; Gogolin, Sina; Friedrich, Mona; Lamprecht, Florian; Westermann, Frank; Höfer, Thomas

    2017-09-27

    While many tumors initially respond to chemotherapy, regrowth of surviving cells compromises treatment efficacy in the long term. The cell-biological basis of this regrowth is not understood. Here, we characterize the response of individual, patient-derived neuroblastoma cells driven by the prominent oncogene MYC to the first-line chemotherapy, doxorubicin. Combining live-cell imaging, cell-cycle-resolved transcriptomics, and mathematical modeling, we demonstrate that a cell's treatment response is dictated by its expression level of MYC and its cell-cycle position prior to treatment. All low-MYC cells enter therapy-induced senescence. High-MYC cells, by contrast, disable their cell-cycle checkpoints, forcing renewed proliferation despite treatment-induced DNA damage. After treatment, the viability of high-MYC cells depends on their cell-cycle position during treatment: newborn cells promptly halt in G1 phase, repair DNA damage, and form re-growing clones; all other cells show protracted DNA repair and ultimately die. These findings demonstrate that fast-proliferating tumor cells may resist cytotoxic treatment non-genetically, by arresting within a favorable window of the cell cycle. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Leslie Chávez-Galán

    2016-01-01

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

  3. Alcoholic beverages and gastric epithelial cell viability: effect on oxidative stress-induced damage.

    Science.gov (United States)

    Loguercio, C; Tuccillo, C; Federico, A; Fogliano, V; Del Vecchio Blanco, C; Romano, M

    2009-12-01

    Alcohol is known to cause damage to the gastric epithelium independently of gastric acid secretion. Different alcoholic beverages exert different damaging effects in the stomach. However, this has not been systematically evaluated. Moreover, it is not known whether the non-alcoholic components of alcoholic beverages also play a role in the pathogenesis of gastric epithelial cell damage. Therefore, this study was designed to evaluate whether different alcoholic beverages, at a similar ethanol concentration, exerted different damaging effect in gastric epithelial cells in vitro. Moreover, we evaluated whether pre-treatment of gastric epithelial cells with alcoholic beverages prevented oxidative stress-induced damage to gastric cells. Cell damage was assessed, in MKN-28 gastric epithelial cells, by MTT assay. Oxidative stress was induced by incubating cells with xanthine and xanthine oxidase. Gastric cell viability was assessed following 30, 60, and 120 minutes incubation with ethanol 17.5-125 mg/ml(-1) or different alcoholic beverages (i.e., beer, white wine, red wine, spirits) at comparable ethanol concentration. Finally, we assessed whether pre-incubation with red wine (with or without ethanol) prevented oxidative stress-induced cell damage. Red wine caused less damage to gastric epithelial cells in vitro compared with other alcoholic beverages at comparable ethanol concentration. Pre-treatment with red wine, but not with dealcoholate red wine, significantly and time-dependently prevented oxidative stress-induced cell damage. 1) red wine is less harmful to gastric epithelial cells than other alcoholic beverages; 2) this seems related to the non-alcoholic components of red wine, because other alcoholic beverages with comparable ethanol concentration exerted more damage than red wine; 3) red wine prevents oxidative stress-induced cell damage and this seems to be related to its ethanol content.

  4. Intracellular magnesium level determines cell viability in the MPP(+) model of Parkinson's disease.

    Science.gov (United States)

    Shindo, Yutaka; Yamanaka, Ryu; Suzuki, Koji; Hotta, Kohji; Oka, Kotaro

    2015-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder resulting from mitochondrial dysfunction in dopaminergic neurons. Mitochondria are believed to be responsible for cellular Mg²⁺ homeostasis. Mg²⁺ is indispensable for maintaining ordinal cellular functions, hence perturbation of the cellular Mg²⁺ homeostasis may be responsible for the disorders of physiological functions and diseases including PD. However, the changes in intracellular Mg²⁺ concentration ([Mg²⁺]i) and the role of Mg²⁺ in PD have still been obscure. In this study, we investigated [Mg²⁺]i and its effect on neurodegeneration in the 1-methyl-4-phenylpyridinium (MPP⁺) model of PD in differentiated PC12 cells. Application of MPP⁺ induced an increase in [Mg²⁺]i immediately via two different pathways: Mg²⁺ release from mitochondria and Mg²⁺ influx across cell membrane, and the increased [Mg²⁺]i sustained for more than 16 h after MPP⁺ application. Suppression of Mg²⁺ influx decreased the viability of the cells exposed to MPP⁺. The cell viability correlated highly with [Mg²⁺]i. In the PC12 cells with suppressed Mg²⁺ influx, ATP concentration decreased and the amount of reactive oxygen species (ROS) increased after an 8h exposure to MPP⁺. Our results indicate that the increase in [Mg²⁺]i inhibited cellular ROS generation and maintained ATP production, which resulted in the protection from MPP⁺ toxicity. Copyright © 2015. Published by Elsevier B.V.

  5. Mast cells dysregulate apoptotic and cell cycle genes in mucosal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Davis Paul

    2006-12-01

    Full Text Available Abstract Background Mucosal squamous cell carcinoma of the head and neck is a disease of high mortality and morbidity. Interactions between the squamous cell carcinoma and the host's local immunity, and how the latter contributes to the biological behavior of the tumor are unclear. In vivo studies have demonstrated sequential mast cell infiltration and degranulation during squamous cell carcinogenesis. The degree of mast cell activation correlates closely with distinct phases of hyperkeratosis, dysplasia, carcinoma in-situ and invasive carcinoma. However, the role of mast cells in carcinogenesis is unclear. Aim This study explores the effects of mast cells on the proliferation and gene expression profile of mucosal squamous cell carcinoma using human mast cell line (HMC-1 and human glossal squamous cell carcinoma cell line (SCC25. Methods HMC-1 and SCC25 were co-cultured in a two-compartment chamber, separated by a polycarbonate membrane. HMC-1 was stimulated to degranulate with calcium ionophore A23187. The experiments were done in quadruplicate. Negative controls were established where SCC25 were cultured alone without HMC-1. At 12, 24, 48 and 72 hours, proliferation and viability of SCC25 were assessed with MTT colorimetric assay. cDNA microarray was employed to study differential gene expression between co-cultured and control SCC25. Results HMC-1/SCC25 co-culture resulted in suppression of growth rate for SCC-25 (34% compared with 110% for the control by 72 hours, p Conclusion We show that mast cells have a direct inhibitory effect on the proliferation of mucosal squamous cell carcinoma in vitro by dysregulating key genes in apoptosis and cell cycle control.

  6. K+ channels and cell cycle progression in tumor cells

    Directory of Open Access Journals (Sweden)

    HALIMA eOUADID-AHIDOUCH

    2013-08-01

    Full Text Available K+ ions play a major role in many cellular processes. The deregulation of K+ signaling is associated with a variety of diseases such as hypertension, atherosclerosis, or diabetes. K+ ions are important for setting the membrane potential, the driving force for Ca2+ influx, and regulate volume of growing cells. Moreover, it is increasingly recognized that K+ channels control cell proliferation through a novel signaling mechanisms triggered and modulated independently of ion fluxes. In cancer, aberrant expression, regulation and/or sublocalization of K+ channels can alter the downstream signals that converge on the cell cycle machinery. Various K+ channels are involved in cell cycle progression and are needed only at particular stages of the cell cycle. Consistent with this idea, the expression of Eag1 and HERG channels fluctuate along the cell cycle. Despite of acquired knowledge, our understanding of K+ channels functioning in cancer cells requires further studies. These include identifying the molecular mechanisms controling the cell cycle machinery. By understanding how K+ channels regulate cell cycle progression in cancer cells, we will gain insights into how cancer cells subvert the need for K+ signal and its downstream targets to proliferate.

  7. Metyrapone, an inhibitor of cytochrome oxidases, does not affect viability in a neuroblastoma cell model of bilirubin toxicity

    Directory of Open Access Journals (Sweden)

    Maria N. Naguib Leerberg

    2014-01-01

    Conclusion: Herein we have shown that metyrapone does not increase cell death in neuroblastoma cells in culture exposed to bilirubin. Our results question the relationship between the oxidative mechanism evaluated by spectrophotometry and cell viability. Our findings add to the discussion on whether bilirubin oxidation represents a potentially important protective mechanism in neurons challenged by hyperbilirubinemia.

  8. Constant, cycling, hot and cold thermal environments: strong effects on mean viability but not on genetic estimates.

    Science.gov (United States)

    Ketola, T; Kellermann, V; Kristensen, T N; Loeschcke, V

    2012-06-01

    It has frequently been suggested that trait heritabilities are environmentally sensitive, and there are genetic trade-offs between tolerating different environments such as hot and cold or constant and fluctuating temperatures. Future climate predictions suggest an increase in both temperatures and their fluctuations. How species will respond to these changes is uncertain, particularly as there is a lack of studies which compare genetic performances in constant vs. fluctuating environments. In this study, we used a nested full-sib/half-sib breeding design to examine how the genetic variances and heritabilities of egg-to-adult viability differ at high and low temperatures with and without daily fluctuations in temperatures using Drosophila melanogaster as a model organism. Although egg-to-adult viability was clearly sensitive to developmental temperatures, heritabilities were not particularly sensitive to developmental temperatures. Moreover, we found that egg-to-adult viabilities at different developmental temperatures were positively correlated, suggesting a common genetic background for egg-to-adult viability at different temperatures. Finding both a uniform genetic background coupled with rather low heritabilities insensitive to temperatures, our results suggest evolutionary responses are unlikely to be limited by temperature effects on genetic parameters or negative genetic correlations, but by the direct effects of stressful temperatures on egg-to-adult viability accompanied with low heritabilities. © 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.

  9. Introductory review of computational cell cycle modeling.

    Science.gov (United States)

    Kriete, Andres; Noguchi, Eishi; Sell, Christian

    2014-01-01

    Recent advances in the modeling of the cell cycle through computer simulation demonstrate the power of systems biology. By definition, systems biology has the goal to connect a parts list, prioritized through experimental observation or high-throughput screens, by the topology of interactions defining intracellular networks to predict system function. Computer modeling of biological systems is often compared to a process of reverse engineering. Indeed, designed or engineered technical systems share many systems-level properties with biological systems; thus studying biological systems within an engineering framework has proven successful. Here we review some aspects of this process as it pertains to cell cycle modeling.

  10. The effect of pH on cell viability, cell migration, cell proliferation, wound closure, and wound reepithelialization

    DEFF Research Database (Denmark)

    Kruse, Carla R; Singh, Mansher; Targosinski, Stefan

    2017-01-01

    have investigated the role of pH in relation to pathogens but studies concentrating on the effects of pH on wound healing itself are inconclusive. The purpose of this study was to comprehensively and in a controlled fashion investigate the effect of pH on wound healing by studying its effect on human......Wound microenvironment plays a major role in the process of wound healing. It contains various external and internal factors that participate in wound pathophysiology. The pH is an important factor that influences wound healing by changing throughout the healing process. Several previous studies...... primary keratinocyte and fibroblast function in vitro and on wound healing in vivo. In vitro, primary human keratinocytes and fibroblasts were cultured in different levels of pH (5.5-12.5) and the effect on cell viability, proliferation, and migration was studied. A rat full-thickness wound model was used...

  11. Parthenolide Induces Apoptosis and Cell Cycle Arrest of Human 5637 Bladder Cancer Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Guang Cheng

    2011-08-01

    Full Text Available Parthenolide, the principal component of sesquiterpene lactones present in medical plants such as feverfew (Tanacetum parthenium, has been reported to have anti-tumor activity. In this study, we evaluated the therapeutic potential of parthenolide against bladder cancer and its mechanism of action. Treatment of bladder cancer cells with parthenolide resulted in a significant decrease in cell viability. Parthenolide induced apoptosis through the modulation of Bcl-2 family proteins and poly (ADP-ribose polymerase degradation. Treatment with parthenolide led to G1 phase cell cycle arrest in 5637 cells by modulation of cyclin D1 and phosphorylated cyclin-dependent kinase 2. Parthenolide also inhibited the invasive ability of bladder cancer cells. These findings suggest that parthenolide could be a novel therapeutic agent for treatment of bladder cancer.

  12. Analysis of tumor and endothelial cell viability and survival using sulforhodamine B and clonogenic assays.

    Science.gov (United States)

    Woolston, Caroline; Martin, Stewart

    2011-01-01

    A variety of assays, and rationales for their use, exist to monitor viability and/or survival following cellular exposure to insult. Two commonly used in vitro assays are the sulforhodamine B assay and the clonogenic survival assay which can be used to monitor the efficacy of anticancer agents, either via direct tumor cell cytotoxicity or antiangiogenic mechanisms. The techniques described are suitable for studying survival in a number of different cell types; however, this chapter describes how they may be used in the assessment of chemo-/radiosensitivity. The methods are uncomplicated and robust as long as attention is paid to key optimization steps. Except for a multiwell plate reader they do not require any specialized equipment other than that found in a typical tissue-culture laboratory.

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

    Energy Technology Data Exchange (ETDEWEB)

    Barkan, Daniel; Liu, Zhen; Sacchettini, James C.; Glickman, Michael S.; (MSKCC); (TAM)

    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.

  14. CGGBP1 regulates cell cycle in cancer cells

    Directory of Open Access Journals (Sweden)

    Uhrbom Lene

    2011-07-01

    Full Text Available Abstract Background CGGBP1 is a CGG-triplet repeat binding protein, which affects transcription from CGG-triplet-rich promoters such as the FMR1 gene and the ribosomal RNA gene clusters. Earlier, we reported some previously unknown functions of CGGBP1 in gene expression during heat shock stress response. Recently we had found CGGBP1 to be a cell cycle regulatory midbody protein required for normal cytokinetic abscission in normal human fibroblasts, which have all the cell cycle regulatory mechanisms intact. Results In this study we explored the role of CGGBP1 in the cell cycle in various cancer cell lines. CGGBP1 depletion by RNA interference in tumor-derived cells caused an increase in the cell population at G0/G1 phase and reduced the number of cells in the S phase. CGGBP1 depletion also increased the expression of cell cycle regulatory genes CDKN1A and GAS1, associated with reductions in histone H3 lysine 9 trimethylation in their promoters. By combining RNA interference and genetic mutations, we found that the role of CGGBP1 in cell cycle involves multiple mechanisms, as single deficiencies of CDKN1A, GAS1 as well as TP53, INK4A or ARF failed to rescue the G0/G1 arrest caused by CGGBP1 depletion. Conclusions Our results show that CGGBP1 expression is important for cell cycle progression through multiple parallel mechanisms including the regulation of CDKN1A and GAS1 levels.

  15. α-Mangostin Suppresses the Viability and Epithelial-Mesenchymal Transition of Pancreatic Cancer Cells by Downregulating the PI3K/Akt Pathway

    Directory of Open Access Journals (Sweden)

    Qinhong Xu

    2014-01-01

    Full Text Available α-Mangostin, a natural product isolated from the pericarp of the mangosteen fruit, has been shown to inhibit the growth of tumor cells in various types of cancers. However, the underlying molecular mechanisms are largely unclear. Here, we report that α-mangostin suppressed the viability and epithelial-mesenchymal transition (EMT of pancreatic cancer cells through inhibition of the PI3K/Akt pathway. Treatment of pancreatic cancer BxPc-3 and Panc-1 cells with α-mangostin resulted in loss of cell viability, accompanied by enhanced cell apoptosis, cell cycle arrest at G1 phase, and decrease of cyclin-D1. Moreover, Transwell and Matrigel invasion assays showed that α-mangostin significantly reduced the migration and invasion of pancreatic cancer cells. Consistent with these results, α-mangostin decreased the expression of MMP-2, MMP-9, N-cadherin, and vimentin and increased the expression of E-cadherin. Furthermore, we found that α-mangostin suppressed the activity of the PI3K/Akt pathway in pancreatic cancer cells as demonstrated by the reduction of the Akt phosphorylation by α-mangostin. Finally, α-mangostin significantly inhibited the growth of BxPc-3 tumor mouse xenografts. Our results suggest that α-mangostin may be potentially used as a novel adjuvant therapy or complementary alternative medicine for the management of pancreatic cancers.

  16. Loss of rachis cell viability is associated with ripening disorders in grapes.

    Science.gov (United States)

    Hall, Geoffrey E; Bondada, Bhaskar R; Keller, Markus

    2011-01-01

    Rachises of grape (Vitis vinifera L.) clusters that appeared healthy or displayed symptoms of the ripening disorders berry shrivel (BS) or bunch-stem necrosis (BSN) were treated with the cellular viability stain fluorescein diacetate and examined by confocal microscopy. Clusters with BS and BSN symptoms experienced a decrease of cell viability throughout the rachis, and their berries contained 70-80% less sugar than healthy berries. The xylem-mobile dye basic fuchsin, infiltrated via the cut base of shoots with one healthy and one BS cluster, moved to all berries on the healthy cluster but generally failed to move into the peduncle of the BS cluster. Peduncle girdling did not interrupt dye movement in the xylem, but stopped solute accumulation in berries and led to berry shrinkage. In contrast, surgically destroying the peduncle xylem at the onset of ripening did not affect berry growth and solute accumulation. These results indicate that cessation of sugar and water accumulation in BS and BSN is associated with phloem death in the rachis. Although xylem flow to the berries may also cease, a functional xylem connection to the shoot may not be required for normal ripening, while water loss from berries by transpiration and xylem efflux may explain the characteristic berry shrinkage that is associated with these ripening disorders. The similarity of internal tissue breakdown in BS and BSN rachises and the correlation observed here between the proportion of shrinking berries on a cluster and the severity of rachis necrosis suggest that there may be a gradual transition between the two ripening disorders. Seeds from healthy and BS clusters showed no differences in colour, morphology, weight, viability, and ability to germinate, which indicates that the disorder may not appear until seeds are mature.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Hong-Hua Zheng

    2016-01-01

    Full Text Available 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.

  19. Redefining the effect of salt on thermophilic starter cell viability, culturability and metabolic activity in cheese.

    Science.gov (United States)

    Hickey, C D; Fallico, V; Wilkinson, M G; Sheehan, J J

    2018-02-01

    This study investigated the differential effect of salt concentration in the outside and inside layers of brine salted cheeses on viability, culturability and enzyme activity of starter bacteria. The high-salt environment of the outside layer caused a sharp decrease in L. helveticus viability as measured by traditional plate counts. Remarkably, this was associated with lower release of intracellular enzymes (LDH), reduced levels of proteolysis and larger membrane integrity as measured by flow cytometry (FC) following classical Live/Dead staining. FC analysis of light scattering properties highlighted a significant reduction in size and granularity of the microbiota located in the cheese surface, suggestive of cell shrinkage and condensation of internal macromolecules probably due to hyperosmotic stress. The microbiota of the cheese surface were found to experience greater oxidative stress, as measured by FC analysis of the total levels of reactive oxygen species, compared to that of the interior layer. These results lead us to postulate that the physiology and health status of the microbiota were significantly different in the outer and inner layers of the cheese. The hyperosmotic environment of the outer layer resulted in reduced cell lysis, as measurable by assays based upon membrane integrity, but rather triggered cell death via mechanisms involving cell shrinkage and ROS-mediated damage of vital intracellular components. This study challenges the current thinking on how salt controls microbial activity in ripening cheese, especially in cheeses which are brine salted as local variations in biochemical ripening indices can differ significantly from the outside to the inside of a ripening cheese. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Sabine S Lange

    2016-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  2. Aluminum oxide nanoparticles alter cell cycle progression through CCND1 and EGR1 gene expression in human mesenchymal stem cells.

    Science.gov (United States)

    Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A

    2016-05-01

    Aluminum oxide nanoparticles (Al2 O3 -NPs) are important ceramic materials that have been used in a variety of commercial and industrial applications. However, the impact of acute and chronic exposure to Al2 O3 -NPs on the environment and on human health has not been well studied. In this investigation, we evaluated the cytotoxic effects of Al2 O3 -NPs on human mesenchymal stem cells (hMSCs) by using a cell viability assay and observing cellular morphological changes, analyzing cell cycle progression, and monitoring the expression of cell cycle response genes (PCNA, EGR1, E2F1, CCND1, CCNC, CCNG1, and CYCD3). The Al2 O3 -NPs reduced hMSC viability in a dose- and time-dependent manner. Nuclear condensation and fragmentation, chromosomal DNA fragmentation, and cytoplasmic vacuolization were observed in Al2 O3 -NP-exposed cells. The nuclear morphological changes indicated that Al2 O3 -NPs alter cell cycle progression and gene expression. The cell cycle distribution revealed that Al2 O3 -NPs cause cell cycle arrest in the sub-G0-G1 phase, and this is associated with a reduction in the cell population in the G2/M and G0/G1 phases. Moreover, Al2 O3 -NPs induced the upregulation of cell cycle response genes, including EGR1, E2F1, and CCND1. Our results suggested that exposure to Al2 O3 -NPs could cause acute cytotoxic effects in hMSCs through cell cycle regulatory genes. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  3. MicroRNA-194 Regulates Lipopolysaccharide-Induced Cell Viability by Inactivation of Nuclear Factor-κ B Pathway

    Directory of Open Access Journals (Sweden)

    Fei  Xie

    2017-10-01

    Full Text Available The present study explored the functional role of microRNA (miR-194 in lipopolysaccharide (LPS induced lung cell injury, along with the underlying mechanisms and to reveal the potential role in infantile pneumonia. Human fibroblasts WI38 cells were transfected with miR-194 mimic or miR-194 inhibitor, and the transfection efficiency was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR. Thereafter, the cells were treated with or without LPS, and then cell viability, cell apoptosis and mRNA and protein expressions of key proteins of nuclear factor kappa B (NF-κB pathway including inhibitor of NF-κB (IκB α, p-65, and B-cell CLL/lymphoma (Bcl 3 were analyzed. Results showed that overexpression and suppression of miR-194 was effective. Administration of LPS significantly decreased the cell viability and statistically promoted the percentages of apoptotic cells and increased the mRNA and protein expressions of p-65 and Bcl-3 but downregulated IκBα compared to the control group (P < 0.05 or P < 0.01. LPS in combination with miR-194 suppression further enhanced the effects of LPS on cell viability and cell apoptosis compared to the LPS group (P < 0.05. In contrast, LPS in combination with miR-194 overexpression observably reversed the effects of LPS on cell viability, cell apoptosis and mRNA and protein expressions of the key proteins (P < 0.05 or P < 0.01. In conclusion, miR-194 increases the LPS-induced the inhibition of cell viability and increasing of the cell apoptosis by inhibition of NF-κB pathway in WI38 cells. MiR-194 might be a potential targeted therapy for treatment of infantile pneumonia.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Tudor Braniste

    2016-09-01

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

  6. Docetaxel-Chitosan nanoparticles for breast cancer treatment: cell viability and gene expression study.

    Science.gov (United States)

    Mirzaie, Zahra H; Irani, Shiva; Mirfakhraie, Reza; Atyabi, Seyed Mohammad; Dinarvand, Meshkat; Dinarvand, Rassoul; Varshochian, Reyhaneh; Atyabi, Fatemeh

    2016-12-01

    Docetaxel acts through the inhibition of tubulin polymerization and reduction in the expression of BCL-2 gene. In this study, nanoparticles containing Docetaxel were prepared and their effects on the gene expression levels of BCL-2 and BAX genes were investigated. The drug was first conjugated to chitosan, and the nanoparticles were assembled in the presence of hyaluronic acid. Conjugations were confirmed by (1) H-NMR, and the obtained nanoparticles were characterized by dynamic light scattering and SEM. Cytotoxicity of the nanoparticles, cellular uptake, and cell death were evaluated. Finally, the effect of nanoparticles on the expression of BAX and BCL-2 genes in MCF-7 cells were investigated through real-time PCR. The results revealed that the prepared NPs had spherical shape with narrow size distribution of nanoparticles and free Docetaxel investigations revealed that increasing the treatment time with nanoparticles led to decrease in the rate of cell viability. BAX and BCL-2 gene expressions were decreased in nanoparticle-treated cells in comparison with intact cells, while the BAX/BCL-2 ratio was significantly elevated compared with free drug-treated cells after 72 h. Docetaxel-conjugated NPs may offer a promising treatment with low off-target toxicity for breast cancer. © 2016 John Wiley & Sons A/S.

  7. Cytotoxic Effects of Alcoholic Extract of Dorema Glabrum Seed on Cancerous Cells Viability

    Directory of Open Access Journals (Sweden)

    Maryam Bannazadeh Amirkhiz

    2013-08-01

    Full Text Available Purpose: In the present study cytotoxic effects of the alcoholic extract of Dorema Glabrum seed on viability of WEHI-164 cells, mouse Fibrosarcoma cell line and L929 normal cells were compared with the cytotoxic effects of Taxol (anticancer and apoptosis inducer drug. Methods: To find out the plant extract cytotoxic effects, MTT test and DNA fragmentation assay, the biochemical hallmark of apoptosis were performed on cultured and treated cells. Results: According to the findings the alcoholic extract of Dorema Glabrum seed can alter cells morphology and because of chromatin condensation and other changes they shrink and take a spherical shape, and lose their attachment too. So the plant extract inhibits cell growth albeit in a time and dose dependent manner and results in degradation of chromosomal DNA. Conclusion: Our data well established the anti-proliferative effect of methanolic extract of Dorema Glabrum seed and clearly showed that the plant extract can induce apoptosis and not necrosis in vitro, but the mechanism of its activities remained unknown. These results demonstrated that Dorema Glabrum seed might be a novel and attractive therapeutic candidate for tumor treatment in clinical practices.

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

    Directory of Open Access Journals (Sweden)

    Edna Ribeiro-Varandas

    2014-09-01

    Full Text Available 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.

  9. Control points within the cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Van' t Hof, J.

    1984-01-01

    Evidence of the temporal order of chromosomal DNA replication argues favorably for the view that the cell cycle is controlled by genes acting in sequence whose time of expression is determined by mitosis and the amount of nuclear DNA (2C vs 4C) in the cell. Gl and G2 appear to be carbohydrate dependent in that cells starved of either carbohydrate of phosphate fail to make these transitions. Cells deprived of nitrate, however, fail only at Gl to S transition indicating that the controls that operate in G1 differ from those that operate in G2. 46 references, 5 figures.

  10. Diesel exhaust particulate extracts inhibit transcription of nuclear respiratory factor-1 and cell viability in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mattingly, Kathleen A.; Klinge, Carolyn M. [University of Louisville School of Medicine, Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, Louisville, KY (United States)

    2012-04-15

    Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1-regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17{beta}-estradiol (E{sub 2}), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription, and this suppression was not ablated by concomitant treatment with E{sub 2}, 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E{sub 2} increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects. (orig.)

  11. Human amniotic epithelial cells inhibit growth of epithelial ovarian cancer cells via TGF‑β1-mediated cell cycle arrest.

    Science.gov (United States)

    Bu, Shixia; Zhang, Qiuwan; Wang, Qian; Lai, Dongmei

    2017-11-01

    It is reported that human amniotic epithelial cells (hAECs) endow intrinsic antitumor effects on certain kinds of cancer. This research was designed to evaluate whether hAECs endowed potential anticancer properties on epithelial ovarian cancer (EOC) cells in vivo and in vitro, which has not been reported before. In this study, we established a xenografted BALB/c nude mouse model by subcutaneously co-injecting ovarian cancer cell line, SK-OV-3, and hAECs for 28 days. In ex vivo experiments, CCK‑8 cell viability assay, real-time PCR, cell counting assay, cell cycle analysis and immunohistochemistry (IHC) assay were used to detect the effects of hAEC‑secreted factors on the proliferation and cell cycle progression of EOC cells. A cytokine array was conducted to detect anticancer-related cytokines released from hAECs. Human recombinant TGF‑β1 and TGF‑β1 antibody were used to treat EOC cells and analyzed whether TGF‑β1 contributed to the cell cycle arrest. Results from in vivo and ex vivo experiments showed that hAEC-secreted factors and rhTGF‑β1 decreased proliferation of EOC cells and induced G0/G1 cell cycle arrest in cancer cells, which could be partially reversed by excess TGF‑β1 antibody. These data indicate that hAECs endow potential anticancer properties on epithelial ovarian cancer in vivo and in vitro which is partially mediated by hAEC‑secreted TGF‑β1-induced cell cycle arrest. This study suggests a potential application of hAEC‑based therapy against epithelial ovarian cancer.

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

    Science.gov (United States)

    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 (Pbisphenol A+ICI (Pbisphenol A and estradiol promote HepG2 cell proliferation in vitro by inhibition of apoptosis and stimulation of telomerase activity via an estrogen receptor-dependent pathway.

  13. Evaluation of resistance development and viability recovery by toxigenic and non-toxigenic Staphylococcus aureus strains after repeated cycles of high hydrostatic pressure.

    Science.gov (United States)

    Baptista, Inês; Queirós, Rui P; Cunha, Angela; Rocha, Sílvia M; Saraiva, Jorge A; Almeida, Adelaide

    2015-04-01

    In this work, the development of resistance and the recovery of growth after several consecutive cycles of high hydrostatic pressure (HPP) were for the first time evaluated in different strains of Staphylococcus aureus. Three strains of this important and highly resilient to HPP foodborne pathogen were used: a non-enterotoxigenic ATCC 6538 strain, treated with 600 MPa for 30 min at 20 °C, and the toxigenic strains 2153 MA (with enterotoxin A) and 2065 MA (with the enterotoxins A, G and I), treated with 600 MPa for 15 min at 20 °C. After the first treatment, surviving colonies were used to produce new bacterial cultures. This procedure was repeated nine times more for each bacterium or until total inactivation occurred. The inactivation profile of non-enterotoxic strain and the two enterotoxic strains did not change after consecutive cycles, but the toxic strain with three enterotoxins was completely inactivated after the fourth cycle. The three strains did not recover their viability after 14 days. The results indicate that HPP effectively inactivates non-toxigenic and toxigenic strains of S. aureus after a single treatment. The surviving bacteria did not develop resistance after 10 cycles of pressurization and did not recover their viability after 14 days of incubation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Adiponectin Effect on The Viability of Human Endometrial Stromal Cells and mRNA Expression of Adiponectin Receptors

    Directory of Open Access Journals (Sweden)

    Somayeh Bohlouli

    2013-01-01

    Full Text Available Background: Adiponectin is one of the most important adipokines secreted from fattytissue that has a direct inhibitory effect on the development of cancer cells. Adiponectinplays an important role in human reproduction system and fertility of women. Adiponectinconcentration decreases in women with endometriosis and endometrial cancer.The aim of the present study was to investigate the effect of adiponectin on humanendometrial stromal cell (HESC viability as well as mRNA expression of Adipo R1and Adipo R2 receptors.Materials and Methods: In this experimental study, eight endometrial biopsies weretaken and stromal cells were separated by enzymatic digestion and cell filtrations. Stromalcells of each biopsy were divided into four groups: control, 10, 100, and 200 ng/mladiponectin concentrations. The effect of adiponectin on viability of the normal HESCswas studied by trypan blue staining and the relative expression levels of Adipo R1 andR2 were analyzed by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR. Data were analyzed by one way ANOVA and unpaired student’s t test andp<0.05 was considered significant.Results: Adiponectin decreased viability of normal human endometrial stromal cells ina dose and time dependent manner. Expression of Adipo R1 and Adipo R2 receptors didnot change in the presence of adiponectin.Conclusion: Adiponectin can directly influence the viability of HESCs and decreasetheir viability, but it didn’t change expression of adiponectin receptors.

  15. Comparison of Transfection Agents in Forming Complexes with Ferumoxides, Cell Labeling Efficiency, and Cellular Viability

    Directory of Open Access Journals (Sweden)

    Ali Syed Arbab

    2004-01-01

    Full Text Available By complexing ferumoxides or superparamagnetic iron oxide (SPIO to transfection agents (TAs, it is possible to magnetically label mammalian cells. There has been no systematic study comparing TAs complexed to SPIO as far as cell labeling efficiency and viability. This study investigates the toxicity and labeling efficiency at various doses of FEs complexed to different TAs in mammalian cells. Different classes of TAs were used, such as polycationic amines, dendrimers, and lipid-based agents. Cellular toxicity was measured using doses of TAs from 1 to 50 μg/mL in incubation media. Iron incorporation efficiency was measured by combining various amounts of FEs and different doses of TAs. Lipofectamine2000 showed toxicity at lowest dose (1 μg/mL, whereas FuGENE6 and low molecular weight poly-L-lysine (PLI. showed the least toxicity. SPIO labeling efficiency was similar with high-molecular-weight PIX (388.1 kDa and superfect, whereas FuGENE6 and low-molecular-weight PLL were inefficient in labeling cells. Concentrations of 25 to 50 μg/mL of FEs complexed to TAs in media resulted in sufficient endocytosis of the SPIO into endosomes to detect cells on cellular magnetic resonance imaging.

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

    Directory of Open Access Journals (Sweden)

    Xuan Zhou

    2013-01-01

    Full Text Available 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.

  17. Sterilization effects on starPEG coated polymer surfaces: characterization and cell viability.

    Science.gov (United States)

    Lleixà Calvet, Júlia; Grafahrend, Dirk; Klee, Doris; Möller, Martin

    2008-04-01

    Sterilization is frequently an issue for polymeric biomaterials including hydrogels, where autoclaving needs to be discarded, and gamma-irradiation and low temperature hydrogen peroxide gas plasma sterilization are already important alternatives. Coatings based on poly(ethylene glycol) are a well-known strategy to reduce unspecific protein interactions on biomaterial surfaces. Dense, ultrathin coatings of isocyanate terminated star-shaped poly(ethylene glycol) (starPEG) molecules have proven to be resistant to unspecific adsorption of proteins and enable direct biofunctionalization. The effectivity and stability of the starPEG coatings on poly(vinylidene fluoride) (PVDF) were studied after gamma-irradiation (normed dosis 25 kGy) and plasma sterilization (Sterrad 100S). The selected surface properties determined were: surface composition (X-ray photoelectron spectroscopy, XPS), wettability (sessile drop contact angle) and protein adsorption by fluorescence microscopy (Avidin-TexasRed, Bovine Serum Albumin-Rhodamin). Preliminary cell experiments with the cell line L929 were performed prior and after sterilization to investigate the cell repellence of the starPEG coatings as well as cell viability and specific cell adhesion on GRGDS-modified coatings. The starPEG coating undergoes a slight oxidation due to plasma and gamma-sterilization; this represents a minor variation confirmed by XPS and contact angle results. The non-sterilized starPEG and the plasma-sterilized coatings are protein repellent, however the protein adsorption on starPEG coated substrates is much stronger after gamma-sterilization for both avidin and bovine serum albumin. The cell experiments indicate that the starPEG coatings are appliable homogeneously by incubation and are non-cell adherent. Moreover, after both sterilization processes the starPEG coatings remain cell repellent and the GRGDS-modified coatings presented vital cells. Thus we conclude that the plasma sterilization is more convenient

  18. CELL VIABILITY IN X-RAY IRRADIATED HUMAN LENS EPITHELIAL CELL (HLEC CULTURE BY USING TRYPAN BLUE EXCLUSION TEST

    Directory of Open Access Journals (Sweden)

    Rajesh

    2015-12-01

    Full Text Available INTRODUCTION Cataract is one of major cause of blindness, 44% cases of blindness is due to cataract only. In developing countries such as India cataract is more common and it manifest earlier in life than the most developed countries of the world. Patients receiving therapeutic radiation may have developed cataracts as well as radiation retinopathy and optic nerve damage. In this study we are aimed to compare effects of X-ray irradiation on cell viability of HLEC (Human lens epithelial cell culture by using trypan blue dye exclusion test against the unexposed control HLEC culture. Total 20 cataract patients from Dept. of Ophthalmology of Lata Mangeshkar Hospital, Nagpur were included in this study. The capsulorhexis sample from cataract surgery taken for study from eye OT. The capsulorhexis sample without any x-ray exposure taken as control. For culture of HLEC’s RPMI 1640 medium containing 10% fetal calf serum used. The HLEC cultures were X-ray irradiated with increasing count of skull limit parameters of range 150kV, 250mAs and 50mA for 5sec using GE® 650 M.A. X-ray machine. The Trypan blue dye exclusion test was used to analyze cell viability. The slides were analyzed using Olympus® BX51 Research microscope with oil immersion and 100 were observed per sample. The cell viability was calculated using Epi info® (version 6.0, as subject means with standard deviation. Statistical significance will be defined at P≤0.001. Out of 100 cases the 12 were female and 8 were male. Mean age of both male and female patients were 62.45±3.18(SD. A positive linear correlation was found (r:0.819, p<0.001 between the No. of X-Ray exposure and death cells per 100 observed LECs.

  19. Freezing behavior of adherent neuron-like cells and morphological change and viability of post-thaw cells.

    Science.gov (United States)

    Uemura, Makoto; Ishiguro, Hiroshi

    2015-04-01

    Freezing of nerve cells forming a neuronal network has largely been neglected, despite the fact that the cryopreservation of nerve cells benefits the study of cells in the areas of medicine and poison screening. Freezing of nerve cells is also attractive for studying cell morphology because of the characteristic long, thread-like neurites extending from the cell body. In the present study, freezing of neuron-like cells adhering to the substrate (differentiated PC12 cells), in physiological saline, was investigated in order to understand the fundamental freezing and thawing characteristics of nerve cells with neurites. The microscopic freezing behavior of cells under different cooling rates was observed. Next, the post-thaw morphological changes in the cells, including the cytoskeleton, were investigated and post-thaw cell viability was evaluated by dye exclusion using propidium iodide. Two categories of morphological changes, beading and shortening of the neurites, were found and quantified. Also, the morphological changes of neurites due to osmotic stress from sodium chloride were studied to gain a better understanding of causation. The results showed that morphological changes and cell death were promoted with a decrease in end temperature during freezing. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    Science.gov (United States)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

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

    Science.gov (United States)

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

    2013-07-15

    l-Glutamic acid (l-glutamate) is used to induce excitotoxicity and test neuroprotective compounds in cell cultures. However, because l-glutamate powder is nearly insoluble in water, many manufacturers recommend reconstituting l-glutamate in hydrochloric acid (HCl) prior to successive dilutions. Nevertheless, HCl, even at low concentrations, may alter the pH of the cell culture medium and interfere with cell activity. Thus, the aim of this study was to evaluate whether the reconstitution of l-glutamate powder in HCl alters its capacity to induce neurotoxicity in different human neuroblastoma cell lines. SH-SY5Y, IMR-32 and SK-N-BE(2) cells were exposed to various concentrations of l-glutamate, which was either reconstituted in HCl (1M) or post re-equilibrated to the pH of the culture medium (7.5). After 24 and 48h of incubation, changes in the cell viability of treated versus untreated cells were evaluated. The effect of an identical amount of HCl present in the l-glutamate dilutions on neuroblastoma cell survival was also investigated. Our data showed that the neurotoxicity of glutamate reconstituted in HCl was comparable to that of HCl alone. Moreover, the pH variations induced by glutamate or HCl in the culture medium were similar. When the pH of the glutamate stock solution was re-equilibrated, l-glutamate induced variation in cell viability to a lower extent and after a longer incubation time. This study demonstrated that HCl used to reconstitute l-glutamate powder might alter the effect of glutamate itself in neuroblastoma cell cultures. Thus, this information might be useful to scientists who use l-glutamate to induce excitotoxicity or to test neuroprotective agents. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Measurement of bioimpedance and cell viability during ischemia-reperfusion in the rat liver.

    Science.gov (United States)

    Ahn, H; Shin, H; Yun, S; Kim, J; Choi, J

    2005-01-01

    During liver resection and liver transplant, liver is damaged by ischemia-reperfusion injury. Until now, there is no approved method to measure or predict the extent of liver injury during the operation. This is the preliminary study to make the real time monitoring system by quantification of bioimpedance and ischemiareperfusion reperfusion injury in liver. Sprague-Dawley rats were subjected to different periods of 70% partial hepatic ischemia (30, 60, 90 and 120minutes ischemia) and reperfusion. We measured changes of liver tissue bioimpedance (120Hz-100KHz) every five minutes. Cell viability was assessed by metabolic capacity of fatty acid (palmitic acid metabolic rate), ATP content and histological examination (H/E and TUNEL stain) at every 30 minutes interval during ischemia.

  3. Effects of allicin on the proliferation and cell cycle of chondrocytes.

    Science.gov (United States)

    Li, Tao; Shi, Hong-Yan; Hua, Yong-Xin; Gao, Chen; Xia, Qing; Yang, Guang; Li, Bin

    2015-01-01

    The present study demonstrates the effect of allicin on the proliferation and the cell cycle distribution of the chondrocytes. MTT assay and flow cytometry were used for the evaluation of the effect of allicin on cell proliferative and the cell cycle distribution, respectively of the chondrocytes. The reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis were respectively used for the analysis of mRNA and protein expression levels of cyclin D1, CDK4 and CDK6. The results revealed that exposure of the chondrocytes to allicin at a concentration of 40 µM significantly promoted the cell viability. Treatment of the cells with 10, 20, 30, 40, and 50 μg/mL of allicin enhanced the cell viability by 2.5.47 ± 0.86, 5.43 ± 0.66, 10.74 ± 1.48, 35.89 ± 3.78, and 32.21 ± 2.92%, respectively after 36 h compared to control cells. Allicin exposure caused a marked decrease in the percentage of cells in G0/G1 phase with a subsequent increase in the S phase population. Furthermore, allicin treatment enhanced the expression of cyclin D1, CDK4 and CDK6. Therefore, allicin treatment enhances the proliferation of chondrocytes by promoting the transition from G1 to S phase of the cell cycle through increase in the expression of cyclin D1, CDK4 and CDK6 levels.

  4. Cell cycle nucleic acids, polypeptides and uses thereof

    Science.gov (United States)

    Gordon-Kamm, William J.; Lowe, Keith S.; Larkins, Brian A.; Dilkes, Brian R.; Sun, Yuejin

    2007-08-14

    The invention provides isolated nucleic acids and their encoded proteins that are involved in cell cycle regulation. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content, cell cycle progression, cell number and/or composition of plants.

  5. Modeling of SONOS Memory Cell Erase Cycle

    Science.gov (United States)

    Phillips, Thomas A.; MacLeod, Todd C.; Ho, Fat H.

    2011-01-01

    Utilization of Silicon-Oxide-Nitride-Oxide-Silicon (SONOS) nonvolatile semiconductor memories as a flash memory has many advantages. These electrically erasable programmable read-only memories (EEPROMs) utilize low programming voltages, have a high erase/write cycle lifetime, are radiation hardened, and are compatible with high-density scaled CMOS for low power, portable electronics. In this paper, the SONOS memory cell erase cycle was investigated using a nonquasi-static (NQS) MOSFET model. Comparisons were made between the model predictions and experimental data.

  6. Monitoring of cell viability and cell growth in a hollow-fiber bioreactor by use of the dye Alamar Blue.

    Science.gov (United States)

    Gloeckner, H; Jonuleit, T; Lemke, H D

    2001-06-01

    We describe a method for monitoring cell proliferation in a small-scale hollow-fiber bioreactor (culture volume: 1 ml) by use of the Alamar Blue dye. Alamar Blue is a non-fluorescent compound, which yields a fluorescent product after reduction, e.g. by living cells. In contrast to the MTT-assay, the Alamar Blue assay does not lead to cell death. However, when not removed from the cells, the Alamar Blue dye shows a reversible, time- and concentration-dependent growth inhibition as observed for the leukemic cell lines CCRF-CEM, HL-60 and REH. When applied in the medium compartment of a hollow-fiber bioreactor system, the dye is delivered to the cells across the hollow-fiber membrane, reduced by the cells and released from the cell into the medium compartment back again. Thus, fluorescence intensity can be measured in medium samples reflecting growth of the cells in the cell compartment. This procedure offers several advantages. First, exposure of the cells to the dye can be reduced compared to conventional culture in plates. Second, handling steps are minimized since no sample of the cells needs to be taken for readout. Moreover, for the exchange of medium, a centrifugation step can be avoided and the cells can be cultivated further. Third, the method allows discriminating between cell densities of 10(5), 10(6) and 10(7) of proliferating HL-60 cells cultivated in the cell compartment of the bioreactor. Measurement of fluorescence in the medium compartment is more sensitive compared to glucose or lactate measurement for cell counts below 10(6) cells/ml, in particular. We conclude that the Alamar Blue-assay combined with a hollow-fiber bioreactor offers distinct advantages for the non-invasive monitoring of cell viability and proliferation in a closed system.

  7. Radiobiological Effectiveness of Ultrashort Laser-Driven Electron Bunches: Micronucleus Frequency, Telomere Shortening and Cell Viability.

    Science.gov (United States)

    Andreassi, Maria Grazia; Borghini, Andrea; Pulignani, Silvia; Baffigi, Federica; Fulgentini, Lorenzo; Koester, Petra; Cresci, Monica; Vecoli, Cecilia; Lamia, Debora; Russo, Giorgio; Panetta, Daniele; Tripodi, Maria; Gizzi, Leonida A; Labate, Luca

    2016-09-01

    Laser-driven electron accelerators are capable of producing high-energy electron bunches in shorter distances than conventional radiofrequency accelerators. To date, our knowledge of the radiobiological effects in cells exposed to electrons using a laser-plasma accelerator is still very limited. In this study, we compared the dose-response curves for micronucleus (MN) frequency and telomere length in peripheral blood lymphocytes exposed to laser-driven electron pulse and X-ray radiations. Additionally, we evaluated the effects on cell survival of in vitro tumor cells after exposure to laser-driven electron pulse compared to electron beams produced by a conventional radiofrequency accelerator used for intraoperative radiation therapy. Blood samples from two different donors were exposed to six radiation doses ranging from 0 to 2 Gy. Relative biological effectiveness (RBE) for micronucleus induction was calculated from the alpha coefficients for electrons compared to X rays (RBE = alpha laser/alpha X rays). Cell viability was monitored in the OVCAR-3 ovarian cancer cell line using trypan blue exclusion assay at day 3, 5 and 7 postirradiation (2, 4, 6, 8 and 10 Gy). The RBE values obtained by comparing the alpha values were 1.3 and 1.2 for the two donors. Mean telomere length was also found to be reduced in a significant dose-dependent manner after irradiation with both electrons and X rays in both donors studied. Our findings showed a radiobiological response as mirrored by the induction of micronuclei and shortening of telomere as well as by the reduction of cell survival in blood samples and cancer cells exposed in vitro to laser-generated electron bunches. Additional studies are needed to improve preclinical validation of the radiobiological characteristics and efficacy of laser-driven electron accelerators in the future.

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

    Directory of Open Access Journals (Sweden)

    Chang-Fang Chiu

    2016-08-01

    Full Text Available 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.

  9. Cell cycle regulation of hematopoietic stem or progenitor cells.

    Science.gov (United States)

    Hao, Sha; Chen, Chen; Cheng, Tao

    2016-05-01

    The highly regulated process of blood production is achieved through the hierarchical organization of hematopoietic stem cell (HSC) subsets and their progenies, which differ in self-renewal and differentiation potential. Genetic studies in mice have demonstrated that cell cycle is tightly controlled by the complex interplay between extrinsic cues and intrinsic regulatory pathways involved in HSC self-renewal and differentiation. Deregulation of these cellular programs may transform HSCs or hematopoietic progenitor cells (HPCs) into disease-initiating stem cells, and can result in hematopoietic malignancies such as leukemia. While previous studies have shown roles for some cell cycle regulators and related signaling pathways in HSCs and HPCs, a more complete picture regarding the molecular mechanisms underlying cell cycle regulation in HSCs or HPCs is lacking. Based on accumulated studies in this field, the present review introduces the basic components of the cell cycle machinery and discusses their major cellular networks that regulate the dormancy and cell cycle progression of HSCs. Knowledge on this topic would help researchers and clinicians to better understand the pathogenesis of relevant blood disorders and to develop new strategies for therapeutic manipulation of HSCs.

  10. Dual excitation multi-fluorescence flow cytometry for detailed analyses of viability and apoptotic cell transition

    Directory of Open Access Journals (Sweden)

    G Mazzini

    2009-06-01

    Full Text Available The discrimination of live/dead cells as well as the detection of apoptosis is a frequent need in many areas of experimental biology. Cell proliferation is linked to apoptosis and controlled by several genes. During the cell life, specific events can stimulate proliferation while others may trigger the apoptotic pathway. Very few methods (i.e. TUNEL are now available for studies aimed at correlation between apoptosis and proliferation. Therefore, there is interest in developing new methodological approaches that are able to correlate apoptosis to the cell cycle phases. Recently new approaches have been proposed to detect and enumerate apoptotic cells by flow cytometry. Among these, the most established and applied are those based on the cell membrane modifications induced in the early phases of the apoptotic process. The dye pair Hoechst 33342 (HO and Propidium Iodide (PI, thanks to their peculiar characteristics to be respectively permeable and impermeable to the intact cell membrane, seems to be very useful. Unfortunately the spectral interaction of these dyes generates a consistent “energy transfer” from HO to PI. The co-presence of the dyes in a nucleus results in a modification in the intensity of both the emitted fluorescences. In order to designate the damaged cells (red fluorescence to the specific cell cycle phases (blue fluorescence, we have tested different staining protocols aimed to minimize the interference of these dyes as much as possible. In cell culture models, we are able to detect serum-starved apoptotic cells as well as to designate their exact location in the cell cycle phases using a very low PI concentration. Using a Partec PAS flow cytometer equipped with HBO lamp and argon ion laser, a double UV/blue excitation has been performed. This analytical approach is able to discriminate live blue cells from the damaged (blue-red ones even at 0.05 ?g/mL PI. The same instrumental setting allows performing other multi

  11. Induction of apoptosis and antiproliferative activity of naringenin in human epidermoid carcinoma cell through ROS generation and cell cycle arrest.

    Directory of Open Access Journals (Sweden)

    Md Sultan Ahamad

    Full Text Available A natural predominant flavanone naringenin, especially abundant in citrus fruits, has a wide range of pharmacological activities. The search for antiproliferative agents that reduce skin carcinoma is a task of great importance. The objective of this study was to analyze the anti-proliferative and apoptotic mechanism of naringenin using MTT assay, DNA fragmentation, nuclear condensation, change in mitochondrial membrane potential, cell cycle kinetics and caspase-3 as biomarkers and to investigate the ability to induce reactive oxygen species (ROS initiating apoptotic cascade in human epidermoid carcinoma A431 cells. Results showed that naringenin exposure significantly reduced the cell viability of A431 cells (p<0.01 with a concomitant increase in nuclear condensation and DNA fragmentation in a dose dependent manner. The intracellular ROS generation assay showed statistically significant (p<0.001 dose-related increment in ROS production for naringenin. It also caused naringenin-mediated epidermoid carcinoma apoptosis by inducing mitochondrial depolarization. Cell cycle study showed that naringenin induced cell cycle arrest in G0/G1 phase of cell cycle and caspase-3 analysis revealed a dose dependent increment in caspase-3 activity which led to cell apoptosis. This study confirms the efficacy of naringenin that lead to cell death in epidermoid carcinoma cells via inducing ROS generation, mitochondrial depolarization, nuclear condensation, DNA fragmentation, cell cycle arrest in G0/G1 phase and caspase-3 activation.

  12. The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

    Science.gov (United States)

    El-Badawy, Ahmed; El-Badri, Nagwa

    2016-01-13

    The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

  13. In vitro effects of fetal rat cerebrospinal fluid on viability and neuronal differentiation of PC12 cells

    Directory of Open Access Journals (Sweden)

    Nabiuni Mohammad

    2012-06-01

    Full Text Available Abstract Background Fetal cerebrospinal fluid (CSF contains many neurotrophic and growth factors and has been shown to be capable of supporting viability, proliferation and differentiation of primary cortical progenitor cells. Rat pheochromocytoma PC12 cells have been widely used as an in vitro model of neuronal differentiation since they differentiate into sympathetic neuron-like cells in response to growth factors. This study aimed to establish whether PC12 cells were responsive to fetal CSF and therefore whether they might be used to investigate CSF physiology in a stable cell line lacking the time-specific response patterns of primary cells previously described. Methods In vitro assays of viability, proliferation and differentiation were carried out after incubation of PC12 cells in media with and without addition of fetal rat CSF. An MTT tetrazolium assay was used to assess cell viability and/or cell proliferation. Expression of neural differentiation markers (MAP-2 and β-III tubulin was determined by immunocytochemistry. Formation and growth of neurites was measured by image analysis. Results PC12 cells differentiate into neuronal cell types when exposed to bFGF. Viability and cell proliferation of PC12 cells cultured in CSF-supplemented medium from E18 rat fetuses were significantly elevated relative to the control group. Neuronal-like outgrowths from cells appeared following the application of bFGF or CSF from E17 and E19 fetuses but not E18 or E20 CSF. Beta-III tubulin was expressed in PC12 cells cultured in any media except that supplemented with E18 CSF. MAP-2 expression was found in control cultures and in those with E17 and E19 CSF. MAP2 was located in neurites except in E17 CSF when the whole cell was positive. Conclusions Fetal rat CSF supports viability and stimulates proliferation and neurogenic differentiation of PC12 cells in an age-dependent way, suggesting that CSF composition changes with age. This feature may be important

  14. Analysis of the effect of cryoprotectant medium composition to viability of autologous hematopoietic cells collected by leukapheresis.

    Science.gov (United States)

    Kozlowska-Skrzypczak, M; Kubiak, A; Bembnista, E; Matuszak, P; Komarnicki, M

    2014-10-01

    Cryopreservation of hematopoietic stem cells intended for autologous transplantation is a crucial element of the banking process. Although cryopreservation techniques are well known, improvement is needed. This study was designed to optimize cryopreservation to improve the quantitative and qualitative parameters of hematopoietic stem cells in the material intended for transplantation. We used available opportunities to provide the best quantitative and qualitative parameters of hematopoietic stem cell transplants processed in a closed system. Two hundred forty-eight products of hematopoietic stem cells collected by leukapheresis from patients with lymphoproliferative disorders create the basis of this report. The material was frozen in a controlled-rate freezer and stored in containers in the vapor phase of LN2 (-160°C). The composition of a cryoprotectant medium was modified. For freezing, 192 probes were used with a cryoprotective medium containing 20% dimethyl sulfoxide (DMSO) and enriched RPMI 1640. For 56 samples, we used 20% DMSO in autologous plasma harvested during leukapheresis. Products of hematopoietic stem cells and cryoprotectant medium were combined in a 1:1 ratio. The final number of nuclear cells did not exceed 2 × 10(8)/mL. Analysis was performed after thawing the probes. Viability of nuclear cells has been assessed using the microscopic technique after incubation in Trypan blue and the CD34+ cells by flow cytometry using the 7-aminoactynomycin D. A statistical analysis has been conducted using the Statistica program (StatSoft, Cracow, Poland). The results show that the application of autologous plasma is linked with higher viability of nuclear cells and CD34+ cells. Moreover, statistical analysis of the nuclear cells and CD34+ cells viability differs significantly between groups frozen using RPMI 1640 and autologous plasma (P < .05). To assess the viability of CD34+, cells frozen using RPMI 1640 results showed a large span of at 16.4% to 99

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

    Science.gov (United States)

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

    2016-01-01

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

  16. Ionizing radiation damage to cells: effects of cell cycle redistribution.

    Science.gov (United States)

    Chen, P L; Brenner, D J; Sachs, R K

    1995-04-01

    If a population of cycling cells is exposed to a fixed dose of ionizing radiation delivered over time T, it is sometimes observed that increasing T increases the amount of cell killing. This is essentially because at first the radiation preferentially kills cells in a sensitive portion of the cycle and the surviving, more resistant cells then have time to reach more sensitive stages. We refer to this effect as population resensitization, caused by redistribution within the cell cycle. We investigate the effect theoretically by employing the McKendrick-von Foerster equation for age-structured proliferating cell populations, generalized by introducing a radiation damage term. Within our formalism, we show that population resensitization occurs whenever: (a) prior to irradiation the cell population has the stable age-distribution approached asymptotically by an unirradiated population, and (b) T is sufficiently small. Examples and other cases are outlined. The methods of Volterra integral equations, renewal theory, and positive semigroup theory are applied. The effect of varying T is evaluated by considering the ultimate amplitude of the stable age-distribution population at times much greater than both the irradiation duration and the average cell-cycle time. The main biological limitations of the formalism are the following: considering only radiation damage which is not subject to enzymatic repair or quadratic misrepair, using an overly naive method of ensuring loss of cell cycle synchrony, neglecting nonlinear effects such as density inhibition of growth, and neglecting radiatively induced perturbations of the cell cycle. Possible methods for removing these limitations are briefly discussed.

  17. A thermodynamic cycle for the solar cell

    Science.gov (United States)

    Alicki, Robert; Gelbwaser-Klimovsky, David; Jenkins, Alejandro

    2017-03-01

    A solar cell is a heat engine, but textbook treatments are not wholly satisfactory from a thermodynamic standpoint, since they present solar cells as directly converting the energy of light into electricity, and the current in the circuit as maintained by an electrostatic potential. We propose a thermodynamic cycle in which the gas of electrons in the p phase serves as the working substance. The interface between the p and n phases acts as a self-oscillating piston that modulates the absorption of heat from the photons so that it may perform a net positive work during a complete cycle of its motion, in accordance with the laws of thermodynamics. We draw a simple hydrodynamical analogy between this model and the "putt-putt" engine of toy boats, in which the interface between the water's liquid and gas phases serves as the piston. We point out some testable consequences of this model.

  18. Oxidative stress and DNA lesions: the role of 8-oxoguanine lesions in Trypanosoma cruzi cell viability.

    Directory of Open Access Journals (Sweden)

    Pedro H N Aguiar

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

  19. Targeting cell cycle regulators in hematologic malignancies

    Directory of Open Access Journals (Sweden)

    Eiman eAleem

    2015-04-01

    Full Text Available Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia, and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219, pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638 as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed.

  20. Mechanisms of G1 cell cycle arrest and apoptosis in myeloma cells induced by hybrid-compound histone deacetylase inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Fujii, Seiko [Division of Infections and Molecular Biology, Kyushu Dental University (Japan); Division of Maxillofacial Surgery, Kyushu Dental University (Japan); Okinaga, Toshinori; Ariyoshi, Wataru [Division of Infections and Molecular Biology, Kyushu Dental University (Japan); Oral Biology Research Center, Kyushu Dental University (Japan); Takahashi, Osamu; Iwanaga, Kenjiro [Division of Maxillofacial Surgery, Kyushu Dental University (Japan); Nishino, Norikazu [Oral Biology Research Center, Kyushu Dental University (Japan); Tominaga, Kazuhiro [Division of Maxillofacial Surgery, Kyushu Dental University (Japan); Nishihara, Tatsuji, E-mail: tatsujin@kyu-dent.ac.jp [Division of Infections and Molecular Biology, Kyushu Dental University (Japan); Oral Biology Research Center, Kyushu Dental University (Japan)

    2013-05-10

    Highlights: •Novel histone deacetylase inhibitor Ky-2, remarkably inhibits myeloma cell growth. •Ky-2 demonstrates no cytotoxicity against normal lymphocytic cells. •Ky-2 induces cell cycle arrest through the cell cycle-associated proteins. •Ky-2 induces Bcl-2-inhibitable apoptosis through a caspase-dependent cascade. -- Abstract: Objectives: Histone deacetylase (HDAC) inhibitors are new therapeutic agents, used to treat various types of malignant cancers. In the present study, we investigated the effects of Ky-2, a hybrid-compound HDAC inhibitor, on the growth of mouse myeloma cells. Materials and methods: Myeloma cells, HS-72, P3U1, and mouse normal cells were used in this study. Effect of HDAC inhibitors on cell viability was determined by WST-assay and trypan blue assay. Cell cycle was analyzed using flow cytometer. The expression of cell cycle regulatory and the apoptosis associated proteins were examined by Western blot analysis. Hoechst’s staining was used to detect apoptotic cells. Results: Our findings showed that Ky-2 decreased the levels of HDACs, while it enhanced acetylation of histone H3. Myeloma cell proliferation was inhibited by Ky-2 treatment. Interestingly, Ky-2 had no cytotoxic effects on mouse normal cells. Ky-2 treatment induced G1-phase cell cycle arrest and accumulation of a sub-G1 phase population, while Western blotting analysis revealed that expressions of the cell cycle-associated proteins were up-regulated. Also, Ky-2 enhanced the cleavage of caspase-9 and -3 in myeloma cells, followed by DNA fragmentation. In addition, Ky-2 was not found to induce apoptosis in bcl-2 overexpressing myeloma cells. Conclusion: These findings suggest that Ky-2 induces apoptosis via a caspase-dependent cascade and Bcl-2-inhibitable mechanism in myeloma cells.

  1. Arctigenin Inhibits Lung Metastasis of Colorectal Cancer by Regulating Cell Viability and Metastatic Phenotypes.

    Science.gov (United States)

    Han, Yo-Han; Kee, Ji-Ye; Kim, Dae-Seung; Mun, Jeong-Geon; Jeong, Mi-Young; Park, Sang-Hyun; Choi, Byung-Min; Park, Sung-Joo; Kim, Hyun-Jung; Um, Jae-Young; Hong, Seung-Heon

    2016-08-27

    Arctigenin (ARC) has been shown to have an anti-cancer effect in various cell types and tissues. However, there have been no studies concerning metastatic colorectal cancer (CRC). In this study, we investigated the anti-metastatic properties of ARC on colorectal metastasis and present a potential candidate drug. ARC induced cell cycle arrest and apoptosis in CT26 cells through the intrinsic apoptotic pathway via MAPKs signaling. In several metastatic phenotypes, ARC controlled epithelial-mesenchymal transition (EMT) through increasing the expression of epithelial marker E-cadherin and decreasing the expressions of mesenchymal markers; N-cadherin, vimentin, β-catenin, and Snail. Moreover, ARC inhibited migration and invasion through reducing of matrix metalloproteinase-2 (MMP-2) and MMP-9 expressions. In an experimental metastasis model, ARC significantly inhibited lung metastasis of CT26 cells. Taken together, our study demonstrates the inhibitory effects of ARC on colorectal metastasis.

  2. Arctigenin Inhibits Lung Metastasis of Colorectal Cancer by Regulating Cell Viability and Metastatic Phenotypes

    Directory of Open Access Journals (Sweden)

    Yo-Han Han

    2016-08-01

    Full Text Available 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.

  3. Effect of Sodium Dodecyl Sulfate (SDS) and Tween 80 on Cell Viability in an Air-Cathode Microbial Fuel Cell

    KAUST Repository

    Fregoso, Luisa

    2011-07-01

    Microbial fuel cells (MFCs) generate current via electrochemical reactions produced by bacteria attached to the anode that oxidize organic matter. Due to their high volume use in household products, some concentration of surfactant will reach wastewater treatment plants. The average surfactant concentration in wastewater ranges from 10 to 20 mg L-1, and up to 300 mg L-1, for domestic and industrial wastewaters, respectively. This study aimed to demonstrate the feasibility of enhancing power production by adding Tween 80 and SDS surfactants to air-cathode MFCs, and their effect in cell viability at the anodic biofilm. In order to analyze the effect of anionic and nonionic surfactants in MFCs performance, eight MFCs were spiked with two types of surfactants, the anionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant Tween® 80 at two different concentrations 10 and 100 mg L-1. Cell viability at the anodic biofilms was examined using the LIVE/DEAD BacLight viability assay and images were visualized with a confocal laser scanning microscope. The electrochemical results demonstrate that, for an air-cathode MFC operating on 1 g L-1 acetate in a fed-batch mode, reactors where SDS was added show a lower overall performance, maximum PD of 544 mW m-2, CE of 12.3%, Rint of 322 Ω (10 mg L-1) and maximum PD of 265 mW m-2, CE of 9.4%, Rint of 758 Ω (100 mg L-1). Reactors where Tween 80 was added show quite stable performance, maximum PD of 623 mW m-2, CE of 15.4%, Rint of 216 Ω (10 mg L-1) and maximum PD of 591 mW m-2, CE of 10.8%, Rint of 279 Ω (100 mg L-1), compared with reactors operating at only acetate as a substrate, maximum PD of 574 mW m-2. Confocal microscopy images confirm this observation and biofilm viability appeared severely compromised in SDS reactors, especially at high concentrations. This study has opened up a whole new research area in determining which types of surfactants are toxic to the anodic biofilm and to further investigate the

  4. Cell cycle population effects in perturbation studies.

    Science.gov (United States)

    O'Duibhir, Eoghan; Lijnzaad, Philip; Benschop, Joris J; Lenstra, Tineke L; van Leenen, Dik; Groot Koerkamp, Marian J A; Margaritis, Thanasis; Brok, Mariel O; Kemmeren, Patrick; Holstege, Frank C P

    2014-06-21

    Growth condition perturbation or gene function disruption are commonly used strategies to study cellular systems. Although it is widely appreciated that such experiments may involve indirect effects, these frequently remain uncharacterized. Here, analysis of functionally unrelated Saccharyomyces cerevisiae deletion strains reveals a common gene expression signature. One property shared by these strains is slower growth, with increased presence of the signature in more slowly growing strains. The slow growth signature is highly similar to the environmental stress response (ESR), an expression response common to diverse environmental perturbations. Both environmental and genetic perturbations result in growth rate changes. These are accompanied by a change in the distribution of cells over different cell cycle phases. Rather than representing a direct expression response in single cells, both the slow growth signature and ESR mainly reflect a redistribution of cells over different cell cycle phases, primarily characterized by an increase in the G1 population. The findings have implications for any study of perturbation that is accompanied by growth rate changes. Strategies to counter these effects are presented and discussed. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  5. The cell cycle as a brake for ?-cell regeneration from embryonic stem cells

    OpenAIRE

    El-Badawy, Ahmed; El-Badri, Nagwa

    2016-01-01

    The generation of insulin-producing ? cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic ? cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle ...

  6. Studying cell cycle checkpoints using Drosophila cultured cells

    NARCIS (Netherlands)

    Siudeja, Katarzyna; de Jong, Jannie; Sibon, Ody

    2011-01-01

    Drosophila cell lines are valuable tools to study a number of cellular processes, including DNA damage responses and cell cycle checkpoint control. Using an in vitro system instead of a whole organism has two main advantages: it saves time and simple and effective molecular techniques are available.

  7. Lobaplatin arrests cell cycle progression in human hepatocellular carcinoma cells

    Directory of Open Access Journals (Sweden)

    Chen Chang-Jie

    2010-10-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC still is a big burden for China. In recent years, the third-generation platinum compounds have been proposed as potential active agents for HCC. However, more experimental and clinical data are warranted to support the proposal. In the present study, the effect of lobaplatin was assessed in five HCC cell lines and the underlying molecular mechanisms in terms of cell cycle kinetics were explored. Methods Cytotoxicity of lobaplatin to human HCC cell lines was examined using MTT cell proliferation assay. Cell cycle distribution was determined by flow cytometry. Expression of cell cycle-regulated genes was examined at both the mRNA (RT-PCR and protein (Western blot levels. The phosphorylation status of cyclin-dependent kinases (CDKs and retinoblastoma (Rb protein was also examined using Western blot analysis. Results Lobaplatin inhibited proliferation of human HCC cells in a dose-dependent manner. For the most sensitive SMMC-7721 cells, lobaplatin arrested cell cycle progression in G1 and G2/M phases time-dependently which might be associated with the down-regulation of cyclin B, CDK1, CDC25C, phosphorylated CDK1 (pCDK1, pCDK4, Rb, E2F, and pRb, and the up-regulation of p53, p21, and p27. Conclusion Cytotoxicity of lobaplatin in human HCC cells might be due to its ability to arrest cell cycle progression which would contribute to the potential use of lobaplatin for the management of HCC.

  8. In vitro antioxidant and cell viability of Pyrostegia venusta (Ker Gawl. Miers

    Directory of Open Access Journals (Sweden)

    Thales D. P. Altoé

    2015-01-01

    Full Text Available Many diseases are associated with oxidative stress and inflammatory processes. The current research is directed toward evaluating the antioxidant potential and phytochemistry composition of P. venusta leaves. In this study, P. venusta leaves were dried and macerated, and the crude extract was partitioned. Phytochemical analysis was performed using standard methodologies, and the total flavonoid content was measured using a calibration curve with rutin. We evaluated the antioxidant potential of P. venusta leaves using 1,1-Diphenyl-2-picrylhydrazyl (DPPH, 2, 2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, and a Trolox-like standard. Cell viability (CV assays were done using macrophage RAW 264.7 cell lines and compared to four commercial anti-inflammatories (acetylsalicylic acid, Indometacina, Betametasona, and Piroxicam. Phytochemical analysis revealed the presence of steroids, coumarins, and flavone. The flavonoid content was 148.5 ± 7.65 µg as a rutin equivalent/mg of crude extract. The ethyl acetate fraction showed the best antioxidant activity in the methodologies of DPPH inhibition (IC50 = 38.62 µg/mL and ABTS radical (IC50 = 28.58 µg/mL. Samples of P. venusta had CV values that were better than the commercial anti-inflammatory, which showed CV values below the negative control. The crude extract and the ethyl acetate fraction, showed CV values below the negative control and the hexane fraction obtained values above the negative control, these being best results.

  9. Evaluation of goat milk as storage media to preserve viability of human periodontal ligament cells in vitro.

    Science.gov (United States)

    Ulusoy, Ayça Tuba; Kalyoncuoglu, Elif; Kaya, Senay; Cehreli, Zafer Cavit

    2016-08-01

    The purpose of this study was to evaluate the effectiveness of goat milk as a storage media for maintenance of periodontal ligament (PDL) cell viability of avulsed teeth and compare it with commonly used and/or investigated storage media. PDL cells were obtained from the root surface of healthy premolars and were cultured in Eagle's maintenance medium (EMM). Cell cultures were treated with the following storage media: tap water (negative control); EMM (positive control); Hank's balanced salt solution; ultra high temperature (UHT) long-shelf-life lactose-free cow milk; UHT long-shelf-life whole cow milk; UHT long-shelf-life skimmed cow milk; UHT long-shelf-life soy milk; UHT long-shelf-life goat milk, UHT long-shelf-life follow on milk with probiotic, 20% propolis, and egg white. Culture plates were incubated with experimental media at 20°C for 1, 3, 6, 12, and 24 h. PDL cell viability was assessed by tetrazolium salt-based colorimetric (MTT) assay at each test period. One-way anova was used to evaluate the effects of storage solutions at each time point, followed by post hoc Duncan's multiple comparison test (P = 0.05). A dendrogram was constructed to show the arrangement of hierarchical clustering. Goat milk displayed the highest capacity to maintain cell viability at all test intervals (P milk with the probiotic showed the lowest time-dependent PDL cell viability among all test media (P milks, HBSS performed significantly less effectively in maintaining PDL cell viability during the entire test period (P milk can be recommended as a suitable storage medium for avulsed teeth. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Immediate and late analysis of dental pulp stem cells viability after indirect exposition to alternative in-office bleaching strategies.

    Science.gov (United States)

    Soares, Diana Gabriela; Basso, Fernanda Gonçalves; Hebling, Josimeri; de Souza Costa, Carlos Alberto

    2015-06-01

    To evaluate human dental pulp stem cell viability and capacity to recover from experimental dental bleaching techniques. Enamel/dentin disks adapted to trans-wells were positioned on previously cultivated dental pulp stem cells. Bleaching gels containing 35, 17.5, 10, and 8 % hydrogen peroxide (H2O2) were applied one or three times (each application lasting 15 min) on enamel. Cell viability (MTT assay) and morphology (SEM) were evaluated immediately (T1) or 72 h (T2) post-bleaching. The 35 % H2O2 gel promoted intense reduction in viability (93-97 %) and morphological alterations of the cells at T1, irrespective of frequency of application, with absence or limited capacity for recovery being observed at T2. The other bleaching gels presented significant lower toxicity when compared with the 35 % H2O2 gel, in a time/concentration fashion. In T1, no significant difference was observed between the negative control (without bleaching) and the 8 and 10 % H2O2 gels applied on enamel for 15 min, in which the cells presented elevated viability and morphology similar to the negative control at T2. Bleaching gels with 8 and 10 % H2O2 in their composition cause limited immediate toxic effect on pulp stem cells, which recover their viability 3 days after treatment. This study presents proposals for in-office dental bleaching to be performed with limited aggressive effect on dental pulp stem cells. Therefore, we are able to offer interesting clinical alternatives for bleaching vital teeth, under professional supervision, maintaining the integrity and reparative capacity of pulp-dentin complex.

  11. Cell cycle regulation of Golgi membrane dynamics

    Science.gov (United States)

    Tang, Danming; Wang, Yanzhuang

    2013-01-01

    The Golgi apparatus is a membranous organelle in the cell that plays essential roles in protein and lipid trafficking, sorting, processing and modification. Its basic structure is a stack of closely aligned flattened cisternae. In mammalian cells, dozens of Golgi stacks are often laterally linked into a ribbon-like structure. Biogenesis of the Golgi during cell division occurs through a sophisticated disassembly and reassembly process that can be divided into three distinct but cooperative steps, including the deformation and reformation of the Golgi cisternae, stacks and ribbon. Here, we review our current understanding of the protein machineries that control these three steps in the cycle of mammalian cell division: GRASP65 and GRASP55 in Golgi stack and ribbon formation; ubiquitin and AAA ATPases in post-mitotic Golgi membrane fusion; and golgins and cytoskeleton in Golgi ribbon formation. PMID:23453991

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

    Directory of Open Access Journals (Sweden)

    Roberta Lotti

    2016-01-01

    Full Text Available 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.

  13. Cell-cycle-dependent efficacy of photodynamic therapy with ATX-S10(Na).

    Science.gov (United States)

    Sano, Munetaka; Furuta, Takahisa; Takahira, Kenichiro; Kajimura, Masayoshi; Hanai, Hiroyuki; Kohno, Eiji; Hirano, Toru; Hishida, Akira

    2005-01-01

    Photodynamic therapy (PDT) is a useful strategy for treating various cancers. Details of the mechanisms of PDT have not been made clear yet. We intended to study the efficacy of PDT in relation to the cell cycle. HeLa S3 cells were synchronized by the thymidine block method. Cells in different cell cycle phases after release were treated with the water-soluble photosensitizer, ATX-S10(Na). The cellular viability after PDT was determined by the MTT assay. Intracellular levels of ATX-S10(Na) in different cell cycle phases were also determined. We found that cells in the S and G(2)/M phases were hypersensitive to PDT with ATX-S10(Na) in comparison with those in the G(1) phase, and that cellular levels of ATX-S10(Na) were increased in cells in the S and G(2)/M phases compared to those in the G(1) phase. We conclude that cellular ATX-S10(Na) levels differ among the different cell cycle phases, which is associated with the cell-cycle-dependent efficacy of PDT with ATX-S10(Na).

  14. Sirtuin-1 regulates acinar-to-ductal metaplasia and supports cancer cell viability in pancreatic cancer.

    Science.gov (United States)

    Wauters, Elke; Sanchez-Arévalo Lobo, Victor J; Pinho, Andreia V; Mawson, Amanda; Herranz, Daniel; Wu, Jianmin; Cowley, Mark J; Colvin, Emily K; Njicop, Erna Ngwayi; Sutherland, Rob L; Liu, Tao; Serrano, Manuel; Bouwens, Luc; Real, Francisco X; Biankin, Andrew V; Rooman, Ilse

    2013-04-01

    The exocrine pancreas can undergo acinar-to-ductal metaplasia (ADM), as in the case of pancreatitis where precursor lesions of pancreatic ductal adenocarcinoma (PDAC) can arise. The NAD(+)-dependent protein deacetylase Sirtuin-1 (Sirt1) has been implicated in carcinogenesis with dual roles depending on its subcellular localization. In this study, we examined the expression and the role of Sirt1 in different stages of pancreatic carcinogenesis, i.e. ADM models and established PDAC. In addition, we analyzed the expression of KIAA1967, a key mediator of Sirt1 function, along with potential Sirt1 downstream targets. Sirt1 was co-expressed with KIAA1967 in the nuclei of normal pancreatic acinar cells. In ADM, Sirt1 underwent a transient nuclear-to-cytoplasmic shuttling. Experiments where during ADM, we enforced repression of Sirt1 shuttling, inhibition of Sirt1 activity or modulation of its expression, all underscore that the temporary decrease of nuclear and increase of cytoplasmic Sirt1 stimulate ADM. Our results further underscore that important transcriptional regulators of acinar differentiation, that is, Pancreatic transcription factor-1a and β-catenin can be deacetylated by Sirt1. Inhibition of Sirt1 is effective in suppression of ADM and in reducing cell viability in established PDAC tumors. KIAA1967 expression is differentially downregulated in PDAC and impacts on the sensitivity of PDAC cells to the Sirt1/2 inhibitor Tenovin-6. In PDAC, acetylation of β-catenin is not affected, unlike p53, a well-characterized Sirt1-regulated protein in tumor cells. Our results reveal that Sirt1 is an important regulator and potential therapeutic target in pancreatic carcinogenesis. ©2012 AACR.

  15. Emerging Role of Calcium-Activated Potassium Channel in the Regulation of Cell Viability Following Potassium Ions Challenge in HEK293 Cells and Pharmacological Modulation

    Science.gov (United States)

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

    2013-01-01

    Emerging evidences suggest that Ca2+activated-K+-(BK) channel is involved in the regulation of cell viability. The changes of the cell viability observed under hyperkalemia (15 mEq/L) or hypokalemia (0.55 mEq/L) conditions were investigated in HEK293 cells expressing the hslo subunit (hslo-HEK293) in the presence or absence of BK channel modulators. The BK channel openers(10-11-10-3M) were: acetazolamide(ACTZ), Dichlorphenamide(DCP), methazolamide(MTZ), bendroflumethiazide(BFT), ethoxzolamide(ETX), hydrochlorthiazide(HCT), quercetin(QUERC), resveratrol(RESV) and NS1619; and the BK channel blockers(2x10-7M-5x10-3M) were: tetraethylammonium(TEA), iberiotoxin(IbTx) and charybdotoxin(ChTX). Experiments on cell viability and channel currents were performed using cell counting kit-8 and patch-clamp techniques, respectively. Hslo whole-cell current was potentiated by BK channel openers with different potency and efficacy in hslo-HEK293. The efficacy ranking of the openers at -60 mV(Vm) was BFT> ACTZ >DCP ≥RESV≥ ETX> NS1619> MTZ≥ QUERC; HCT was not effective. Cell viability after 24 h of incubation under hyperkalemia was enhanced by 82+6% and 33+7% in hslo-HEK293 cells and HEK293 cells, respectively. IbTx, ChTX and TEA enhanced cell viability in hslo-HEK293. BK openers prevented the enhancement of the cell viability induced by hyperkalemia or IbTx in hslo-HEK293 showing an efficacy which was comparable with that observed as BK openers. BK channel modulators failed to affect cell currents and viability under hyperkalemia conditions in the absence of hslo subunit. In contrast, under hypokalemia cell viability was reduced by -22+4% and -23+6% in hslo-HEK293 and HEK293 cells, respectively; the BK channel modulators failed to affect this parameter in these cells. In conclusion, BK channel regulates cell viability under hyperkalemia but not hypokalemia conditions. BFT and ACTZ were the most potent drugs either in activating the BK current and in preventing the cell

  16. Sevoflurane anesthesia induces apoptosis and cell cycle arrest in ...

    African Journals Online (AJOL)

    Purpose: To determine the effects of sevoflurane on NPC-039 nasopharyngeal carcinoma cells. Methods: WST-8 assays and flow cytometry with annexin V/PI staining were used to analyze the effects of sevoflurane on growth and induction of apoptotic changes in NPC-039 cells. Cell viability was performed on a microplate ...

  17. Evaluation of viability and proliferative activity of human urothelial cells cultured onto xenogenic tissue-engineered extracellular matrices.

    LENUS (Irish Health Repository)

    Davis, Niall F

    2011-04-01

    To evaluate the viability and proliferative activity of human urothelial cells (HUCs) cultured on tissue-engineered extracellular matrix scaffolds and to assess the potential of extracellular matrixes to support the growth of HUCs in their expected in vivo urine environment.

  18. Downregulation of the non-integrin laminin receptor reduces cellular viability by inducing apoptosis in lung and cervical cancer cells.

    Directory of Open Access Journals (Sweden)

    Kiashanee Moodley

    Full Text Available The non-integrin laminin receptor, here designated the 37-kDa/67-kDa laminin receptor (LRP/LR, is involved in many physiologically relevant processes, as well as numerous pathological conditions. The overexpression of LRP/LR on various cancerous cell lines plays critical roles in tumour metastasis and angiogenesis. This study investigated whether LRP/LR is implicated in the maintenance of cellular viability in lung and cervical cancer cell lines. Here we show a significant reduction in cellular viability in the aforementioned cell lines as a result of the siRNA-mediated downregulation of LRP. This reduction in cellular viability is due to increased apoptotic processes, reflected by the loss of nuclear integrity and the significant increase in the activity of caspase-3. These results indicate that LRP/LR is involved in the maintenance of cellular viability in tumorigenic lung and cervix uteri cells through the blockage of apoptosis. Knockdown of LRP/LR by siRNA might represent an alternative therapeutic strategy for the treatment of lung and cervical cancer.

  19. Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

    Directory of Open Access Journals (Sweden)

    Yomo Tetsuya

    2006-06-01

    Full Text Available Abstract Background Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. Results We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea and side scatter (SSheight and SSarea. The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases

  20. The effects of vibration loading on adipose stem cell number, viability and differentiation towards bone-forming cells.

    Science.gov (United States)

    Tirkkonen, Laura; Halonen, Heidi; Hyttinen, Jari; Kuokkanen, Hannu; Sievänen, Harri; Koivisto, Anna-Maija; Mannerström, Bettina; Sándor, George K B; Suuronen, Riitta; Miettinen, Susanna; Haimi, Suvi

    2011-12-07

    Mechanical stimulation is an essential factor affecting the metabolism of bone cells and their precursors. We hypothesized that vibration loading would stimulate differentiation of human adipose stem cells (hASCs) towards bone-forming cells and simultaneously inhibit differentiation towards fat tissue. We developed a vibration-loading device that produces 3g peak acceleration at frequencies of 50 and 100 Hz to cells cultured on well plates. hASCs were cultured using either basal medium (BM), osteogenic medium (OM) or adipogenic medium (AM), and subjected to vibration loading for 3 h d(-1) for 1, 7 and 14 day. Osteogenesis, i.e. differentiation of hASCs towards bone-forming cells, was analysed using markers such as alkaline phosphatase (ALP) activity, collagen production and mineralization. Both 50 and 100 Hz vibration frequencies induced significantly increased ALP activity and collagen production of hASCs compared with the static control at 14 day in OM. A similar trend was detected for mineralization, but the increase was not statistically significant. Furthermore, vibration loading inhibited adipocyte differentiation of hASCs. Vibration did not affect cell number or viability. These findings suggest that osteogenic culture conditions amplify the stimulatory effect of vibration loading on differentiation of hASCs towards bone-forming cells.

  1. Effect of sodium butyrate on cell proliferation and cell cycle in porcine intestinal epithelial (IPEC-J2) cells.

    Science.gov (United States)

    Qiu, Yueqin; Ma, Xianyong; Yang, Xuefen; Wang, Li; Jiang, Zongyong

    2017-04-01

    Conflicting results have been reported that butyrate in normal piglets leads either to an increase or to a decrease of jejunal villus length, implying a possible effect on the proliferation of enterocytes. No definitive study was found for the biological effects of butyrate in porcine jejunal epithelial cells. The present study used IPEC-J2 cells, a non-transformed jejunal epithelial line to evaluate the direct effects of sodium butyrate on cell proliferation, cell cycle regulation, and apoptosis. Low concentrations (0.5 and 1 mM) of butyrate had no effect on cell proliferation. However, at 5 and 10 mM, sodium butyrate significantly decreased cell viability, accompanied by reduced levels of p-mTOR and PCNA protein. Sodium butyrate, in a dose-dependent manner, induced cell cycle arrest in G0/G1 phase and reduced the numbers of cells in S phase. In addition, relative expression of p21, p27, and pro-apoptosis bak genes, and protein levels of p21Waf1/Cip1, p27Kip1, cyclinD3, CDK4, and Cleave-caspase3 were increased by higher concentrations of sodium butyrate (1, 5, 10 mM), and the levels of cyclinD1 and CDK6 were reduced by 5 and 10 mM butyrate. Butyrate increased the phosphorylated form of the signaling molecule p38 and phosphorylated JNK. In conclusion, the present in vitro study indicated that sodium butyrate inhibited the proliferation of IPEC-J2 cells by inducing cell cycle arrest in the G0/G1 phase of cell cycles and by increasing apoptosis at high concentrations.

  2. MiR-15a Decreases Bovine Mammary Epithelial Cell Viability and Lactation and Regulates Growth Hormone Receptor Expression

    Directory of Open Access Journals (Sweden)

    Xue-Jun Gao

    2012-10-01

    Full Text Available MicroRNAs (miRNAs are a class of small non-coding RNAs that regulate the expression of target genes at the post-transcriptional level by transcript degradation or translational inhibition. The role of bta-miR-15a in bovine mammary gland hasn’t been reported. Using miRNAs prediction software, GHR gene was predicted to be a potential target of bta-miR-15a. In this study, bovine mammary epithelial cell line was used as an in vitro cell model to address the function of bta-miR-15a on bovine mammary epithelial cells. The expression changes of bta-miR-15a and Ghr after bta-miR-15a transfection were detected by qRT-PCR; the expression of GHR protein and casein was detected by western blotting. To determine whether bta-miR-15a can affect cell viability, cells were examined using an electronic Coulter counter (CASY-TT. In conclusion, bta-miR-15a inhibited the expression of casein of bovine mammary epithelial cells, and cell number and viability were reduced by bta-miR-15a expression. Bta-miR-15a inhibited the viability of mammary epithelial cells as well as the expression of GHR mRNA and protein level, therefore suggesting that bta-miR-15a may play an important role in mammary gland physiology.

  3. A simple method to measure cell viability in proliferation and cytotoxicity assays.

    Science.gov (United States)

    Borra, Ricardo Carneiro; Lotufo, Mônica Andrade; Gagioti, Sonia Maria; Barros, Fabiana de Mesquita; Andrade, Priscila Maria

    2009-01-01

    Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 etam and resorufin at 570 etam wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 etam) and green (500 to 600 etam) light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r(2) = 0.996; p < 0.01) and with the cellular concentrations (r(2) = 0.965; p < 0.01). We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

  4. Glycerol Monolaurate Inhibits Lipase Production by Clinical Ocular Isolates Without Affecting Bacterial Cell Viability.

    Science.gov (United States)

    Flanagan, Judith Louise; Khandekar, Neeta; Zhu, Hua; Watanabe, Keizo; Markoulli, Maria; Flanagan, John Terence; Papas, Eric

    2016-02-01

    We sought to determine the relative lipase production of a range of ocular bacterial isolates and to assess the efficacy of glycerol monolaurate (GML) in inhibiting this lipase production in high lipase-producing bacteria without affecting bacterial cell growth. Staphylococcus aureus,Staphylococcus epidermidis,Propionibacterium acnes, and Corynebacterium spp. were inoculated at a density of 10(6)/mL in varying concentrations of GML up to 25 μg/mL for 24 hours at 37 °C with constant shaking. Bacterial suspensions were centrifuged, bacterial cell density was determined, and production of bacterial lipase was quantified using a commercial lipase assay kit. Staphylococcus spp. produced high levels of lipase activity compared with P. acnes and Corynebacterium spp. GML inhibited lipase production by Staphylococcal spp. in a dose-dependent manner, with S. epidermidis lipase production consistently more sensitive to GML than S. aureus. Glycerol monolaurate showed significant (P lipase inhibition above concentrations of 15 μg/mL in S. aureus and was not cytotoxic up to 25 μg/mL. For S. epidermidis, GML showed significant (P lipase inhibition above 7.5 μg/mL. Lipase activity varied between species and between strains. Staphylococcal spp. produced higher lipase activity compared with P. acnes and Corynebacterium spp. Glycerol monolaurate inhibited lipase production by S. aureus and S. epidermidis at concentrations that did not adversely affect bacterial cell growth. GML can be used to inhibit ocular bacterial lipase production without proving detrimental to commensal bacteria viability.

  5. A simple method to measure cell viability in proliferation and cytotoxicity assays

    Directory of Open Access Journals (Sweden)

    Ricardo Carneiro Borra

    2009-09-01

    Full Text Available Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 ηm and resorufin at 570 ηm wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 ηm and green (500 to 600 ηm light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r² = 0.996; p < 0.01 and with the cellular concentrations (r² = 0.965; p < 0.01. We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

  6. Cinnamomum cassia essential oil and its major constituent cinnamaldehyde induced cell cycle arrest and apoptosis in human oral squamous cell carcinoma HSC-3 cells.

    Science.gov (United States)

    Chang, Wen-Lun; Cheng, Fu-Chou; Wang, Shu-Ping; Chou, Su-Tze; Shih, Ying

    2017-02-01

    Cinnamomum cassia essential oil (CC-EO) has various functional properties, such as anti-microbial, hypouricemic, anti-tyrosinase and anti-melanogenesis activities. The present study aimed to evaluate the anti-cancer activities of CC-EO and its major constituent, cinnamaldehyde, in human oral squamous cell carcinoma HSC-3 cells. Determination of the cell viability, apoptotic characteristics, DNA damage, cell cycle analysis, reactive oxygen species (ROS) production, mitochondrial membrane potential, cytosolic Ca(2+) level and intracellular redox status were performed. Our results demonstrated that CC-EO and cinnamaldehyde significantly decreased cell viability and caused morphological changes. The cell cycle analysis revealed that CC-EO and cinnamaldehyde induced G2/M cell cycle arrest in HSC-3 cells. The apoptotic characteristics (DNA laddering and chromatin condensation) and DNA damage were observed in the CC-EO-treated and cinnamaldehyde-treated HSC-3 cells. Moreover, CC-EO and cinnamaldehyde promoted an increase in cytosolic Ca(2+) levels, induced mitochondrial dysfunction and activated cytochrome c release. The results of ROS production and intracellular redox status demonstrated that CC-EO and cinnamaldehyde significantly increased the ROS production and thiobarbituric acid reactive substance levels, and the cellular glutathione content and glutathione peroxidase activity were significantly reduced in HSC-3 cells. Our results suggest that CC-EO and cinnamaldehyde may possess anti-oral cancer activity in HSC-3 cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 456-468, 2017. © 2016 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Arteaga-Cardona, Fernando [Universidad de las Américas de Puebla, Departamento de Ciencias Químico-Biológicas (Mexico); Gutiérrez-García, Eric [Instituto Literario, Universidad Autónoma del Estado de México (Mexico); Hidalgo-Tobón, Silvia, E-mail: shid@xanum.uam.mx [Universidad Autónoma Metropolitana, Departamento de Física (Mexico); López-Vasquez, Ciro; Brito-Barrera, Yazmín A. [Universidad de las Américas de Puebla, Departamento de Ciencias Químico-Biológicas (Mexico); Flores-Tochihuitl, Julia [Benemérita Universidad Autónoma de Puebla, Facultad de Estomatología (Mexico); Angulo-Molina, Aracely [Universidad de Sonora, Departamento de Ciencias Químico-Biológicas (Mexico); Reyes-Leyva, Julio R. [Instituto Mexicano del Seguro Social, Centro de Investigación Biomédica de Oriente (CIBIOR) (Mexico); González-Rodríguez, Roberto; Coffer, Jeffery L. [Texas Christian University, Department of Chemistry (United States); Pal, Umapada [Benemérita Universidad Autónoma de Puebla, Apdo, Instituto de Física (Mexico); and others

    2016-11-15

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

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

    Directory of Open Access Journals (Sweden)

    Jennifer Munkley

    2016-06-01

    Full Text Available 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 LewisX (SLeX, 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.

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

    Science.gov (United States)

    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

  11. ATR Kinase Inhibition Protects Non-cycling Cells from the Lethal Effects of DNA Damage and Transcription Stress*

    Science.gov (United States)

    Kemp, Michael G.; Sancar, Aziz

    2016-01-01

    ATR (ataxia telangiectasia and Rad-3-related) is a protein kinase that maintains genome stability and halts cell cycle phase transitions in response to DNA lesions that block DNA polymerase movement. These DNA replication-associated features of ATR function have led to the emergence of ATR kinase inhibitors as potential adjuvants for DNA-damaging cancer chemotherapeutics. However, whether ATR affects the genotoxic stress response in non-replicating, non-cycling cells is currently unknown. We therefore used chemical inhibition of ATR kinase activity to examine the role of ATR in quiescent human cells. Although ATR inhibition had no obvious effects on the viability of non-cycling cells, inhibition of ATR partially protected non-replicating cells from the lethal effects of UV and UV mimetics. Analyses of various DNA damage response signaling pathways demonstrated that ATR inhibition reduced the activation of apoptotic signaling by these agents in non-cycling cells. The pro-apoptosis/cell death function of ATR is likely due to transcription stress because the lethal effects of compounds that block RNA polymerase movement were reduced in the presence of an ATR inhibitor. These results therefore suggest that whereas DNA polymerase stalling at DNA lesions activates ATR to protect cell viability and prevent apoptosis, the stalling of RNA polymerases instead activates ATR to induce an apoptotic form of cell death in non-cycling cells. These results have important implications regarding the use of ATR inhibitors in cancer chemotherapy regimens. PMID:26940878

  12. TLR4- and TLR9-dependent effects on cytokines, cell viability, and invasion in human bladder cancer cells.

    Science.gov (United States)

    Olbert, Peter J; Kesch, Claudia; Henrici, Marcus; Subtil, Florentine S; Honacker, Astrid; Hegele, Axel; Hofmann, Rainer; Hänze, Jörg

    2015-03-01

    Adjuvant immunotherapy of bladder cancer by instillation of bacillus Calmette-Guérin (BCG) is highly recommended within certain groups of non-muscle-invasive stages but only partially effective. Toll-like receptors (TLRs) TLR4 and TLR9 likely mediate BCG effects by triggering innate systemic immune cell responses. In addition, TLR4 and TLR9 expressed in bladder cancer cells may contribute to the outcome of BCG treatment. Here, we studied the expression and function of TLR4 and TLR9 in human bladder cancer cell lines. TLR4 and TLR9 messenger RNA and protein levels were determined by real-time reverse transcription polymerase chain reaction and Western blot. Selected cell lines were analyzed with respect to cytokine induction, proliferation, and cell invasion after addition of BCG, TLR4-specific agonist lipopolysaccharide (LPS), or TLR9 agonist (CpG-oligodeoxynucleotide [ODN]). TLR4 and TLR9 were expressed quite heterogeneously in human bladder cancer cells. BCG caused induction of interleukin (IL)-6 or IL-8 in BFTC905 and T24 cells as representatives for TLR4-/TLR9-expressing cells. The study aimed to dissect TLR4- and TLR9-mediated effects. For functional analysis of TLR4 with LPS, we selected T24 and BFTC905 cells with high and undetectable TLR4 levels, respectively. For TLR9 analysis with CpG-ODN, we selected UMUC3 and RT112 cells with high and low TLR9 levels, respectively. Addition of LPS caused significant induction of TNFα and IL-6 messenger RNA in T24 cells but not in BFTC905 cells. Addition of CpG-ODN induced interferon ß (INFß), IL-8, tumor necrosis factor α (TNFα) and the angiogenic factors vascular endothelial growth factor-A and placental growth factor in UMUC3 cells; whereas in RT112 cells, induction of IL-8 and TNFα was noticed. Interestingly, addition of CpG-ODN significantly reduced cell viability and increased cell invasion in UMUC3 and RT112 cells. Our findings demonstrate that bladder cancer cell lines express functional TLR4 and TLR9 with

  13. Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID.

    Directory of Open Access Journals (Sweden)

    Quy Le

    2015-09-01

    Full Text Available AID (Activation Induced Deaminase deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.

  14. Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing.

    Science.gov (United States)

    Ng, Wei Long; Yeong, Wai Yee; Naing, May Win

    2017-02-16

    Drop-on-demand (DOD) bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP) macromolecules. Different PVP-based bio-inks (0%-3% w/v) were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh), which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v). Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process.

  15. Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing

    Science.gov (United States)

    Ng, Wei Long; Yeong, Wai Yee; Naing, May Win

    2017-01-01

    Drop-on-demand (DOD) bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP) macromolecules. Different PVP-based bio-inks (0%–3% w/v) were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh), which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v). Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process. PMID:28772551

  16. Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing

    Directory of Open Access Journals (Sweden)

    Wei Long Ng

    2017-02-01

    Full Text Available Drop-on-demand (DOD bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP macromolecules. Different PVP-based bio-inks (0%–3% w/v were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh, which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v. Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process.

  17. Effect of photobiomodulation on viability and proliferation of stem cells from exfoliated deciduous teeth under different nutritional conditions

    Science.gov (United States)

    Morato de Souza, Letícia; Guilherme Roque Rinco, Ugo; Aparecida Tavares Aguiar, Daniela; Aparecido de Almeida Junior, Luciano; Cosme-Silva, Leopoldo; Marchini Oliveira, Thais; Teixeira Marques, Nádia Carolina; Thiemy Sakai, Vivien

    2018-02-01

    This study aimed to evaluate the effect of different doses of low-level laser irradiation on the viability and proliferation of stem cells from exfoliated deciduous teeth (SHED) cultured under nutritional deficit (cellular stress) or regular nutritional conditions. SHED underwent irradiation by a red laser between 1.2 and 6.2 J cm‑2. Prior to the irradiation, all groups received culture medium (MEMα, Eagle’s minimum essential medium alpha modification) supplemented with 1% of fetal bovine serum (FBS) for 1 h. After the irradiation, cells received MEMα supplemented with 10% of FBS (regular nutrition) or 1% of FBS (nutritional deficit). Cell viability and proliferation were respectively determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assays 6 and 24 h after irradiation (P  nutritional deficit showed lower viability and proliferation after 1.2 J cm‑2 irradiation. All of the irradiated groups revealed significantly higher viability and proliferation in SHED maintained under nutritional deficit than in regular nutritional conditions, except in the 3.7 and 6.2 J cm‑2 groups by MTT assay. In the crystal violet assay, SHED irradiated with 1.2 J cm‑2 showed no difference between the different nutritional conditions. Decrease of FBS concentration in the culture medium seems to enhance the sensitivity of SHED to the effects of photobiomodulation therapy. Nutritional stress conditions improved cell viability and proliferation of SHED after laser irradiation, except for 1.2 J cm‑2.

  18. Icarisid II inhibits the proliferation of human osteosarcoma cells by inducing apoptosis and cell cycle arrest.

    Science.gov (United States)

    Tang, Yuanyuan; Xie, Mao; Jiang, Neng; Huang, Feifei; Zhang, Xiao; Li, Ruishan; Lu, Jingjing; Liao, Shijie; Liu, Yun

    2017-06-01

    Icarisid II, one of the main active components of Herba Epimedii extracts, shows potent antitumor activity in various cancer cell lines, including osteosarcoma cells. However, the anticancer mechanism of icarisid II against osteosarcoma U2OS needs further exploration. This study aims to investigate further antitumor effects of icarisid II on human osteosarcoma cells and elucidate the underlying mechanism. We cultivated human osteosarcoma USO2 cells in vitro using different concentrations of icarisid II (0-30 µM). Cell viability was detected at 24, 48, and 72 h using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis. Cell cycle was tested by flow cytometry after treatment with icarisid II for 48 h. Annexin V-allophycocyanin and 7-aminoactinomycin D staining were conducted to detect cell apoptosis. Quantitative real-time polymerase chain reaction and Western blot assay were performed to measure the levels of genes and proteins related to cell cycle and apoptosis. Results showed that icarisid II significantly inhibited the proliferation and induced apoptosis of human osteosarcoma U2OS cells. The half maximal inhibitory concentration values were 14.44, 11.02, and 7.37 µM at 24, 48, and 72 h, respectively. Cell cycle was arrested in the G2/M phase in vitro. In addition, icarisid II upregulated the expression levels of P21 and CyclinB1 whereas downregulated the expression levels of CyclinD1, CDC2, and P-Cdc25C, which were related to cell cycle arrest in U2OS cells. The cell apoptotic rate increased in a dose-dependent manner after treatment with icarisid II for 48 h. Icarisid II induced apoptosis by upregulating Bax, downregulating Bcl-2, and activating apoptosis-related proteins, including cleaved caspase-3, caspase-7, caspase-9, and poly (ADP-ribose) polymerase. These data indicate that icarisid II exhibits an antiproliferation effect on human osteosarcoma cells and induces apoptosis by activating the caspase family in a time- and dose

  19. Viability Theory

    CERN Document Server

    Aubin, Jean-Pierre; Saint-Pierre, Patrick

    2011-01-01

    Viability theory designs and develops mathematical and algorithmic methods for investigating the adaptation to viability constraints of evolutions governed by complex systems under uncertainty that are found in many domains involving living beings, from biological evolution to economics, from environmental sciences to financial markets, from control theory and robotics to cognitive sciences. It involves interdisciplinary investigations spanning fields that have traditionally developed in isolation. The purpose of this book is to present an initiation to applications of viability theory, explai

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. A comparison of five treatment protocols for contaminated bone grafts in reference to sterility and cell viability.

    Science.gov (United States)

    Bauer, Jennifer; Liu, Raymond W; Kean, Thomas J; Dennis, James E; Petersilge, William; Gilmore, Allison

    2011-03-02

    Occasionally, a bone graft or comminuted fracture fragment is dropped on the operating-room floor and becomes contaminated. The purpose of this study was to determine an optimal method for sterilizing this bone with the minimum sacrifice of cell viability. A set of discarded bone samples was taken from a series of twenty total knee arthroplasty operations. The bone samples were uniformly contaminated with use of a bacterial broth prepared from culture samples taken from the operating-room floor. The bone samples in each set underwent five different decontamination procedures. Specifically, one sample in each set was autoclaved and four other samples underwent mechanical agitation in normal saline solution, 2% chlorhexidine gluconate, or 10% povidone-iodine (which was either left wet or was dried). Positive and negative controls were used for comparison. Ten sets were then cultured to determine sterility, and ten underwent live/dead trypan blue staining to determine cell viability. Autoclaving, chlorhexidine gluconate, and dry povidone-iodine sterilized all samples; wet povidone-iodine decontaminated four (40%) of ten samples; and saline solution sterilized none. While all decontamination methods reduced the cell count to some extent, autoclaving and chlorhexidine gluconate left no viable cells. When the cell counts were expressed as a percentage of the control value, dry povidone-iodine sterilization maintained significantly fewer live cells than controls (21%; p bone graft in povidone-iodine that is allowed to dry offers the best balance between complete sterilization of contaminated bone and maintenance of tissue viability.

  2. Spiclomazine induces apoptosis associated with the suppression of cell viability, migration and invasion in pancreatic carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Wenjing Zhao

    Full Text Available 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.

  3. Addition of Wollastonite Fibers to Calcium Phosphate Cement Increases Cell Viability and Stimulates Differentiation of Osteoblast-Like Cells

    Directory of Open Access Journals (Sweden)

    Juliana Almeida Domingues

    2017-01-01

    Full Text Available Calcium phosphate cement (CPC that is based on α-tricalcium phosphate (α-TCP is considered desirable for bone tissue engineering because of its relatively rapid degradation properties. However, such cement is relatively weak, restricting its use to areas of low mechanical stress. Wollastonite fibers (WF have been used to improve the mechanical strength of biomaterials. However, the biological properties of WF remain poorly understood. Here, we tested the response of osteoblast-like cells to being cultured on CPC reinforced with 5% of WF (CPC-WF. We found that both types of cement studied achieved an ion balance for calcium and phosphate after 3 days of immersion in culture medium and this allowed subsequent long-term cell culture. CPC-WF increased cell viability and stimulated cell differentiation, compared to nonreinforced CPC. We hypothesize that late silicon release by CPC-WF induces increased cell proliferation and differentiation. Based on our findings, we propose that CPC-WF is a promising material for bone tissue engineering applications.

  4. Molecular Mechanisms by Which a Fucus vesiculosus Extract Mediates Cell Cycle Inhibition and Cell Death in Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ulf Geisen

    2015-07-01

    Full Text Available Pancreatic cancer is one of the most aggressive cancer entities, with an extremely poor 5-year survival rate. Therefore, novel therapeutic agents with specific modes of action are urgently needed. Marine organisms represent a promising source to identify new pharmacologically active substances. Secondary metabolites derived from marine algae are of particular interest. The present work describes cellular and molecular mechanisms induced by an HPLC-fractionated, hydrophilic extract derived from the Baltic brown seaweed Fucus vesiculosus (Fv1. Treatment with Fv1 resulted in a strong inhibition of viability in various pancreatic cancer cell lines. This extract inhibited the cell cycle of proliferating cells due to the up-regulation of cell cycle inhibitors, shown on the mRNA (microarray data and protein level. As a result, cells were dying in a caspase-independent manner. Experiments with non-dividing cells showed that proliferation is a prerequisite for the effectiveness of Fv1. Importantly, Fv1 showed low cytotoxic activity against non-malignant resting T cells and terminally differentiated cells like erythrocytes. Interestingly, accelerated killing effects were observed in combination with inhibitors of autophagy. Our in vitro data suggest that Fv1 may represent a promising new agent that deserves further development towards clinical application.

  5. Molecular Mechanisms by Which a Fucus vesiculosus Extract Mediates Cell Cycle Inhibition and Cell Death in Pancreatic Cancer Cells.

    Science.gov (United States)

    Geisen, Ulf; Zenthoefer, Marion; Peipp, Matthias; Kerber, Jannik; Plenge, Johannes; Managò, Antonella; Fuhrmann, Markus; Geyer, Roland; Hennig, Steffen; Adam, Dieter; Piker, Levent; Rimbach, Gerald; Kalthoff, Holger

    2015-07-20

    Pancreatic cancer is one of the most aggressive cancer entities, with an extremely poor 5-year survival rate. Therefore, novel therapeutic agents with specific modes of action are urgently needed. Marine organisms represent a promising source to identify new pharmacologically active substances. Secondary metabolites derived from marine algae are of particular interest. The present work describes cellular and molecular mechanisms induced by an HPLC-fractionated, hydrophilic extract derived from the Baltic brown seaweed Fucus vesiculosus (Fv1). Treatment with Fv1 resulted in a strong inhibition of viability in various pancreatic cancer cell lines. This extract inhibited the cell cycle of proliferating cells due to the up-regulation of cell cycle inhibitors, shown on the mRNA (microarray data) and protein level. As a result, cells were dying in a caspase-independent manner. Experiments with non-dividing cells showed that proliferation is a prerequisite for the effectiveness of Fv1. Importantly, Fv1 showed low cytotoxic activity against non-malignant resting T cells and terminally differentiated cells like erythrocytes. Interestingly, accelerated killing effects were observed in combination with inhibitors of autophagy. Our in vitro data suggest that Fv1 may represent a promising new agent that deserves further development towards clinical application.

  6. The Efficiency of Methionine as a Radioprotectant of Bacillus anthracis for Cell Viability and Outgrowth Time after UVC and Gamma Irradiation

    Science.gov (United States)

    2015-03-01

    4. TITLE AND SUBTITLE The Efficiency of Methionine as a Radioprotectant of Bacillus anthracis for Cell Viability and Outgrowth Time after UVC and...THE EFFICIENCY OF METHIONINE AS A RADIOPROTECTANT OF BACILLUS ANTHRACIS FOR CELL VIABILITY AND...subject to copyright protection in the United States. AFIT-ENP-MS-15-M-105 THE EFFICIENCY OF METHIONINE AS A RADIOPROTECTANT OF BACILLUS

  7. Preliminary viability studies of fibroblastic cells cultured on microcrystalline and nanocrystalline diamonds produced by chemical vapour deposition method

    Directory of Open Access Journals (Sweden)

    Ana Amélia Rodrigues

    2013-02-01

    Full Text Available Implant materials used in orthopedics surgery have demonstrated some disadvantages, such as metallic corrosion processes, generation of wear particles, inflammation reactions and bone reabsorption in the implant region. The diamond produced through hot-filament chemical vapour deposition method is a new potential biomedical material due to its chemical inertness, extreme hardness and low coefficient of friction. In the present study we analysis two samples: the microcrystalline diamond and the nanocrystalline diamond. The aim of this study was to evaluate the surface properties of the diamond samples by scanning electron microscopy, Raman spectroscopy and atomic force microscopy. Cell viability and morphology were assessed using thiazolyl blue tetrazolium bromide, cytochemical assay and scanning electron microscopy, respectively. The results revealed that the two samples did not interfere in the cell viability, however the proliferation of fibroblasts cells observed was comparatively higher with the nanocrystalline diamond.

  8. Preliminary viability studies of fibroblastic cells cultured on microcrystalline and nanocrystalline diamonds produced by chemical vapour deposition method

    Directory of Open Access Journals (Sweden)

    Ana Amélia Rodrigues

    2012-01-01

    Full Text Available Implant materials used in orthopedics surgery have demonstrated some disadvantages, such as metallic corrosion processes, generation of wear particles, inflammation reactions and bone reabsorption in the implant region. The diamond produced through hot-filament chemical vapour deposition method is a new potential biomedical material due to its chemical inertness, extreme hardness and low coefficient of friction. In the present study we analysis two samples: the microcrystalline diamond and the nanocrystalline diamond. The aim of this study was to evaluate the surface properties of the diamond samples by scanning electron microscopy, Raman spectroscopy and atomic force microscopy. Cell viability and morphology were assessed using thiazolyl blue tetrazolium bromide, cytochemical assay and scanning electron microscopy, respectively. The results revealed that the two samples did not interfere in the cell viability, however the proliferation of fibroblasts cells observed was comparatively higher with the nanocrystalline diamond.

  9. Alteration of cell cycle progression by Sindbis virus infection

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ruirong; Saito, Kengo [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Isegawa, Naohisa [Laboratory Animal Center, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Shirasawa, Hiroshi, E-mail: sirasawa@faculty.chiba-u.jp [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan)

    2015-07-10

    We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G{sub 1} phase preferred to proliferate during S/G{sub 2} phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G{sub 1} phase than in cells infected during S/G{sub 2} phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases.

  10. Cisplatin and photodynamic therapy exert synergistic inhibitory effects on small-cell lung cancer cell viability and xenograft tumor growth.

    Science.gov (United States)

    Cheng, You-Shuang; Peng, Yin-Bo; Yao, Min; Teng, Ji-Ping; Ni, Da; Zhu, Zhi-Jun; Zhuang, Bu-Feng; Yang, Zhi-Yin

    2017-06-03

    Lung cancer is the leading cause of cancer death worldwide. Small-cell lung cancer (SCLC) is an aggressive type of lung cancer that shows an overall 5-year survival rate below 10%. Although chemotherapy using cisplatin has been proven effective in SCLC treatment, conventional dose of cisplatin causes adverse side effects. Photodynamic therapy, a form of non-ionizing radiation therapy, is increasingly used alone or in combination with other therapeutics in cancer treatment. Herein, we aimed to address whether low dose cisplatin combination with PDT can effectively induce SCLC cell death by using in vitro cultured human SCLC NCI-H446 cells and in vivo tumor xenograft model. We found that both cisplatin and PDT showed dose-dependent cytotoxic effects in NCI-H446 cells. Importantly, co-treatment with low dose cisplatin (1 μM) and PDT (1.25 J/cm2) synergistically inhibited cell viability and cell migration. We further showed that the combined therapy induced a higher level of intracellular ROS in cultured NCI-H446 cells. Moreover, the synergistic effect by cisplatin and PDT was recapitulated in tumor xenograft as revealed by a more robust increase in the staining of TUNEL (a marker of cell death) and decrease in tumor volume. Taken together, our findings suggest that low dose cisplatin combination with PDT can be an effective therapeutic modality in the treatment of SCLC patients. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Caroline Calloni

    2016-03-01

    Full Text Available 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.

  12. Low Concentration of Sodium Nitroprusside Promotes Mesenchymal Stem Cell Viability and Proliferation Through Elevation of Metabolic Activity

    Directory of Open Access Journals (Sweden)

    A.Mohammadi

    2017-06-01

    Full Text Available Background: Sodium nitroprusside (SNP releases nitric oxide which has signaling role. Objectives: This study was conducted to understand the role of low concentration of SNP on viability, proliferation and biochemical properties of rat bone marrow mesenchymal stem cells (MSCs. Materials and Methods: MSCs were used to evaluate the viability and morphology in presence of SNP (1 to 100 µM at 12, 24 and 36 hours. Then 10, 50 and 100 µM of SNP as well as 24 hours were selected for further study. Cell proliferation was investigated by colony forming assay and population doubling number (PDN. Calcium (Ca2+ potassium (K+ and sodium (Na+ level as well as activity of alanine transaminase (ALT, aspartate transaminase (AST, alkaline phosphatase (ALP and lactate dehydrogenase (LDH were measured. Results: The MSCs viability increased when treatment with 1 and 10 µM at all the treatment periods while 90 and 100 µM caused significant reduction after 24 and 36 hours. Also 10 µM caused elevation whereas 50 and 100 µM showed reduction of proliferation ability. We observed morphological changes and significant reduction of all the investigated enzymes with 100 µM. Activity of ALT and AST were elevated with 10 µM after 24 hours, whereas LDH and ALP activities were not changed. Na+, K+ and Ca2+ was not changed due to 10 and 50 µM treatments, whereas 100 µM only elevated the level of calcium and sodium ions. Conclusions: Low concentration of SNP caused increase of viability and proliferation due to metabolic activity elevation. But the high concentration of SNP induced cell viability and proliferation reduction caused by metabolic and ionic imbalance as well as infrastructure alteration.

  13. Effect of human autologous serum and fetal bovine serum on human corneal epithelial cell viability, migration and proliferation in vitro

    Directory of Open Access Journals (Sweden)

    Ming-Feng Wu

    2017-06-01

    Full Text Available AIM: To analyze the concentration-dependent effects of autologous serum (AS and fetal bovine serum (FBS on human corneal epithelial cell (HCEC viability, migration and proliferation. METHODS: AS was prepared from 13 patients with non-healing epithelial defects Dulbecco's modified eagle medium/Ham’s F12 (DMEM/F12 with 5% FBS, 0.5% dimethyl sulphoxide (DMSO, 10 ng/mL human epidermal growth factor, 1% insulin-transferrin-selenium, then were incubated in serum media: DMEM/F12 supplemented by 5%, 10%, 15% or 30% AS or FBS. HCEC viability was analyzed using cell proliferation kit XTT, migration using a wound healing assay, proliferation by the cell proliferation enzyme-linked immunosorbent assay (ELISA BrdU kit. Statistical analysis was performed using the generalized linear model, the values at 30% AS or 30% FBS were used as the baselines. RESULTS: HCEC viability was the highest at 30% AS or 15% FBS and the lowest at 10% AS or 30% FBS application. HCEC migration was the quickest through 30% AS or 30% FBS and the slowest through 5% AS or 5% FBS concentrations. Proliferation was the most increased through 15% AS or 5% FBS and the least increased through 30% AS or 30% FBS concentrations. HCEC viability at 10% and 15% AS was significantly worse (P=0.001, P=0.023 compared to baseline and significantly better at 15% FBS (P=0.003 concentrations. HCEC migration was significantly worse (P≤0.007 and HCEC proliferation significantly better (P<0.001 in all concentration groups compared to baseline. CONCLUSION: For the best viability of HCEC 30% AS or 15% FBS, for HCEC migration 30% AS or 30% FBS, for proliferation 15% AS or 5% FBS should be used. Therefore, we suggest the use of 30% AS in clinical practice.

  14. Influence of Arsenic (III, Cadmium (II, Chromium (VI, Mercury (II, and Lead (II Ions on Human Triple Negative Breast Cancer (HCC1806 Cell Cytotoxicity and Cell Viability

    Directory of Open Access Journals (Sweden)

    Tsdale F. Mehari

    2017-01-01

    Full Text Available The hazardous consequences of heavy metal ions (HMIs on human health necessitate the immediate need to probe fundamentally the interactions and cytotoxic effects of HMIs on humans. This study investigated the influence of five toxic HMIs (arsenic (As (III, cadmium (Cd (II, chromium (Cr (VI, mercury (Hg (II, and lead (Pb (II on human TNBC (HCC 1806 cell viability using optical microscopy, trypan blue dye-exclusion assays, and flow cytometry. The TNBC cells were exposed to varying concentrations of HMIs for 24 and 48 hours. We evaluated the influence of the concentrations and duration of HMIs exposure on TNBC cell viability. Light microscopy, cell viability assays, revealed that after 48-hour treatment of TNBC cells with 1 x 10-5 M of As (III, Cd (II, Hg (II, Cr (IV, and Pb (II resulted in cell viabilities of 23%, 34%, 35%, 56%, 91% respectively, suggesting that As (III has the greatest cytotoxicity (77% cell death while Pb (II showed the least (9% cell death. Furthermore, flow cytometry revealed that while Pb (II, As (III and Cr (IV had significant increases in cell death, Hg (II caused a G1 arrest. Together, this study revealed that HMIs cause a differential cytotoxic effect on TNBC cells and suggest that they may have very different genotoxic targets and implications in their mutagenic potential.

  15. Silencing of mutant p53 by siRNA induces cell cycle arrest and apoptosis in human bladder cancer cells

    Directory of Open Access Journals (Sweden)

    Zhu Hai-Bin

    2013-01-01

    Full Text Available Abstract Background p53 is the most frequently mutated tumor-suppressor gene in human cancers. It has been reported that mutations in p53 result not only in the loss of its ability as a tumor suppressor, but also in the gain of novel cancer-related functions that contribute to oncogenesis. The present study evaluated the potential of silencing of mutant p53 by small interfering RNA in the treatment of bladder cancer cells in vitro. Methods We used the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay to assess cell viability and flow cytometry to detect cell cycle alterations and apoptosis. The related molecular mechanisms were assessed by western blotting. We also used the MTT assay and flow cytometry to investigate if silencing of mutant p53 by knockdown with small interfering (siRNA would change the sensitivity to cisplatin treatment. Results Using 5637 and T24 human bladder cancer cell lines characterized by mutations in p53, we found that silencing of the mutant p53 by RNA interference induced evident inhibition of cell proliferation and viability, which was related to the induction of G2 phase cell cycle arrest and apoptosis. Moreover, our study also showed that the p53-targeting siRNA cooperated with cisplatin in the inhibition of bladder cancer cells. Conclusions These findings suggest that RNA interference targeting mutant p53 may be a promising therapeutic strategy for the treatment of bladder cancer.

  16. Overexpression of Midkine promotes the viability of BA/F3 cells.

    Science.gov (United States)

    Wang, Yang; Xing, Haiyan; Tian, Zheng; Tang, Kejing; Wang, Jiying; Xu, Zhifang; Rao, Qing; Wang, Min; Wang, Jianxiang

    2009-07-03

    Midkine (MK), a heparin-binding growth factor, has been reported to be overexpressed in a variety of human solid tumors. In the previous study, we found that MK was overexpressed in bone marrow samples derived from acute leukemia (AL) patients. To elucidate the role of MK, we stably transfected MK in IL-3-dependent BA/F3 cells. The results indicated that the capacity of proliferation and colony formation was significantly increased in the MK-transfected subclones than in the empty vector-transfected subclones. MK potentiated proliferation of BA/F3 cells by promoting cell cycle progression. Apoptosis assays showed a remarkable reduction of apoptosis in MK expressing subclones. Exogenous MK could induce the phosphorylation of Raf-1, and inhibit the expression of Bax in BA/F3 cells. These results indicate that MK might be involved in the pathogenesis of leukemia and could be taken as an ideal diagnostic marker and molecular target for the treatment of acute leukemia.

  17. Encapsulation of factor IX–engineered mesenchymal stem cells in fibrinogen–alginate microcapsules enhances their viability and transgene secretion

    Directory of Open Access Journals (Sweden)

    Bahareh Sayyar

    2012-12-01

    Full Text Available Cell microencapsulation holds significant promise as a strategy for cellular therapies; however, inadequate survival and functionality of the enclosed cells limit its application in hemophilia treatment. Here, we evaluated the use of alginate-based microcapsules to enhance the viability and transgene secretion of human cord blood–derived mesenchymal stem cells in three-dimensional cultures. Given the positive effects of extracellular matrix molecules on mesenchymal stem cell growth, we tested whether fibrinogen-supplemented alginate microcapsules can improve the efficiency of encapsulated factor IX–engineered mesenchymal stem cells as a treatment of hemophilia B. We found that fibrinogen-supplemented alginate microcapsules (a significantly enhanced the viability and proliferation of factor IX–engineered mesenchymal stem cells and (b increased factor IX secretion by mesenchymal stem cells compared to mesenchymal stem cells in nonsupplemented microcapsules. Moreover, we observed the osteogenic, but not chondrogenic or adipogenic, differentiation capability of factor IX–engineered cord blood mesenchymal stem cells and their efficient factor IX secretion while encapsulated in fibrinogen-supplemented alginate microcapsules. Thus, the use of engineered mesenchymal stem cells encapsulated in fibrinogen-modified microcapsules may have potential application in the treatment of hemophilia or other protein deficiency diseases.

  18. Extracts of centipede Scolopendra subspinipes mutilans induce cell cycle arrest and apoptosis in A375 human melanoma cells.

    Science.gov (United States)

    Ma, Weina; Liu, Rui; Qi, Junpeng; Zhang, Yanmin

    2014-07-01

    Extracts from the centipede Scolopendra genus, have been used in traditional medicine for the treatment of various diseases and have been found to exhibit anticancer activity in tumor cells. To investigate the potential and associated antitumor mechanism of alcohol extracts of the centipede Scolopendra subspinipes mutilans (AECS), cell viability, cell cycle and cell apoptosis were studied and the results revealed that AECS inhibits A375 cell proliferation in a dose- and time-dependent manner. In addition, AECS was found to arrest the cell cycle of A375 cells at the S phase, which was accompanied by a marked increase in the protein levels of cyclin E and a decrease in the protein levels of cyclin D1. In a cell culture system, AECS markedly induced the apoptosis of A375 cells, which was closely associated with the effects on the Bcl-2 family, whereby decreased Bcl-2 and increased Bak, Bax and Bad expression levels were observed. The underlying mechanism of AECS inhibiting A375 cell proliferation was associated with the induction of cell cycle arrest and apoptosis, indicating that AECS may present as a potential therapeutic agent for administration in human melanoma cancer intervention.

  19. P27 in cell cycle control and cancer

    DEFF Research Database (Denmark)

    Møller, Michael Boe

    2000-01-01

    In order to survive, cells need tight control of cell cycle progression. The control mechanisms are often lost in human cancer cells. The cell cycle is driven forward by cyclin-dependent kinases (CDKs). The CDK inhibitors (CKIs) are important regulators of the CDKs. As the name implies, CKIs were...

  20. δ-Catenin promotes prostate cancer cell growth and progression by altering cell cycle and survival gene profiles

    Directory of Open Access Journals (Sweden)

    Chen Yan-Hua

    2009-03-01

    Full Text Available Abstract Background δ-Catenin is a unique member of β-catenin/armadillo domain superfamily proteins and its primary expression is restricted to the brain. However, δ-catenin is upregulated in human prostatic adenocarcinomas, although the effects of δ-catenin overexpression in prostate cancer are unclear. We hypothesized that δ-catenin plays a direct role in prostate cancer progression by altering gene profiles of cell cycle regulation and cell survival. Results We employed gene transfection and small interfering RNA to demonstrate that increased δ-catenin expression promoted, whereas its knockdown suppressed prostate cancer cell viability. δ-Catenin promoted prostate cancer cell colony formation in soft agar as well as tumor xenograft growth in nude mice. Deletion of either the amino-terminal or carboxyl-terminal sequences outside the armadillo domains abolished the tumor promoting effects of δ-catenin. Quantitative RT2 Profiler™ PCR Arrays demonstrated gene alterations involved in cell cycle and survival regulation. δ-Catenin overexpression upregulated cyclin D1 and cdc34, increased phosphorylated histone-H3, and promoted the entry of mitosis. In addition, δ-catenin overexpression resulted in increased expression of cell survival genes Bcl-2 and survivin while reducing the cell cycle inhibitor p21Cip1. Conclusion Taken together, our studies suggest that at least one consequence of an increased expression of δ-catenin in human prostate cancer is the alteration of cell cycle and survival gene profiles, thereby promoting tumor progression.

  1. Analysis of Cell Cycle Switches in Drosophila Oogenesis.

    Science.gov (United States)

    Jia, Dongyu; Huang, Yi-Chun; Deng, Wu-Min

    2015-01-01

    The study of Drosophila oogenesis provides invaluable information about signaling pathway regulation and cell cycle programming. During Drosophila oogenesis, a string of egg chambers in each ovariole progressively develops toward maturity. Egg chamber development consists of 14 stages. From stage 1 to stage 6 (mitotic cycle), main-body follicle cells undergo mitotic divisions. From stage 7 to stage 10a (endocycle), follicle cells cease mitosis but continue three rounds of endoreduplication. From stage 10b to stage 13 (gene amplification), instead of whole genome duplication, follicle cells selectively amplify specific genomic regions, mostly for chorion production. So far, Drosophila oogenesis is one of the most well studied model systems used to understand cell cycle switches, which furthers our knowledge about cell cycle control machinery and sheds new light on potential cancer treatments. Here, we give a brief summary of cell cycle switches, the associated signaling pathways and factors, and the detailed experimental procedures used to study the cell cycle switches.

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

    Directory of Open Access Journals (Sweden)

    Leonardo Romorini

    Full Text Available 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.

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

    Science.gov (United States)

    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

  4. Effectiveness of helium-neon laser irradiation on viability and cytotoxicity of diabetic-wounded fibroblast cells.

    Science.gov (United States)

    Houreld, N N; Abrahamse, H

    2007-12-01

    This study investigated the effectiveness of helium-neon (He-Ne) laser irradiation at increasing intervals on diabetic-induced wounded human skin fibroblast cells (WS1) at a morphological, cellular, and molecular level. The controversies over light therapy can be explained by the differing exposure regimens and models used. No therapeutic window for dosimetry and mechanism of action has been determined at the level of individual cell types, particularly in diabetic cells in vitro. WS1 cells were used to simulate an in vitro wounded diabetic model. The effect of the frequency of He-Ne irradiation (632.8 nm) at a fluence of 5 J/cm(2) was determined by analysis of cell morphology, viability, cytotoxicity, and DNA damage. Cells were irradiated using three different protocols: they were irradiated at 30 min only; irradiated twice, at 30 min and at 24 h; or irradiated twice, at 30 min and at 72 h post-wound induction. A single exposure to 5 J/cm(2) 30 min post-wound induction increased cellular damage. Irradiation of cells at 30 min and at 24 h post-wound induction decreased cellular viability, cytotoxicity, and DNA damage. However, complete wound closure as well as an increase in viability and a decrease in cytotoxicity and DNA damage occurs when cells were irradiated at 30 min and at 72 h post-wound induction. Wounded diabetic WS1 cells irradiated to 5 J/cm(2) showed increased cellular repair when irradiated with adequate time between irradiations, allowing time for cellular response mechanisms to take effect. Therefore, the irradiation interval was shown to play an important role in wound healing in vitro and should be taken into account.

  5. Andrographolide inhibits prostate cancer by targeting cell cycle regulators, CXCR3 and CXCR7 chemokine receptors.

    Science.gov (United States)

    Mir, Hina; Kapur, Neeraj; Singh, Rajesh; Sonpavde, Guru; Lillard, James W; Singh, Shailesh

    2016-01-01

    Despite state of the art cancer diagnostics and therapies offered in clinic, prostate cancer (PCa) remains the second leading cause of cancer-related deaths. Hence, more robust therapeutic/preventive regimes are required to combat this lethal disease. In the current study, we have tested the efficacy of Andrographolide (AG), a bioactive diterpenoid isolated from Andrographis paniculata, against PCa. This natural agent selectively affects PCa cell viability in a dose and time-dependent manner, without affecting primary prostate epithelial cells. Furthermore, AG showed differential effect on cell cycle phases in LNCaP, C4-2b and PC3 cells compared to retinoblastoma protein (RB(-/-)) and CDKN2A lacking DU-145 cells. G2/M transition was blocked in LNCaP, C4-2b and PC3 after AG treatment whereas DU-145 cells failed to transit G1/S phase. This difference was primarily due to differential activation of cell cycle regulators in these cell lines. Levels of cyclin A2 after AG treatment increased in all PCa cells line. Cyclin B1 levels increased in LNCaP and PC3, decreased in C4-2b and showed no difference in DU-145 cells after AG treatment. AG decreased cyclin E2 levels only in PC3 and DU-145 cells. It also altered Rb, H3, Wee1 and CDC2 phosphorylation in PCa cells. Intriguingly, AG reduced cell viability and the ability of PCa cells to migrate via modulating CXCL11 and CXCR3 and CXCR7 expression. The significant impact of AG on cellular and molecular processes involved in PCa progression suggests its potential use as a therapeutic and/or preventive agent for PCa.

  6. Human bone marrow-derived mesenchymal cell reactions to 316L stainless steel : An in vitro study on cell viability and interleukin-6 expression

    NARCIS (Netherlands)

    Anwar, I.B.; Santoso, A.; Saputra, E.; Ismail, R.; Jamari, J.; van der Heide, E.

    2017-01-01

    Purpose: Human bone marrow-derived mesenchymal cell (hBMC) reactions to 316L stainless steel (316L-SS) have never been evaluated. The objective of this study was to assess cell viability and interleukin-6 expression of hBMC cultures upon treatment with a 316L-SS implant. Methods: A cytotoxicity

  7. Influence of the mycotoxins alpha- and beta-zearalenol and deoxynivalenol on the cell cycle of cultured porcine endometrial cells.

    Science.gov (United States)

    Tiemann, U; Viergutz, T; Jonas, L; Schneider, F

    2003-01-01

    The present study investigated the effects of the mycotoxins alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) at concentrations of 7.5, 15, and 30 microM, and deoxynivalenol (DON) at concentrations of 0.94, 1.88, and 3.76 microM on cell cycle distribution (propidium iodide, PI staining) in combination with the proliferating cell nuclear antigen (PCNA) by flow cytometry. The viability of porcine uterine cells was not impaired at 30 microM alpha-ZOL, whereas beta-ZOL at this concentration and 3.76 microM DON significantly decreased cell number. Some cells showed ultrastructural features of cell death indicated by swollen mitochondria, disrupted cell membranes, and many vacuoles. After 24 and 48h of exposure to alpha-ZOL (7.5, 15, or 30 microM), the cell cycle distribution was still comparable to the control groups. An anti-proliferative effect of beta-ZOL and DON was detected by a significant reduction in the S-phase together with arrest of cells in the G(0)/G(1)-phase. The results show that beta-ZOL (7.5, 15, or 30 microM) and DON (0.94, 1.88, or 3.76 microM) control the progression of cells through the cycle by decreasing S-phase and arresting cells in the G(0)/G(1)-phase of the cell cycle. A significant decrease in the expression of the proliferation marker PCNA amounts indicates that beta-ZOL and DON disengaged cells from active cycling. We confirm that alpha-ZOL possesses a relative binding affinity to porcine uterine cytoplasmic estrogen receptor.

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

    Energy Technology Data Exchange (ETDEWEB)

    Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas [Laboratory of Biochemistry, School of Medicine, University of Thessaly, BIOPOLIS, 41110 Larissa (Greece); Institute of Biomedical Research and Technology (BIOMED), 51 Papanastasiou str., 41222 Larissa (Greece); Simos, George, E-mail: simos@med.uth.gr [Laboratory of Biochemistry, School of Medicine, University of Thessaly, BIOPOLIS, 41110 Larissa (Greece); Institute of Biomedical Research and Technology (BIOMED), 51 Papanastasiou str., 41222 Larissa (Greece)

    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.

  9. Cell Cycle Arrest and Apoptosis Induced by Kinamycin F in Human Osteosarcoma Cells.

    Science.gov (United States)

    Bavelloni, Alberto; Focaccia, Enrico; Piazzi, Manuela; Errani, Costantino; Blalock, William; Faenza, Irene

    2017-08-01

    Kinamycin F is a bacterial metabolite which contains an unusual and potentially reactive diazo group that is known for its ability to inhibit cell growth. In this study, the potential anti-tumor activity of kinamycin F was investigated in three human osteosarcoma cell lines, MG-63, U-2 OS and HOS as an antitumor agent with a potentially novel target. Proliferation and cell viability were measured in three human osteosarcoma cell lines by commercially available kits. We also evaluated the effects of the drug on cell cycle progression using the Muse™ Cell Analyzer. Caspase-3 activity was determined by a fluorometric EnzChek assay kit. Finally, following treatment with kinamycin F the protein levels of cyclin D3, cyclin A and cdK-2 were examined. Kinamycin F induced a concentration-dependent cell death in all the three cell lines. Flow cytometry revealed that kinamycin F treatment at 1 μM concentration significantly increased the cell population in the G2/M-phase (60-65%). Kinamycin F activated caspase 3 in all the three cell lines, clearly demonstrating that the growth inhibitory effect of kinamycin F can be attributed to apoptosis induction. Finally, kinamycin F suppressed osteosarcoma cell proliferation affecting cyclin A and D3 expression. Understanding the mechanism by which kinamycin F exerts its ability to inhibit cell growth may be a step forward in the development of new therapeutic strategies for the treatment of OS. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Ilaria Roato

    2016-01-01

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

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

    Science.gov (United States)

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

    2017-04-06

    The formation and metastatic colonization of circulating tumor cells (CTCs) are responsible for the vast majority of cancer-related deaths. Over the last decade, drug-delivery systems (DDSs) have rapidly developed with the emergence of nanotechnology; however, most reported tumor-targeting DDSs are able to deliver drugs only to solid tumor cells and not CTCs. Herein, a novel DDS comprising a composite nanofiber film was constructed to inhibit the viability of CTCs. In this system, gold nanoparticles (Au NPs) were functionalized with doxorubicin (DOX) through an acid-responsive cleavable linker to obtain Au-DOX NPs. Then, the Au-DOX NPs were mixed in a solution of an acid-responsive polymer {i.e., poly[2-(dimethylamino)ethyl methacrylate]} to synthesize the nanofiber film through electrospinning technology. After that, the nanofiber film was modified with a specific antibody (i.e., anti-EpCAM) to enrich the concentration of CTCs on the film. Finally, the Au-DOX NPs were released from the nanofiber film, and they consequently inhibited the viability of CTCs by delivering DOX to the enriched CTCs. This composite nanofiber film was able to decrease the viability of CTCs significantly in the suspended and fluid states, and it is expected to limit the migration and proliferation of tumor cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Piperine Triggers Apoptosis of Human Oral Squamous Carcinoma Through Cell Cycle Arrest and Mitochondrial Oxidative Stress.

    Science.gov (United States)

    Siddiqui, Sahabjada; Ahamad, Md Sultan; Jafri, Asif; Afzal, Mohd; Arshad, Md

    2017-07-01

    Piperine is a nitrogenous pungent substance exhibiting multifunctional pharmacological properties. However, the mechanism underlying its anticancer potential is not well elucidated in human oral squamous carcinoma (KB) cell line. The anticancer potential of piperine was evaluated through potent biomarkers viz. reactive oxygen species (ROS), cellular apoptosis, and loss of mitochondrial membrane potential (MMP). In addition, cell cycle kinetics and caspases-3 activity were also carried out to confirm anticancer activity of piperine. Results showed that various concentrations (25-300 μM) of piperine exposure reduced the cell viability of KB cells significantly (P Piperine induced significant (P piperine stimulated cell death by inducing loss of MMP, and caspase-3 activation. Cell cycle study revealed that piperine arrested the cells in G2/M phase and decreased the DNA content. Findings of this study suggest the efficacy of piperine in inducing cell death via the decrease in MMP and ROS liberation followed by caspase-3 activation and cell cycle arrest. Further assessment of the anticancer potency of piperine is needed for anticancer drug development.

  13. Effect of Lactobacillus reuteri on Cell Viability and PGE2 Production in Human Gingival Fibroblasts

    DEFF Research Database (Denmark)

    Castiblanco, Gina A.; Yucel-Lindberg, Tulay; Roos, Stefan

    2017-01-01

    Emerging evidence suggests that probiotic therapy can play a role in the prevention and management of oral inflammatory diseases through immunomodulation and down-regulation of the inflammatory cascade. The aim of this in vitro study was to investigate the viability of human gingival fibroblasts...

  14. The Effect of Histone Hyperacetylation on Viability of Basal-Like Breast Cancer Cells MDA-MB-231

    Directory of Open Access Journals (Sweden)

    Aliasghar Rahimian

    2017-06-01

    Full Text Available Background The Basal-Like breast cancer, is always known for lack of expression of estrogen receptor (ER, progesterone receptor (PR and as well, absence of epidermal growth factor receptor 2 (HER2 gene amplification. Improper expression pattern of ER, PR, and Her2, makes Basal-Like breast tumors resistant to the current hormonal and anti HER2 treatments. In recent decades, several studies have been conducted to investigate the regulatory role of chemical modifications of core histones in gene expression. Their results have shown that histone acetylation is involved in regulation of cell survival. Acetylation of core histones is regulated by the epigenetic-modifying enzymes named Histone Deacetylases (HDACs. As a new approach to control the viability of breast tumor cells resistant to the hormonal and anti-HER2 treatments, we have targeted the HDACs. Using Trichostatin A (TSA as a known HDACs inhibitor, we have tried to hyperacetylate the core histones of MDA-MB-231 cells as an in vitro model of Basal-Like breast tumors. Then we have investigated the effect of histone hyperacetylation on viability of MDA-MB-231 cells. Methods MDA-MB-231 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS and were incubated at 37°C, in a humidified incubator with 5% CO2 atmosphere. Then cells were treated with different concentrations of TSA including: 50, 100, 200, 400, 800 and 1000 nM or control (1% DMSO. After 24 and 48 hours, viability of cells was evaluated by MTT assay. Results After 24 and 48h exposure to different concentrations of TSA, MDA-MB-231 cells showed a maximum tolerable dose. At higher concentrations, TSA decreased the percentage of cell viability through a time-dose dependent manner. IC50 value for 48h treatment was 600 nM. Conclusions Our results indicate that HDACs inhibition and subsequently hyperacetylation of histones, leads to cytotoxic effects on breast tumor cells resistant to the current treatments. Following

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

    Science.gov (United States)

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

    2017-04-01

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

  16. RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Houcai; Yu, Jing; Zhang, Lixia; Xiong, Yuanyuan; Chen, Shuying; Xing, Haiyan; Tian, Zheng; Tang, Kejing; Wei, Hui; Rao, Qing; Wang, Min; Wang, Jianxiang, E-mail: wangjx@ihcams.ac.cn

    2014-04-18

    Highlights: • RPS27a expression was up-regulated in advanced-phase CML and AL patients. • RPS27a knockdown changed biological property of K562 and K562/G01 cells. • RPS27a knockdown affected Raf/MEK/ERK, P21 and BCL-2 signaling pathways. • RPS27a knockdown may be applicable for new combination therapy in CML patients. - Abstract: Ribosomal protein S27a (RPS27a) could perform extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. The high expression level of RPS27a was reported in solid tumors, and we found that the expression level of RPS27a was up-regulated in advanced-phase chronic myeloid leukemia (CML) and acute leukemia (AL) patients. In this study, we explored the function of RPS27a in leukemia cells by using CML cell line K562 cells and its imatinib resistant cell line K562/G01 cells. It was observed that the expression level of RPS27a was high in K562 cells and even higher in K562/G01 cells. Further analysis revealed that RPS27a knockdown by shRNA in both K562 and K562G01 cells inhibited the cell viability, induced cell cycle arrest at S and G2/M phases and increased cell apoptosis induced by imatinib. Combination of shRNA with imatinib treatment could lead to more cleaved PARP and cleaved caspase-3 expression in RPS27a knockdown cells. Further, it was found that phospho-ERK(p-ERK) and BCL-2 were down-regulated and P21 up-regulated in RPS27a knockdown cells. In conclusion, RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. It appears that drugs targeting RPS27a combining with tyrosine kinase inhibitor (TKI) might represent a novel therapy strategy in TKI resistant CML patients.

  17. Effect of human autologous serum and fetal bovine serum on human corneal epithelial cell viability, migration and proliferation in vitro.

    Science.gov (United States)

    Wu, Ming-Feng; Stachon, Tanja; Seitz, Berthold; Langenbucher, Achim; Szentmáry, Nóra

    2017-01-01

    To analyze the concentration-dependent effects of autologous serum (AS) and fetal bovine serum (FBS) on human corneal epithelial cell (HCEC) viability, migration and proliferation. AS was prepared from 13 patients with non-healing epithelial defects Dulbecco's modified eagle medium/Ham's F12 (DMEM/F12) with 5% FBS, 0.5% dimethyl sulphoxide (DMSO), 10 ng/mL human epidermal growth factor, 1% insulin-transferrin-selenium, then were incubated in serum media: DMEM/F12 supplemented by 5%, 10%, 15% or 30% AS or FBS. HCEC viability was analyzed using cell proliferation kit XTT, migration using a wound healing assay, proliferation by the cell proliferation enzyme-linked immunosorbent assay (ELISA) BrdU kit. Statistical analysis was performed using the generalized linear model, the values at 30% AS or 30% FBS were used as the baselines. HCEC viability was the highest at 30% AS or 15% FBS and the lowest at 10% AS or 30% FBS application. HCEC migration was the quickest through 30% AS or 30% FBS and the slowest through 5% AS or 5% FBS concentrations. Proliferation was the most increased through 15% AS or 5% FBS and the least increased through 30% AS or 30% FBS concentrations. HCEC viability at 10% and 15% AS was significantly worse (P=0.001, P=0.023) compared to baseline and significantly better at 15% FBS (P=0.003) concentrations. HCEC migration was significantly worse (P≤0.007) and HCEC proliferation significantly better (Pmigration 30% AS or 30% FBS, for proliferation 15% AS or 5% FBS should be used. Therefore, we suggest the use of 30% AS in clinical practice.

  18. Indirect-fired gas turbine dual fuel cell power cycle

    Science.gov (United States)

    Micheli, Paul L.; Williams, Mark C.; Sudhoff, Frederick A.

    1996-01-01

    A fuel cell and gas turbine combined cycle system which includes dual fuel cell cycles combined with a gas turbine cycle wherein a solid oxide fuel cell cycle operated at a pressure of between 6 to 15 atms tops the turbine cycle and is used to produce CO.sub.2 for a molten carbonate fuel cell cycle which bottoms the turbine and is operated at essentially atmospheric pressure. A high pressure combustor is used to combust the excess fuel from the topping fuel cell cycle to further heat the pressurized gas driving the turbine. A low pressure combustor is used to combust the excess fuel from the bottoming fuel cell to reheat the gas stream passing out of the turbine which is used to preheat the pressurized air stream entering the topping fuel cell before passing into the bottoming fuel cell cathode. The CO.sub.2 generated in the solid oxide fuel cell cycle cascades through the system to the molten carbonate fuel cell cycle cathode.

  19. Cell cycle checkpoints: reversible when possible, irreversible when needed

    NARCIS (Netherlands)

    Krenning, L.

    2015-01-01

    Cell cycle checkpoints are reversible in nature, and can prevent progression into the next cell cycle phase if needed. In the case of DNA damage, cells can prevent progression from G1 into S phase, and from G2 into mitosis in the presence of DNA double strand breaks. Following DNA repair, these

  20. Pore architecture and cell viability on freeze dried 3D recombinant human collagen-peptide (RHC)–chitosan scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jing; Zhou, Aimei; Deng, Aipeng [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Yang [Faculty of Engineering, University of Nottingham, Nottingham NG7 2RD (United Kingdom); Gao, Lihu; Zhong, Zhaocai [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Shulin, E-mail: yshulin@njust.edu.cn [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China)

    2015-04-01

    Pore architecture of 3D scaffolds used in tissue engineering plays a critical role in the maintenance of cell survival, proliferation and further promotion of tissue regeneration. We investigated the pore size and structure, porosity, swelling as well as cell viability of a series of recombinant human collagen-peptide–chitosan (RHCC) scaffolds fabricated by lyophilization. In this paper, freezing regime containing a final temperature of freezing (T{sub f}) and cooling rates was applied to obtain scaffolds with pore size ranging from 100 μm to 120 μm. Other protocols of RHC/chitosan suspension concentration and ratio modification were studied to produce more homogenous and appropriate structural scaffolds. The mean pore size decreased along with the decline of T{sub f} at a slow cooling rate of 0.7 °C/min; a more rapid cooling rate under 5 °C/min resulted to a smaller pore size and more homogenous microstructure. High concentration could reduce pore size and lead to thick well of scaffold, while improved the ratio of RHC, lamellar and fiber structure coexisted with cellular pores. Human umbilical vein endothelial cells (HUVECs) were seeded on these manufactured scaffolds, the cell viability represented a negative correlation to the pore size. This study provides an alternative method to fabricate 3D RHC–chitosan scaffolds with appropriate pores for potential tissue engineering. - Highlights: • Fabrication of recombinant human collagen-chitosan scaffolds by freezing drying • Influence of freeze drying protocols on lyophilized scaffolds • Pore size, microstructure, porosity, swelling and cell viability were compared. • The optimized porous scaffold is suitable for cell (HUVEC) seeding.

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

    Directory of Open Access Journals (Sweden)

    Arkadiusz Dziedzic

    2014-10-01

    Full Text Available 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.

  2. Combined application of arsenic trioxide and lithium chloride augments viability reduction and apoptosis induction in human rhabdomyosarcoma cell lines.

    Directory of Open Access Journals (Sweden)

    Sabine B Schleicher

    Full Text Available Rhabdomyosarcomas (RMS are the most prevalent soft tissue sarcomas affecting children and adolescents. Despite intensive treatment consisting of multimodal chemotherapy and surgery RMS patients diagnosed with metastatic disease expect long term survival rates of only 20%. Often multidrug resistance arises upon initial response emphasizing the need for new therapeutic drugs to improve treatment efficiency. Previously, we demonstrated the efficacy of the FDA approved drug arsenic trioxide (ATO specifically inhibiting viability and clonal growth as well as inducing cell death in human RMS cell lines of different subtypes. In this study, we combined low dose ATO with lithium chloride (LiCl, which is approved as mood stabilizer for the treatment of bipolar disorder, but also inhibits growth and survival of different cancer cell types in pre-clinical research. Indeed, we could show additive effects of LiCl and ATO on viability reduction, decrease of colony formation as well as cell death induction. In the course of this, LiCl induced inhibitory glycogen synthase kinase-3β (GSK-3β serine 9 phosphorylation, whereas glioma associated oncogene family 1 (GLI1 protein expression was particularly reduced by combined ATO and LiCl treatment in RD and RH-30 cell lines, showing high rates of apoptotic cell death. These results imply that combination of ATO with LiCl or another drug targeting GSK-3 is a promising strategy to enforce the treatment efficiency in resistant and recurrent RMS.

  3. Assessment of Radiobiological α/β Ratio in Lung Cancer and Fibroblast Cell Lines Using Viability Assays.

    Science.gov (United States)

    Karagounis, Ilias V; Skourti, Eleni K; Liousia, Maria V; Koukourakis, Michael I

    2017-01-01

    Altered fractionation is an area of intense clinical research in radiation oncology. Estimation of the α/β ratio of individual carcinomas after establishment of primary cell cultures from tumor biopsies may prove of importance in the individualization of radiotherapy schemes. Here we proposed a simple method to estimate the α/β ratio in cultured cell lines (two lung carcinomas: A549 and H1299; one lung fibroblast cell line: MRC5), using viability assays. For the A549 cell line, the α/β ratio ranged from 14-25 Gy, for H1299 from 11-43 Gy and for the MRC5 fibroblast cell line this was far lower, ranging from 0.69 to 6 Gy. The α/β ratio decreased when extracted from comparisons of lower dose per fraction schemes. The α/β ratio of a cell line can be easily defined after simple viability/dose fractionation experiments. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  4. Iodine-131 treatment of thyroid cancer cells leads to suppression of cell proliferation followed by induction of cell apoptosis and cell cycle arrest by regulation of B-cell translocation gene 2-mediated JNK/NF-κB pathways

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, L.M.; Pang, A.X., E-mail: zhaoliming515@126.com [Department of Nuclear Medicine, Linyi People' s Hospital, Linyi (China); Department of Urology, Linyi People' s Hospital, Linyi (China)

    2017-10-01

    Iodine-131 ({sup 131}I) is widely used for the treatment of thyroid-related diseases. This study aimed to investigate the expression of p53 and BTG2 genes following {sup 131}I therapy in thyroid cancer cell line SW579 and the possible underlying mechanism. SW579 human thyroid squamous carcinoma cells were cultured and treated with {sup 131}I. They were then assessed for {sup 131}I uptake, cell viability, apoptosis, cell cycle arrest, p53 expression, and BTG2 gene expression. SW579 cells were transfected with BTG2 siRNA, p53 siRNA and siNC and were then examined for the same aforementioned parameters. When treated with a JNK inhibitor of SP600125 and {sup 131}I or with a NF-kB inhibitor of BMS-345541 and {sup 131}I, non-transfected SW579 cells were assessed in JNK/NFkB pathways. It was observed that {sup 131}I significantly inhibited cell proliferation, promoted cell apoptosis and cell cycle arrest. Both BTG2 and p53 expression were enhanced in a dose-dependent manner. An increase in cell viability by up-regulation in Bcl2 gene, a decrease in apoptosis by enhanced CDK2 gene expression and a decrease in cell cycle arrest at G{sub 0}/G{sub 1} phase were also observed in SW579 cell lines transfected with silenced BTG2 gene. When treated with SP600125 and {sup 131}I, the non transfected SW579 cell lines significantly inhibited JNK pathway, NF-kB pathway and the expression of BTG2. However, when treated with BMS-345541 and {sup 131}I, only the NF-kB pathway was suppressed. {sup 131}I suppressed cell proliferation, induced cell apoptosis, and promoted cell cycle arrest of thyroid cancer cells by up-regulating B-cell translocation gene 2-mediated activation of JNK/NF--κB pathways. (author)

  5. Low-level laser therapy: Effects on human face aged skin and cell viability of HeLa cells exposed to UV radiation

    Directory of Open Access Journals (Sweden)

    Mezghani Sana

    2015-01-01

    Full Text Available Chronic and excessive exposure to UV radiation leads to photoaging and photocarcinogenesis. Adequate protection of the skin against the deleterious effects of UV irradiation is essential. Low-level laser therapy (LLLT is a light source in the red to near-infrared range that has been accepted in a variety of medical applications. In this study, we explored the effect of LLLT in human face aged skin and the cell viability of HeLa cells exposed to UV radiation. We found that LLLT significantly reduced visible wrinkles and the loss of firmness of facial skin in aging subjects. Additionally, treatment of cultured HeLa cells with LLLT prior to or post UVA or UVB exposure significantly protected cells from UV-mediated cell death. All results showed the beneficial effects of LLLT on relieving signs of skin aging and its prevention and protection of the cell viability against UV-induced damage.

  6. Viability and DNA Maintenance in Nonculturable Spiral Campylobacter jejuni Cells after Long-Term Exposure to Low Temperatures

    Science.gov (United States)

    Lázaro, Beatriz; Cárcamo, Jose; Audícana, Ana; Perales, Ildefonso; Fernández-Astorga, Aurora

    1999-01-01

    Survival of Campylobacter jejuni at 4 and 20°C was investigated by using cellular integrity, respiratory activity, two-dimensional (2D) protein profile, and intact DNA content as indicators of potential viability of nonculturable cells. Intact DNA content after 116 days, along with cellular integrity and respiring cells, was detected for up to 7 months at 4°C by pulsed-field gel electrophoresis. Most changes in 2D protein profiles involved up- or down-regulation. PMID:10508106

  7. The potential role of polyphenols in the modulation of skin cell viability by Aspalathus linearis and Cyclopia spp. herbal tea extracts in vitro.

    Science.gov (United States)

    Magcwebeba, Tandeka Unathi; Riedel, Sylvia; Swanevelder, Sonja; Swart, Pieter; De Beer, Dalene; Joubert, Elizabeth; Andreas Gelderblom, Wentzel Christoffel

    2016-11-01

    The relationship between polyphenol constituents, antioxidant properties of aqueous and methanol extracts of green tea (Camellia sinensis), the herbal teas, rooibos (Aspalathus linearis) and honeybush (Cyclopia spp.), against skin cell viability was investigated in vitro. The effect of extracts, characterised in terms of polyphenol content and antioxidant properties, on cell viability of premalignant, normal and malignant skin cells was determined. Phenolic composition, particularly high levels of potent antioxidants, of rooibos and green tea methanol extracts was associated with a strong reduction in cell viability specifically targeting premalignant cells. In contrast, the aqueous extracts of Cyclopia spp. were more effective in reducing cell viability. This correlated with a relatively high flavanol/proanthocyanidin content and ABTS radical cation scavenging capacity. The major green tea flavanol (epigallocatechin gallate) and rooibos dihydrochalcone (aspalathin) exhibited differential effects against cell viability, while the major honeybush xanthone (mangiferin) and flavanone (hesperidin) lacked any effect presumably due to a cytoprotective effect. The underlying mechanisms against skin cell viability are likely to involve mitochondrial dysfunction resulting from polyphenol-iron interactions. The polyphenol constituents and antioxidant parameters of herbal tea extracts are useful tools to predict their activity against skin cell survival in vitro and potential chemopreventive effects in vivo. © 2016 Royal Pharmaceutical Society.

  8. Flow cytometry analysis of cell cycle and specific cell synchronization with butyrate

    Science.gov (United States)

    Synchronized cells have been invaluable in many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. The possibility of using butyrate-blocked cells to obtain synchronized cells was explored and the properties of butyrate-induced cell ...

  9. Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle.

    Science.gov (United States)

    Ahn, Eunyong; Kumar, Praveen; Mukha, Dzmitry; Tzur, Amit; Shlomi, Tomer

    2017-11-06

    Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal-fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse-chase LC-MS-based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine-derived fluxes: Oxidation of glucose-derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  10. 2001 Volvo Award Winner in Basic Science Studies: Effect of nutrient supply on the viability of cells from the nucleus pulposus of the intervertebral disc.

    Science.gov (United States)

    Horner, H A; Urban, J P

    2001-12-01

    Disc cell viability was analyzed in relation to nutrient supply and cellular demand in vitro in a diffusion chamber. To determine relations among nutrient supply, nutrient concentrations. and cell viability. Although a fall in nutrient supply has long been thought the cause of disc degeneration in vivo, little information exists about the effects of nutrient levels or supply on cell viability and metabolism. Isolated bovine nucleus cells were cultured in agarose gels in a diffusion chamber up to 13 days. Nutrients were supplied to the open sides of the chamber and diffused through the gel to the center, 12.5 mm away from the nutrient supply, in a configuration analogous to that of the disc in vivo. Profiles of cell viability and concentration of glycosaminoglycans across the chamber were measured in relation to cell density and medium composition. Cells remained viable across the chamber at low cell densities. However, at higher densities, cells in the center of the chamber died. The viable distance from the nutrient supply fell with an increase in cell density. Glucose was a critical nutrient. Survival was also poor at acidic pH (6.0). At 0% oxygen, disc cells survived up to 13 days with no loss of viability, but produced very little proteoglycan. The results support the idea that maximum cell density in the disc is regulated by nutritional constraints, and that a fall in nutrient supply reduces the number of viable cells in the disc and thus leads to degeneration.

  11. Analysis and integration of fuel cell combined cycles for development of low-carbon energy technologies

    Energy Technology Data Exchange (ETDEWEB)

    Varbanov, Petar [Marie Curie ERG ESCHAINS, Research Institute of Chemical and Process Engineering, University of Pannonia, Egyetem u. 10, Veszprem H-8200 (Hungary); Klemes, Jiri [EC Marie Curie Chair (EXC) ' ' INEMAGLOW' ' , Research Institute of Chemical Technology and Process Engineering, FIT, University of Pannonia, Egyetem u. 10, Veszprem H-8200 (Hungary)

    2008-10-15

    Integrated and combined cycles (ICC, CC) traditionally involve gas and steam turbines only. The paper analyses the further integration of high-temperature fuel cells (FC) having high electrical efficiency reaching up to 60% compared with 30-35% for most gas turbines. The previous research on FC hybrids indicates achieving high efficiencies and economic viability is possible. The ICC of various FC types - their performance and the potential for utilisation of renewables - are analysed considering also power generation capacity and site heat integration context. Further research and development with industrial relevance are outlined focusing on CO{sub 2} emissions reduction. (author)

  12. Quantitative Characterization of Cell Behaviors through Cell Cycle Progression via Automated Cell Tracking

    Science.gov (United States)

    Wang, Yuliang; Jeong, Younkoo; Jhiang, Sissy M.; Yu, Lianbo; Menq, Chia-Hsiang

    2014-01-01

    Cell behaviors are reflections of intracellular tension dynamics and play important roles in many cellular processes. In this study, temporal variations in cell geometry and cell motion through cell cycle progression were quantitatively characterized via automated cell tracking for MCF-10A non-transformed breast cells, MCF-7 non-invasive breast cancer cells, and MDA-MB-231 highly metastatic breast cancer cells. A new cell segmentation method, which combines the threshold method and our modified edge based active contour method, was applied to optimize cell boundary detection for all cells in the field-of-view. An automated cell-tracking program was implemented to conduct live cell tracking over 40 hours for the three cell lines. The cell boundary and location information was measured and aligned with cell cycle progression with constructed cell lineage trees. Cell behaviors were studied in terms of cell geometry and cell motion. For cell geometry, cell area and cell axis ratio were investigated. For cell motion, instantaneous migration speed, cell motion type, as well as cell motion range were analyzed. We applied a cell-based approach that allows us to examine and compare temporal variations of cell behavior along with cell cycle progression at a single cell level. Cell body geometry along with distribution of peripheral protrusion structures appears to be associated with cell motion features. Migration speed together with motion type and motion ranges are required to distinguish the three cell-lines examined. We found that cells dividing or overlapping vertically are unique features of cell malignancy for both MCF-7 and MDA-MB-231 cells, whereas abrupt changes in cell body geometry and cell motion during mitosis are unique to highly metastatic MDA-MB-231 cells. Taken together, our live cell tracking system serves as an invaluable tool to identify cell behaviors that are unique to malignant and/or highly metastatic breast cancer cells. PMID:24911281

  13. Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays.

    Science.gov (United States)

    Limame, Ridha; Wouters, An; Pauwels, Bea; Fransen, Erik; Peeters, Marc; Lardon, Filip; De Wever, Olivier; Pauwels, Patrick

    2012-01-01

    Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (patho)biological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions. Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB) assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0-100 nM) correlated well with SRB (Rho>0.95) with similar IC(50) values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90) and optical density (OD) measurement of extracted dye (Rho>0.95). Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95). Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method. The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on different platforms applying only adapted matrix surface densities. The increased

  14. Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays.

    Directory of Open Access Journals (Sweden)

    Ridha Limame

    Full Text Available BACKGROUND: Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (pathobiological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions. METHODOLOGY/PRINCIPAL FINDINGS: Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer and A549 (lung cancer cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964. Cytotoxic action by paclitaxel (0-100 nM correlated well with SRB (Rho>0.95 with similar IC(50 values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90 and optical density (OD measurement of extracted dye (Rho>0.95. Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95. Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method. CONCLUSIONS/SIGNIFICANCE: The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on

  15. Decreased MiR-17 in glioma cells increased cell viability and migration by increasing the expression of Cyclin D1, p-Akt and Akt.

    Directory of Open Access Journals (Sweden)

    Guangwei Sun

    Full Text Available The activating mutations of micro RNA (miR-17 have been revealed in tumors such as human non-Hodgkin's lymphoma and T cell leukemia. However, it is unclear about the role of miR-17 in glioma cells. The current study aimed to investigate effects of miR-17 mimics or inhibitor on the viability and migration of rat glioma C6 cells, and explore possible mechanisms.The expression of miR-17 in rat glioma C6 cells and normal brain tissue was detected by quantitative PCR. Protein expression of Cyclin D1 in rat glioma C6 cells and normal brain tissue was measured by Western Blot. Glioma C6 cells were transfected with MiR-17 mimics or inhibitor. Cells that were not transfected (Lipofectamine only and cells that were transfected with nonsense RNA negative control served as control. MTT assay was utilized to detect cell viability, and cell wound scratch assay was utilized to examine the migration index. In addition, protein expression of Cyclin D1, p-Akt and Akt in MiR-17 mimics or inhibitor-transfected glioma C6 cells was detected by Western Blot. This study had been approved by the Medical Ethics Committee of the First Affiliated Hospital of Soochow University. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.The expression of miR-17 was significantly lower, whereas the expression of Cyclin D1 was significantly higher in glioma C6 cells compared to normal brain tissue. MiR-17 mimics decreased the viability and migration of glioma C6 cells markedly at 48 h. In addition, MiR-17 inhibitor increased the viability and migration of glioma C6 cells at 24 and 48 h. The protein expression of Cyclin D1, p-Akt and Akt in glioma C6 cells decreased after transfection with miR-17 mimics for 72 h, and increased after transfection with miR-17 inhibitor for 72 h.The reduced miR-17 levels in glioma cells increased cell viability and migration, which correlates with increased expression of Cyclin D1, p

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

    Directory of Open Access Journals (Sweden)

    Elisavet Vasilopoulou

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

  17. Using a full annual cycle model to evaluate long-term population viability of the conservation-reliant Kirtland's warbler after successful recovery

    Science.gov (United States)

    Brown, Donald J.; Ribic, Christine; Donner, Deahn M.; Nelson, Mark D.; Bocetti, Carol I.; Deloria-Sheffield, Christie M.

    2017-01-01

    Long-term management planning for conservation-reliant migratory songbirds is particularly challenging because habitat quality in different stages and geographic locations of the annual cycle can have direct and carry-over effects that influence the population dynamics. The Neotropical migratory songbird Kirtland's warbler Setophaga kirtlandii (Baird 1852) is listed as endangered under the U.S. Endangered Species Act and Near Threatened under the IUCN Red List. This conservation-reliant species is being considered for U.S. federal delisting because the species has surpassed the designated 1000 breeding pairs recovery threshold since 2001.To help inform the delisting decision and long-term management efforts, we developed a population simulation model for the Kirtland's warbler that incorporated both breeding and wintering grounds habitat dynamics, and projected population viability based on current environmental conditions and potential future management scenarios. Future management scenarios included the continuation of current management conditions, reduced productivity and carrying capacity due to the changes in habitat suitability from the creation of experimental jack pine Pinus banksiana (Lamb.) plantations, and reduced productivity from alteration of the brown-headed cowbird Molothrus ater (Boddaert 1783) removal programme.Linking wintering grounds precipitation to productivity improved the accuracy of the model for replicating past observed population dynamics. Our future simulations indicate that the Kirtland's warbler population is stable under two potential future management scenarios: (i) continuation of current management practices and (ii) spatially restricting cowbird removal to the core breeding area, assuming that cowbirds reduce productivity in the remaining patches by ≤41%. The additional future management scenarios we assessed resulted in population declines.Synthesis and applications. Our study indicates that the Kirtland's warbler population

  18. Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis

    Science.gov (United States)

    Ingber, D. E.; Prusty, D.; Sun, Z.; Betensky, H.; Wang, N.

    1995-01-01

    Capillary endothelial cells can be switched between growth and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its growth-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G0-synchronized cells were cultured in basic fibroblast growth factor (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthetic integrin ligand (RGD-containing peptide), cell spreading, nuclear extension, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12-15 h and hence, most of G1, in order to enter S phase. After this restriction point was passed, normally 'anchorage-dependent' endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent growth control was confirmed by culturing cells in the presence of cytochalasin D (25-1000 ng ml-1): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml-1) resulted in potent inhibition of both spreading and growth, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed

  19. Combined EGFR- and notch inhibition display additive inhibitory effect on glioblastoma cell viability and glioblastoma-induced endothelial cell sprouting in vitro

    DEFF Research Database (Denmark)

    Staberg, Mikkel; Michaelsen, Signe Regner; Olsen, Louise Stobbe

    2016-01-01

    BACKGROUND: For Glioblastoma (GBM) patients, a number of anti-neoplastic strategies using specifically targeting drugs have been tested; however, the effects on survival have been limited. One explanation could be treatment resistance due to redundant signaling pathways, which substantiates the n...... and cell viability, thereby stressing the importance of further evaluation of this targeting approach in a clinical setting....

  20. Viability and proliferation of pluripotential cells delivered to tendon repair sites using bioactive sutures--an in vitro study.

    Science.gov (United States)

    Yao, Jeffrey; Korotkova, Tatiana; Smith, R Lane

    2011-02-01

    We evaluated the fate of pluripotential stem cells adherent to a suture carrier after being passed through tendon tissue in vitro. FiberWire suture segments were coated with poly-L-lysine (PLL) and a 2 × 10(6) C3H10T1/2 (a mouse embryo pluripotential cell line) cell suspension. The sutures were incubated for 7 days, passed through two 1-cm segments of acellularized rabbit Achilles tendons and tied (horizontal mattress). The repairs were frozen and sectioned (6 μm). The sections were stained with 4',6-diamidino-2-phenylindole and a live/dead viability/cytotoxicity (calcein/ethidium homodimer) kit and examined with fluorescent microscopy to evaluate cell presence and viability. Alamar Blue was used in parallel to assess metabolic activity. PLL-coated sutures showed a 3-fold increase in fluorescence when compared with the phosphate-buffered saline-coated controls. At day 3, fluorescence was 2.2 times greater. At day 5, a 2-fold increase was found, and at day 8 there was no significant difference in values. Furthermore, after delivery of the cells into tendon, fluorescence readings for the samples (n = 19) showed 9450 compared with the positive control at 21,218. At 96 hours the mean was 27,609 compared with 34,850 for the positive control. The difference in fluorescence means at 48 hours and 96 hours were significant (p cells at the tendon repair site. Sutures seeded with pluripotential embryonic cells deliver cells to a tendon repair site. The cells deposited at the repair site survive the trauma of passage and remain metabolically active, as seen in staining and metabolic assay studies. Use of bioactive sutures leads to repopulation of the acellular zone surrounding sutures within the tendon. Copyright © 2011 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.

  1. Jaceosidin inhibits proliferation of human bladder cancer T24 cells through induction of cell cycle arrest and apoptosis

    Directory of Open Access Journals (Sweden)

    Yong Li

    2013-08-01

    Full Text Available Jaceosidin, isolated from Artemesia argyi, has been shown to possess promising anticancer potential against various cancer cells. However, its effect against bladder cancer cells remained unknown. In this study, for the first time, we investigated the effects of jaceosidin on cell proliferation, cell cycle, and apoptosis in bladder cancer T24 cells by using MTT assay and flow cytometric analysis. The results revealed that jaceosidin decreased the cell viability of bladder cancer T24 cells in a dose- and time-dependent manner. Flow cytometric analysis demonstrated that jaceosidin significantly triggered apoptosis in T24 cells and arrested cell cycle at G2/M phase in a time-dependent manner. Further characterization showed that jaceosidin-induced apoptosis is associated with dissipation in mitochondrial membrane potential (ΔΨm, up-regulation of Bax and down-regulation of Bcl-2 in jaceosidin-treated T24 cells. These in vitro results suggested that jaceosidin should be further examined for in vivo activity and molecular mechanism in human bladder cancer.

  2. Tracing Conidial Fate and Measuring Host Cell Antifungal Activity Using a Reporter of Microbial Viability in the Lung

    Directory of Open Access Journals (Sweden)

    Anupam Jhingran

    2012-12-01

    Full Text Available Fluorescence can be harnessed to monitor microbial fate and to investigate functional outcomes of individual microbial cell-host cell encounters at portals of entry in native tissue environments. We illustrate this concept by introducing fluorescent Aspergillus reporter (FLARE conidia that simultaneously report phagocytic uptake and fungal viability during cellular interactions with the murine respiratory innate immune system. Our studies using FLARE conidia reveal stepwise and cell-type-specific requirements for CARD9 and Syk, transducers of C-type lectin receptor and integrin signals, in neutrophil recruitment, conidial uptake, and conidial killing in the lung. By achieving single-event resolution in defined leukocyte populations, the FLARE method enables host cell profiling on the basis of pathogen uptake and killing and may be extended to other pathogens in diverse model host organisms to query molecular, cellular, and pharmacologic mechanisms that shape host-microbe interactions.

  3. Multiple Applications of Alamar Blue as an Indicator of Metabolic Function and Cellular Health in Cell Viability Bioassays

    Science.gov (United States)

    Rampersad, Sephra N.

    2012-01-01

    Accurate prediction of the adverse effects of test compounds on living systems, detection of toxic thresholds, and expansion of experimental data sets to include multiple toxicity end-point analysis are required for any robust screening regime. Alamar Blue is an important redox indicator that is used to evaluate metabolic function and cellular health. The Alamar Blue bioassay has been utilized over the past 50 years to assess cell viability and cytotoxicity in a range of biological and environmental systems and in a number of cell types including bacteria, yeast, fungi, protozoa and cultured mammalian and piscine cells. It offers several advantages over other metabolic indicators and other cytotoxicity assays. However, as with any bioassay, suitability must be determined for each application and cell model. This review seeks to highlight many of the important considerations involved in assay use and design in addition to the potential pitfalls. PMID:23112716

  4. The effect of spiritual healing on in vitro tumour cell proliferation and viability--an experimental study

    DEFF Research Database (Denmark)

    Zachariae, R; Højgaard, L; Zachariae, C

    2005-01-01

    of the recipient's conscious awareness of the healer's intention. The aim of this study was to test the hypothesis that spiritual healing will reduce proliferation and viability of two cancer cell lines in vitro. Three controlled experiments were conducted with three different healers and randomised allocation...... three experimental days, doses, assays, and cells, 34 (51.6%) of 66 independent comparisons showed differences in the hypothesised direction (P = 0.90). The average effect size across cell lines, days, assays, and doses approached zero (Cohen's d = -0.01). The results do not support previous reports...... of beneficial effects of spiritual healing on malignant cell growth in vitro. Reported beneficial effects of spiritual healing on the well-being of cancer patients seem more likely to be mediated by psychosocial and psychophysiological effects of the healer-patient relationship....

  5. Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy.

    Science.gov (United States)

    Wu, Feng-Hua; Mu, Lei; Li, Xiao-Lan; Hu, Yi-Bing; Liu, Hui; Han, Lin-Tao; Gong, Jian-Ping

    2017-10-03

    The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo . Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo . Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro . Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.

  6. Carfilzomib induces G2/M cell cycle arrest in human endometrial cancer cells via upregulation of p21Waf1/Cip1and p27Kip1.

    Science.gov (United States)

    Zhou, Yuanyuan; Wang, Ke; Zhen, Shuai; Wang, Ruili; Luo, Wenjuan

    2016-12-01

    Carfilzomib is a second-generation tetrapeptide epoxyketone proteasome inhibitor used in current clinical therapy of hematologic malignancies. The mechanism of proteasome inhibition in endometrial cancer is not very clear. Carfilzomib inhibition of type I endometrial carcinoma cell proliferation by inducing cell cycle arrest at the G2/M phase was investigated in our study. HEC-1-A and Ishikawa endometrial carcinoma cell lines and three tumor cell lines were treated by different concentrations of carfilzomib. Methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability. Flow cytometry was used to analyze the cell cycle. Western blot was used to detect proteins involved in cell cycle progression. Carfilzomib impaired viability of myelogenous leukemia cell line K562, cervical cancer cell line HeLa, hepatocellular carcinoma cell line SMCC-7721, and endometrial carcinoma cell lines HEC-1-A and Ishikawa. The cell cycle was arrested at the G2/M phase in carfilzomib-treated HEC-1-A endometrial carcinoma cells, while it was arrested at both S and G2/M phases in carfilzomib-treated Ishikawa cells. Carfilzomib treatment significantly induced p21 Waf1/ Cip1 and p27, while substantially reduced cyclin D3 and cyclin-dependent kinase 1. This study showed that carfilzomib inhibited endometrial cancer proliferation by upregulating cyclin-dependent kinase inhibitors p21 Waf1/Cip1 and p27 Kip1 , and reducing cyclin-dependent kinase 1 to arrest the cell cycle at the G2/M phase. Copyright © 2016. Published by Elsevier B.V.

  7. Calmodulin antagonists decrease glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, ATP and viability of melanoma cells.

    Science.gov (United States)

    Glass-Marmor, L; Morgenstern, H; Beitner, R

    1996-10-17

    Glycolysis is known to be the primary energy source in cancer cells. We investigated here the effect of four different calmodulin antagonists: thioridazine (10-[2-(1-methyl-2-piperidyl) ethyl]-2-methylthiophenothiazine), CGS 9343B (1,3-dihydro-1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a] [4,1]-benzoxazepin-4-yl)methyl]-4-piperidinyl]-2 H-benzimidazol-2-one (1:1) maleate), clotrimazole (1-(alpha-2-chlorotrityl)imidazole) and bifonazole (1-(alpha-biphenyl-4-ylbenzyl)imidazole), on the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis, and on ATP content and cell viability in B16 melanoma cells. We found that all four substances significantly reduced the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate and ATP, in a dose- and time-dependent manner. Cell viability was reduced in a close correlation with the fall in ATP. The decrease in glucose 1,6-bisphosphate and fructose 1,6-bisphosphate did not result from the cytotoxic effects of the calmodulin antagonists, since their content was already reduced before any cytotoxic effect was observed. These findings suggest that the fall in the levels of the two signal molecules of glycolysis, induced by the calmodulin antagonists, causes a reduction in glycolysis and ATP levels, which eventually leads to cell death. Since cell proliferation was also reported to be inhibited by calmodulin antagonists, these substances are most promising agents in treatment of cancer by inhibiting both cell proliferation and the glycolytic supply of ATP required for cell growth.

  8. How does vitrification affect oocyte viability in oocyte donation cycles? A prospective study to compare outcomes achieved with fresh versus vitrified sibling oocytes.

    Science.gov (United States)

    Solé, M; Santaló, J; Boada, M; Clua, E; Rodríguez, I; Martínez, F; Coroleu, B; Barri, P N; Veiga, A

    2013-08-01

    How does vitrification affect oocyte viability? Vitrification does not affect oocyte viability in oocyte donation cycles. Oocyte vitrification is performed routinely and successfully in IVF and oocyte donation programs. This is a prospective study performed between June 2009 and February 2012 to compare ongoing pregnancy rates and other indices of viability between fresh and vitrified oocytes. A total of 99 donations with more than 16 oocytes (MII) in which oocytes were allocated both to a synchronous recipient (fresh oocytes) and to an asynchronous recipient (vitrified oocytes) were included. The participants were consenting couples (donors and recipients) from the oocyte donation program. On the day of retrieval, the oocytes allocated to the synchronous recipient were inseminated and those allocated for banking were denuded of cumulus and vitrified. Vitrified oocytes were microinjected with spermatozoa 2 h after warming. Embryo transfer was performed on Day 2 of development in both groups, and the remaining embryos were cryopreserved on Day 3. Clinical pregnancy was defined by a positive fetal heartbeat at 6 weeks. A total of 989 oocytes were warmed and 85.6% survived. No significant differences were observed between fresh and vitrified oocytes: fertilization rate (80.7 versus 78.2%), ongoing embryo rate (71.0 versus 68.2%) or good-quality embryo rate (54.1 versus 49.8%). The mean number of embryos transferred was similar in both groups (1.82 ± 0.44 versus 1.90 ± 0.34). The implantation rate (33.3 versus 34.0%) and the multiple pregnancy rate (27.7 versus 20.8) were also similar between both groups (P > 0.05). The live birth rate per cycle was 38.4% in the recipients of fresh oocytes and 43.4% in the recipients of vitrified oocytes (P > 0.05). Eighty five frozen embryo transfers were also evaluated. Comparing embryos from fresh and vitrified oocytes there were no significant differences in the embryo survival rate (70.1 versus 65.8%), clinical pregnancy rate

  9. Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

    Energy Technology Data Exchange (ETDEWEB)

    Byrne, Ann; McLaren, Rajashree P.; Mason, Paul; Chai, Lilly; Dufault, Michael R.; Huang, Yinyin; Liang, Beirong; Gans, Joseph D.; Zhang, Mindy; Carter, Kara; Gladysheva, Tatiana B.; Teicher, Beverly A.; Biemann, Hans-Peter N.; Booker, Michael; Goldberg, Mark A.; Klinger, Katherine W.; Lillie, James [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Madden, Stephen L., E-mail: steve.madden@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Jiang, Yide, E-mail: yide.jiang@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States)

    2010-01-15

    The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21{sup /Cip} and p27{sup /Kip1}. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

  10. Rethinking cell-cycle-dependent gene expression in Schizosaccharomyces pombe.

    Science.gov (United States)

    Cooper, Stephen

    2017-11-01

    Three studies of gene expression during the division cycle of Schizosaccharomyces pombe led to the proposal that a large number of genes are expressed at particular times during the S. pombe cell cycle. Yet only a small fraction of genes proposed to be expressed in a cell-cycle-dependent manner are reproducible in all three published studies. In addition to reproducibility problems, questions about expression amplitudes, cell-cycle timing of expression, synchronization artifacts, and the problem with methods for synchronizing cells must be considered. These problems and complications prompt the idea that caution should be used before accepting the conclusion that there are a large number of genes expressed in a cell-cycle-dependent manner in S. pombe.

  11. Toll-like receptor 4 (TLR4 expression in human and murine pancreatic beta-cells affects cell viability and insulin homeostasis

    Directory of Open Access Journals (Sweden)

    Santos Icaro A

    2011-02-01

    Full Text Available Abstract Background Toll-like receptor 4 (TLR4 is widely recognized as an essential element in the triggering of innate immunity, binding pathogen-associated molecules such as Lipopolysaccharide (LPS, and in initiating a cascade of pro-inflammatory events. Evidence for TLR4 expression in non-immune cells, including pancreatic β-cells, has been shown, but, the functional role of TLR4 in the physiology of human pancreatic β-cells is still to be clearly established. We investigated whether TLR4 is present in β-cells purified from freshly isolated human islets and confirmed the results using MIN6 mouse insulinoma cells, by analyzing the effects of TLR4 expression on cell viability and insulin homeostasis. Results CD11b positive macrophages were practically absent from isolated human islets obtained from non-diabetic brain-dead donors, and TLR4 mRNA and cell surface expression were restricted to β-cells. A significant loss of cell viability was observed in these β-cells indicating a possible relationship with TLR4 expression. Monitoring gene expression in β-cells exposed for 48h to the prototypical TLR4 ligand LPS showed a concentration-dependent increase in TLR4 and CD14 transcripts and decreased insulin content and secretion. TLR4-positive MIN6 cells were also LPS-responsive, increasing TLR4 and CD14 mRNA levels and decreasing cell viability and insulin content. Conclusions Taken together, our data indicate a novel function for TLR4 as a molecule capable of altering homeostasis of pancreatic β-cells.

  12. Variety in intracellular diffusion during the cell cycle

    DEFF Research Database (Denmark)

    Selhuber-Unkel, C.; Yde, P.; Berg-Sørensen, Kirstine

    2009-01-01

    During the cell cycle, the organization of the cytoskeletal network undergoes dramatic changes. In order to reveal possible changes of the viscoelastic properties in the intracellular space during the cell cycle we investigated the diffusion of endogenous lipid granules within the fission yeast...... Schizosaccharomyces Pombe using optical tweezers. The cell cycle was divided into interphase and mitotic cell division, and the mitotic cell division was further subdivided in its stages. During all stages of the cell cycle, the granules predominantly underwent subdiffusive motion, characterized by an exponent...... a that is also linked to the viscoelastic moduli of the cytoplasm. The exponent a was significantly smaller during interphase than during any stage of the mitotic cell division, signifying that the cytoplasm was more elastic during interphase than during division. We found no significant differences...

  13. Changes of Constituents and Activity to Apoptosis and Cell Cycle During Fermentation of Tea

    Directory of Open Access Journals (Sweden)

    Wei Shi

    2011-03-01

    Full Text Available Tea is believed to be beneficial for health, and the effects of the fermentation process on its contributions to apoptosis and cell cycle arrest of gastric cancer cells have not been completely investigated. In this study, the chemical components in green tea, black tea and pu-erh tea aqueous extracts were analyzed and compared. The polysaccharide and caffeine levels were substantially higher in the fermented black tea and pu-erh tea, while the polyphenol level was higher in the unfermented green tea. Hence, a treatment of tea aqueous extract and the components, which are emerging as promising anticancer agents, were pursued to determine whether this treatment could lead to enhance apoptosis and cell cycle arrest. In the human gastric cancer cell line SGC-7901, the cell viability and flow cytometry analysis for apoptotic cells indicated effects in a dose-dependent inhibition manner for the three tea treatment groups. The apoptosis rates were found to be elevated after 48 h of treatment with 31.2, 125, and 500 μg/mL of green tea extract, the higher catechins content may be involved in the mechanism. Cell cycle was arrested in S phase in the fermented black tea and pu-erh tea, and the populations were significantly decreased in G2/M phases, possibly due to the oxidation of tea polyphenols, which causes an increase of theabrownins. CCC-HEL-1 normal cells were not sensitive to tea extract. These findings suggest that the fermentation process causes changes of the compounds which might be involved in the changes of cell proliferation inhibition, apoptosis induction and cell cycle arrest.

  14. Changes of Constituents and Activity to Apoptosis and Cell Cycle During Fermentation of Tea

    Science.gov (United States)

    Zhao, Hang; Zhang, Min; Zhao, Lu; Ge, Ya-kun; Sheng, Jun; Shi, Wei

    2011-01-01

    Tea is believed to be beneficial for health, and the effects of the fermentation process on its contributions to apoptosis and cell cycle arrest of gastric cancer cells have not been completely investigated. In this study, the chemical components in green tea, black tea and pu-erh tea aqueous extracts were analyzed and compared. The polysaccharide and caffeine levels were substantially higher in the fermented black tea and pu-erh tea, while the polyphenol level was higher in the unfermented green tea. Hence, a treatment of tea aqueous extract and the components, which are emerging as promising anticancer agents, were pursued to determine whether this treatment could lead to enhance apoptosis and cell cycle arrest. In the human gastric cancer cell line SGC-7901, the cell viability and flow cytometry analysis for apoptotic cells indicated effects in a dose-dependent inhibition manner for the three tea treatment groups. The apoptosis rates were found to be elevated after 48 h of treatment with 31.2, 125, and 500 μg/mL of green tea extract, the higher catechins content may be involved in the mechanism. Cell cycle was arrested in S phase in the fermented black tea and pu-erh tea, and the populations were significantly decreased in G2/M phases, possibly due to the oxidation of tea polyphenols, which causes an increase of theabrownins. CCC-HEL-1 normal cells were not sensitive to tea extract. These findings suggest that the fermentation process causes changes of the compounds which might be involved in the changes of cell proliferation inhibition, apoptosis induction and cell cycle arrest. PMID:21673927

  15. Cytokine-induced impairment of short-chain fatty acid oxidation and viability in human colonic epithelial cells

    DEFF Research Database (Denmark)

    Pedersen, G; Saermark, T; Horn, T

    2000-01-01

    Pro-inflammatory cytokines may directly influence the viability and metabolic function of colonic epithelial cells (CEC) as an early event in the development of inflammatory bowel disease. We report here that TNF-alpha+IFN-gamma induced a synergistic, concentration-dependent decline in butyrate o...... was independent on NO formation and involved the IFN-gamma signalling pathway as well as induction of apoptosis. If cytokines have similar effects in vivo, these may lead to energy deficiency and thus contribute to CEC damage and disturbance of the epithelial integrity....

  16. Sesquiterpene lactones from Ambrosia spp. are active against a murine lymphoma cell line by inducing apoptosis and cell cycle arrest.

    Science.gov (United States)

    Martino, Renzo; Beer, María Florencia; Elso, Orlando; Donadel, Osvaldo; Sülsen, Valeria; Anesini, Claudia

    2015-10-01

    Sesquiterpene lactones (STLs) are natural terpenoid compounds. They have been recognized as antitumor agents. The purpose of this investigation was to explore the antiproliferative effects of psilostachyin, psilostachyin C, peruvin and cumanin on the murine lymphoma cell line BW5147. Cells were treated with the STLs at different concentrations. Tritiated thymidine uptake was employed to determine cell proliferation. MTT assay was used to analyze cell viability. Flow cytometry assay with annexin V-FITC and propidium iodide was employed to evaluate cell death. Reactive oxygen species (ROS), mitochondrial membrane potential and cell cycle analysis were also evaluated by flow cytometry. Antioxidant enzymes activities were determined spectrophotometrically by kinetic assays. Results showed that these STLs inhibited cell proliferation in a concentration-dependent manner by exerting cytotoxicity through apoptosis. Psilostachyin C was the most active and the less toxic compound. This STL induced apoptosis with an impairment in mitochondrial membrane potential. Psilostachyin C was able to induce ROS generation, related to a modulation of the antioxidant enzymes activity. In addition, it induced cell cycle arrest in S phase. In conclusion, psilostachyin C was found to be active against lymphoma cells exerting both cytostatic and cytotoxic effects. These findings may provide a novel approach for lymphoma treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Systems Level Modeling of the Cell Cycle Using Budding Yeast

    Directory of Open Access Journals (Sweden)

    D.R. Kim

    2007-01-01

    Full Text Available Proteins involved in the regulation of the cell cycle are highly conserved across all eukaryotes, and so a relatively simple eukaryote such as yeast can provide insight into a variety of cell cycle perturbations including those that occur in human cancer. To date, the budding yeast Saccharomyces cerevisiae has provided the largest amount of experimental and modeling data on the progression of the cell cycle, making it a logical choice for in-depth studies of this process. Moreover, the advent of methods for collection of high-throughput genome, transcriptome, and proteome data has provided a means to collect and precisely quantify simultaneous cell cycle gene transcript and protein levels, permitting modeling of the cell cycle on the systems level. With the appropriate mathematical framework and suffi cient and accurate data on cell cycle components, it should be possible to create a model of the cell cycle that not only effectively describes its operation, but can also predict responses to perturbations such as variation in protein levels and responses to external stimuli including targeted inhibition by drugs. In this review, we summarize existing data on the yeast cell cycle, proteomics technologies for quantifying cell cycle proteins, and the mathematical frameworks that can integrate this data into representative and effective models. Systems level modeling of the cell cycle will require the integration of high-quality data with the appropriate mathematical framework, which can currently be attained through the combination of dynamic modeling based on proteomics data and using yeast as a model organism.

  18. Chromatin association of UHRF1 during the cell cycle

    KAUST Repository

    Al-Gashgari, Bothayna

    2017-05-01

    Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a nuclear protein that associates with chromatin. Regardless of the various functions of UHRF1 in the cell, one of its more important functions is its role in the maintenance of DNA methylation patterns by the recruitment of DNMT1. Studies on UHRF1 based on this function have revealed the importance of UHRF1 during the cell cycle. Moreover, based on different studies various factors were described to be involved in the regulation of UHRF1 with different functionalities that can control its binding affinity to different targets on chromatin. These factors are regulated differently in a cell cycle specific manner. In light of this, we propose that UHRF1 has different binding behaviors during the cell cycle in regard to its association with chromatin. In this project, we first analyzed the binding behavior of endogenous UHRF1 from different unsynchronized cell systems in pull-down assays with peptides and oligonucleotides. Moreover, to analyze UHRF1 binding behavior during the cell cycle, we used two different approaches. First we sorted Jurkat and HT1080 cells based on their cell cycle stage using FACS analysis. Additionally, we synchronized HeLa cells to different stages of the cell cycle by chemical treatments, and used extracts from cellsorting and cell synchronization experiments for pull-down assays. We observed that UHRF1 in different cell systems has different preferences in regard to its binding to H3 unmodified and H3K9me3. Moreover, we detected that UHRF1, in general, displays different patterns between different stages of cell cycle; however, we cannot draw a final model for UHRF1 binding pattern during cell cycle.

  19. The Study of Alginate and Whey Protein Hydrolyzed Suplementation Utilization for Cell Release and Microencapsulated Lactobacillus Acidophilus Viability in Probiotic Ice Cream

    Directory of Open Access Journals (Sweden)

    Purwadi Purwadi

    2013-10-01

    Full Text Available The objectives of this research were to increase viability and activity of L. acidophilus encapsulated with alginate and whey protein hydrolyzed for cell release and microencapsulated Lactobacillus acidophilus viability in probiotic ice cream. The methods used were factorial experiment using Completely Randomized Design. Data was analysed with Variance Analysis. The results showed that the interaction between alginate and whey protein hydrolyzed supplemented could be increased the function of CaCl2 and also encapsulated L. acidophilus viability. The used alginate of 1% and whey protein hydrolyzed supplemented of 0,5% produced encapsulated L. acidophilus viability higher than before, but however, the utilization of alginate of 1% and whey protein hydrolyzed supplemented of 0% could release a few cell. Therefore, the utilization of alginate 1% and whey protein hydrolyzed supplemented 0,5% in ice cream produced L. acidophilus highest than other.   Keywords :   Lactobacillus acidophilus, microencapsulation, alginate, whey protein hydrolyzed, cell release, ice cream

  20. Comparative cell cycle transcriptomics reveals synchronization of developmental transcription factor networks in cancer cells

    Science.gov (United States)

    Johard, Helena; Mahdessian, Diana; Fedr, Radek; Marks, Carolyn; Medalová, Jiřina; Souček, Karel; Lundberg, Emma; Linnarsson, Sten; Bryja, Vítězslav; Sekyrova, Petra; Altun, Mikael; Andäng, Michael

    2017-01-01

    The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development. PMID:29228002

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

    Science.gov (United States)

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

    2017-03-31

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

  2. The Cell Cycle: An Activity Using Paper Plates to Represent Time Spent in Phases of the Cell Cycle

    Science.gov (United States)

    Scherer, Yvette D.

    2014-01-01

    In this activity, students are given the opportunity to combine skills in math and geometry for a biology lesson in the cell cycle. Students utilize the data they collect and analyze from an online onion-root-tip activity to create a paper-plate time clock representing a 24-hour cell cycle. By dividing the paper plate into appropriate phases of…

  3. Effect of thymol on Ca²⁺ homeostasis and viability in PC3 human prostate cancer cells.

    Science.gov (United States)

    Yeh, Jeng-Hsien; Chou, Chiang-Ting; Chen, I-Shu; Lu, Ti; Lin, Ko-Long; Yu, Chia-Cheng; Liang, Wei-Zhe; Chang, Hong-Tai; Kuo, Chun-Chi; Ho, Chin-Man; Chang, Wen-Teng; Shieh, Pochuen; Jan, Chung-Ren

    2017-02-28

    Thymol is a phenolic compound that affects physiology in different cell models. However, whether thymol affects Ca²⁺ homeostasis in prostate cancer cells is unknown. The action of this compound on cytosolic Ca²⁺ concentrations ([Ca²⁺]i) and viability in PC3 human prostate cancer cells was explored. The results show that thymol at concentrations of 100-1500 μM caused [Ca²⁺]i rises in a concentration-dependent manner. Removal of extracellular Ca²⁺ reduced thymol’s effect by approximately 80%. Thymol-induced Ca²⁺ entry was confirmed by Mn²⁺ entry-induced quench of fura-2 fluorescence, and was inhibited by approximately 30% by Ca²⁺ entry modulators (nifedipine, econazole, SKF96365), and the protein kinase C (PKC) inhibitor GF109203X. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished thymol-induced [Ca²⁺]i rises. Treatment with thymol also abolished thapsigargin-induced [Ca²⁺]i rises. Thymol-induced Ca²⁺ release from the endoplasmic reticulum was abolished by the phospholipase C (PLC) inhibitor U73122. Thymol at 100-900 μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in PC3 cells, thymol induced [Ca²⁺]i rises by inducing PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ channels and other unknown channels. Thymol also induced Ca²⁺-dissociated cell death.

  4. The cell-cycle state of stem cells determines cell fate propensity.

    Science.gov (United States)

    Pauklin, Siim; Vallier, Ludovic

    2013-09-26

    Self-renewal and differentiation of stem cells are fundamentally associated with cell-cycle progression to enable tissue specification, organ homeostasis, and potentially tumorigenesis. However, technical challenges have impaired the study of the molecular interactions coordinating cell fate choice and cell-cycle progression. Here, we bypass these limitations by using the FUCCI reporter system in human pluripotent stem cells and show that their capacity of differentiation varies during the progression of their cell cycle. These mechanisms are governed by the cell-cycle regulators cyclin D1-3 that control differentiation signals such as the TGF-β-Smad2/3 pathway. Conversely, cell-cycle manipulation using a small molecule directs differentiation of hPSCs and provides an approach to generate cell types with a clinical interest. Our results demonstrate that cell fate decisions are tightly associated with the cell-cycle machinery and reveal insights in the mechanisms synchronizing differentiation and proliferation in developing tissues. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. The Cell-Cycle State of Stem Cells Determines Cell Fate Propensity

    Science.gov (United States)

    Pauklin, Siim; Vallier, Ludovic

    2013-01-01

    Summary Self-renewal and differentiation of stem cells are fundamentally associated with cell-cycle progression to enable tissue specification, organ homeostasis, and potentially tumorigenesis. However, technical challenges have impaired the study of the molecular interactions coordinating cell fate choice and cell-cycle progression. Here, we bypass these limitations by using the FUCCI reporter system in human pluripotent stem cells and show that their capacity of differentiation varies during the progression of their cell cycle. These mechanisms are governed by the cell-cycle regulators cyclin D1–3 that control differentiation signals such as the TGF-β-Smad2/3 pathway. Conversely, cell-cycle manipulation using a small molecule directs differentiation of hPSCs and provides an approach to generate cell types with a clinical interest. Our results demonstrate that cell fate decisions are tightly associated with the cell-cycle machinery and reveal insights in the mechanisms synchronizing differentiation and proliferation in developing tissues. PMID:24074866

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

    Science.gov (United States)

    McKnight, Timothy E [Greenback, TN; Melechko, Anatoli V [Oak Ridge, TN; Simpson, Michael L [Knoxville, TN

    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.

  7. Fucoidan from Sargassum sp. and Fucus vesiculosus reduces cell viability of lung carcinoma and melanoma cells in vitro and activates natural killer cells in mice in vivo.

    Science.gov (United States)

    Ale, Marcel Tutor; Maruyama, Hiroko; Tamauchi, Hidekazu; Mikkelsen, Jørn D; Meyer, Anne S

    2011-10-01

    Fucoidan is known to exhibit crucial biological activities, including anti-tumor activity. In this study, we examined the influence of crude fucoidan extracted from Sargassum sp. (MTA) and Fucus vesiculosus (SIG) on Lewis lung carcinoma cells (LCC) and melanoma B16 cells (MC). In vitro studies were performed using cell viability analysis and showed that SIG and MTA fucoidans significantly decreased the viable number of LCC and MC cells in a dose-response fashion. Histochemical staining showed morphological changes of melanoma B16 cells after exposure to fucoidan. The observed changes were indicative of crude fucoidan induced apoptosis. Male C57BL/6JJCL mice were subjected to daily i.p. injections over 4 days with either SIG or MTA fucoidan (50mg/kg body wt.). The cytolytic activity of natural killer (NK) cells was enhanced by crude fucoidan in a dose-dependent manner as indicated by (51)Cr labeled YAC-1 target cell release. This study provides substantial indications that crude fucoidan exerts bioactive effects on lung and skin cancer model cells in vitro and induces enhanced natural killer cell activity in mice in vivo. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. EBV-encoded EBNA1 regulates cell viability by modulating miR34a-NOX2-ROS signaling in gastric cancer cells.

    Science.gov (United States)

    Kim, Seung-Mi; Hur, Dae Young; Hong, Seung-Woo; Kim, Ji Hyun

    2017-12-16

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a viral protein expressed in all EBV-infected cells that induces malignant transformation. EBNA1 is reported to contribute to tumor progression through an increase in reactive oxygen species via nicotinamide adenine dinucleotide phosphate oxidase. However, the underlying molecular mechanism of EBNA1-induced ROS accumulation in gastric cancer is poorly understood. Here, we demonstrated that miR34a regulation by EBNA1 determined cell fate in EBV-infected gastric cancer cells. ROS content and NOX2 expression were higher in EBNA1-expressing SNU719 cells than in EBNA1-nonexpressing SNU638 cells. Downregulation of NOX2 using siRNA technology in SNU719 cells decreased cell viability and ROS content. Regulation of EBNA1 expression in EBV-associated gastric cancers modulated NOX2 expression, ROS content and cell viability. We also showed that upregulation of NOX2 by EBNA1 was mediated by downregulating miRNA34a. Finally, overexpression of miR34a in EBNA1-expressing SNU719 cells induced typical apoptosis, suggesting that reactivation of miR34a in EBNA1-expressing gastric cancer cells could be a strategy for treatment of EBV-infected gastric cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Apigenin promotes apoptosis, inhibits invasion and induces cell cycle arrest of T24 human bladder cancer cells

    Science.gov (United States)

    2013-01-01

    Background Apigenin (4’,5,7-trihydroxyflavone) was recently shown effective in inhibiting several cancers. The aim of this study was to investigate the effect and mechanism of apigenin in the human bladder cancer cell line T24 for the first time. Methods T24 cells were treated with varying concentrations and time of apigenin. Cell viability was evaluated by MTT assay. Cell motility and invasiveness were assayed by Matrigel migration and invasion assay. Flow cytometry and western blot analysis were used to detect cell apoptosis, cell cycle and signaling pathway. Results The results demonstrated that apigenin suppressed proliferation and inhibited the migration and invasion potential of T24 bladder cancer cells in a dose- and time-dependent manner, which was associated with induced G2/M Phase cell cycle arrest and apoptosis. The mechanism of action is like to involve PI3K/Akt pathway and Bcl-2 family proteins. Apigenin increased caspase-3 activity and PARP cleavage, indicating that apigenin induced apoptosis in a caspase-dependent way. Conclusions These findings suggest that apigenin may be an effective way for treating human bladder cancer. PMID:23724790

  10. Apigenin promotes apoptosis, inhibits invasion and induces cell cycle arrest of T24 human bladder cancer cells.

    Science.gov (United States)

    Zhu, Yi; Mao, Yeqing; Chen, Hong; Lin, Yiwei; Hu, Zhenghui; Wu, Jian; Xu, Xin; Xu, Xianglai; Qin, Jie; Xie, Liping

    2013-06-01

    Apigenin (4',5,7-trihydroxyflavone) was recently shown effective in inhibiting several cancers. The aim of this study was to investigate the effect and mechanism of apigenin in the human bladder cancer cell line T24 for the first time. T24 cells were treated with varying concentrations and time of apigenin. Cell viability was evaluated by MTT assay. Cell motility and invasiveness were assayed by Matrigel migration and invasion assay. Flow cytometry and western blot analysis were used to detect cell apoptosis, cell cycle and signaling pathway. The results demonstrated that apigenin suppressed proliferation and inhibited the migration and invasion potential of T24 bladder cancer cells in a dose- and time-dependent manner, which was associated with induced G2/M Phase cell cycle arrest and apoptosis. The mechanism of action is like to involve PI3K/Akt pathway and Bcl-2 family proteins. Apigenin increased caspase-3 activity and PARP cleavage, indicating that apigenin induced apoptosis in a caspase-dependent way. These findings suggest that apigenin may be an effective way for treating human bladder cancer.

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

    Science.gov (United States)

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

    2014-02-01

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

  12. The oil-resin of the tropical rainforest tree Copaifera langsdorffii reduces cell viability, changes cell morphology and induces cell death in human endometriotic stromal cultures.

    Science.gov (United States)

    Henriques da Silva, Julianna; Borges, Vinicius Raphael de Almeida; Pereira, Leonardo da Cunha Boldrini; Ferrari, Renato; de Mattos, Rômulo Medina; Barros, Eliane Gouveia de Oliveira; Palmero, Celia Yelimar; Fernandes, Patricia Dias; de Carvalho, Patricia Ribeiro; Pereira de Sousa, Valeria; Cabral, Lucio Mendes; Nasciutti, Luiz Eurico

    2015-12-01

    The hormonal treatment for endometriosis frequently fails to completely eradicate endometriotic implants. A new therapeutic treatment is needed. This study investigates the in-vitro effect of Copaifera langsdorffii oil-resin on human eutopic and ectopic endometrium stromal cell cultures (EuESCs and EctESCs). A nanocomposite system containing the copaiba oil-resin (NanoCOR) was developed and acute toxicity test was performed. Endometrial stromal cells (ESCs) from non-endometriotics controls (CESCs), EuESCs and EctESCs were isolated and treated with different concentrations of NanoCOR, at different time intervals to evaluate its effect on cell morphology, proliferation, viability, necrosis and apoptosis induction. When treated with 50 μg/ml of NanoCOR, the morphology of EctESCs changed, as the actin microfilaments were disorganized, disassembled or disrupted. Moreover, at 24 h of treatment with NanoCOR, the EctESCs viability was inhibited, and a significant number of these cells underwent apoptosis. In EuESCs, these effects were observed only at 48 h. Finally, the treatment of EctESCs with NanoCOR increased the lactate dehydrogenase release into the extracellular medium more than in EuESCs. Our data indicate that NanoCOR has a greater impact on the behaviour of human endometriotic stromal cells than on the eutopic endometrium stromal cells, supporting the idea that NanoCOR should be further investigated as a novel and valuable alternative to treat endometriosis. © 2015 Royal Pharmaceutical Society.

  13. Complete human serum maintains viability and chondrogenic potential of human synovial stem cells: suitable conditions for transplantation.

    Science.gov (United States)

    Mizuno, Mitsuru; Katano, Hisako; Otabe, Koji; Komori, Keiichiro; Kohno, Yuji; Fujii, Shizuka; Ozeki, Nobutake; Horie, Masafumi; Tsuji, Kunikazu; Koga, Hideyuki; Muneta, Takeshi; Sekiya, Ichiro

    2017-06-13

    In our clinical practice, we perform transplantations of autologous synovial mesenchymal stem cells (MSCs) for cartilage and meniscus regenerative medicine. One of the most important issues to ensuring clinical efficacy involves the transport of synovial MSCs from the processing facility to the clinic. Complete human serum (100% human serum) is an attractive candidate material in which to suspend synovial MSCs for their preservation during transport. The purpose of this study was to investigate whether complete human serum maintained MSC viability and chondrogenic potential and to examine the optimal temperature conditions for the preservation of human synovial MSCs. Human synovium was harvested from the knees of 14 donors with osteoarthritis during total knee arthroplasty. Passage 2 synovial MSCs were suspended at 2 million cells/100 μL in Ringer's solution or complete human serum at 4, 13, and 37 °C for 48 h. These cells were analyzed for live cell rates, cell surface marker expression, metabolic activity, proliferation, and adipogenic, calcification, and chondrogenic differentiation potentials before and after preservation. After preservation, synovial MSCs maintained higher live cell rates in human serum than in Ringer's solution at 4 and 13 °C. Synovial MSCs preserved in human serum at 4 and 13 °C also maintained high ratios of propidium iodide(-) and annexin V(-) cells. MSC surface marker expression was not altered in cells preserved at 4 and 13 °C. The metabolic activities of cells preserved in human serum at 4 and 13 °C was maintained, while significantly reduced in other conditions. Replated MSCs retained their proliferation ability when preserved in human serum at 4 and 13 °C. Adipogenesis and calcification potential could be observed in cells preserved in each condition, whereas chondrogenic potential was retained only in cells preserved in human serum at 4 and 13 °C. The viability and chondrogenic potential of synovial MSCs were

  14. Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

    Energy Technology Data Exchange (ETDEWEB)

    Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey; Dooner, Gerri J.; Abedi, Mehrdad; Colvin, Gerald; Liu, Qin; Weier, Heinz-Ulli; Dooner, Mark S.; Quesenberry, Peter J.

    2007-12-31

    Green-fluorescent protein (GFP) labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit cell cycle by exposure to IL-3, IL-6, IL-11 and steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. These cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. Thus marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine induced cell cycle transit. Previous studies have shown the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.

  15. When does the lung die? Kfc, cell viability, and adenine nucleotide changes in the circulation-arrested rat lung.

    Science.gov (United States)

    Jones, D R; Becker, R M; Hoffmann, S C; Lemasters, J J; Egan, T M

    1997-07-01

    Lungs harvested from cadaveric circulation-arrested donors may increase the donor pool for lung transplantation. To determine the degree and time course of ischemia-reperfusion injury, we evaluated the effect of O2 ventilation on capillary permeability [capillary filtration coefficient (Kfc)], cell viability, and total adenine nucleotide (TAN) levels in in situ circulation-arrested rat lungs. Kfc increased with increasing postmortem ischemic time (r = 0.88). Lungs ventilated with O2 1 h postmortem had similar Kfc and wet-to-dry ratios as controls. Nonventilated lungs had threefold (P Kfc at 30 and 60 min postmortem compared with controls. Cell viability decreased in all groups except for 30-min postmortem O2-ventilated lungs. TAN levels decreased with increasing ischemic time, particularly in nonventilated lungs. Loss of adenine nucleotides correlated with increasing Kfc values (r = 0.76). This study indicates that lungs retrieved 1 h postmortem may have normal Kfc with preharvest O2 ventilation. The relationship between Kfc and TAN suggests that vascular permeability may be related to lung TAN levels.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Procalcitonin Impairs Liver Cell Viability and Function In Vitro: A Potential New Mechanism of Liver Dysfunction and Failure during Sepsis?

    Directory of Open Access Journals (Sweden)

    Martin Sauer

    2017-01-01

    Full Text Available Purpose. Liver dysfunction and failure are severe complications of sepsis and result in poor outcome and increased mortality. The underlying pathologic mechanisms of hepatocyte dysfunction and necrosis during sepsis are only incompletely understood. Here, we investigated whether procalcitonin, a biomarker of sepsis, modulates liver cell function and viability. Materials and Methods. Employing a previously characterized and patented biosensor system evaluating hepatocyte toxicity in vitro, human hepatocellular carcinoma cells (HepG2/C3A were exposed to 0.01–50 ng/mL procalcitonin for 2×72 h and evaluated for proliferation, necrosis, metabolic activity, cellular integrity, microalbumin synthesis, and detoxification capacity. Acetaminophen served as positive control. For further standardization, procalcitonin effects were confirmed in a cellular toxicology assay panel employing L929 fibroblasts. Data were analyzed using ANOVA/Tukey’s test. Results. Already at concentrations as low as 0.25 ng/mL, procalcitonin induced HepG2/C3A necrosis (P<0.05 and reduced metabolic activity, cellular integrity, synthesis, and detoxification capacity (all P<0.001. Comparable effects were obtained employing L929 fibroblasts. Conclusion. We provide evidence for procalcitonin to directly impair function and viability of human hepatocytes and exert general cytotoxicity in vitro. Therapeutical targeting of procalcitonin could thus display a novel approach to reduce incidence of liver dysfunction and failure during sepsis and lower morbidity and mortality of septic patients.

  18. Cell viability, collagen synthesis and cytokine expression in human osteoblasts following incubation with generated wear particles using different bone cements.

    Science.gov (United States)

    Schulze, Christoph; Lochner, Katrin; Jonitz, Anika; Lenz, Robert; Duettmann, Oliver; Hansmann, Doris; Bader, Rainer

    2013-07-01

    In total hip arthroplasty, wear particles generated at articulating surfaces and interfaces between bone, cement and implants have a negative impact on osteoblasts, leading to osteolysis and implant loosening. The aim of this experimental study was to determine the effects of particulate wear debris generated at the interface between straight stainless steel hip stems (Exeter(®)) and three different bone cements (Palacos(®) R, Simplex™ P and Cemex(®) Genta) on cell viability, collagen synthesis and cytokine expression in human osteoblasts. Primary osteoblasts were treated with various concentrations of wear particles. The synthesis of procollagen type I and different cytokines was analysed, and markers for apoptosis and necrosis were also detected. The cytokine synthesis rates in the osteoblasts were initially increased and varied, depending on incubation time and particle concentration. Specific differences in the synthesis rates of interleukin (IL)‑6, IL-8, vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) were observed with the different bone cements examined. The negative effect of the particles on the synthesis of procollagen type I and increased rates of cell apoptosis and necrosis were observed with all three cements analysed. Our present data suggest that wear particles from the interface between the total hip stem and bone cement have a significant effect on viability, cytokine expression and collagen synthesis in human osteoblasts, depending on the bone cement used.

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

    Energy Technology Data Exchange (ETDEWEB)

    Genz, Berit [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany); Thomas, Maria [Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart (Germany); Pützer, Brigitte M. [Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical Center, Rostock (Germany); Siatkowski, Marcin; Fuellen, Georg [Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Center, Rostock (Germany); Vollmar, Brigitte [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany); Abshagen, Kerstin, E-mail: kerstin.abshagen@uni-rostock.de [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany)

    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.

  20. mTOR inhibition increases cell viability via autophagy induction during endoplasmic reticulum stress – An experimental and modeling study

    Directory of Open Access Journals (Sweden)

    Orsolya Kapuy

    2014-01-01

    Full Text Available Unfolded or misfolded proteins in the endoplasmic reticulum (ER trigger an adaptive ER stress response known as unfolded protein response (UPR. Depending on the severity of ER stress, either autophagy-controlled survival or apoptotic cell death can be induced. The molecular mechanisms by which UPR controls multiple fate decisions have started to emerge. One such molecular mechanism involves a master regulator of cell growth, mammalian target of rapamycin (mTOR, which paradoxically is shown to have pro-apoptotic role by mutually interacting with ER stress response. How the interconnections between UPR and mTOR influence the dynamics of autophagy and apoptosis activation is still unclear. Here we make an attempt to explore this problem by using experiments and mathematical modeling. The effect of perturbed mTOR activity in ER stressed cells was studied on autophagy and cell viability by using agents causing mTOR pathway inhibition (such as rapamycin or metyrapone. We observed that mTOR inhibition led to an increase in cell viability and was accompanied by an increase in autophagic activity. It was also shown that autophagy was activated under conditions of severe ER stress but that in the latter phase of stress it was inhibited at the time of apoptosis activation. Our mathematical model shows that both the activation threshold and temporal dynamics of autophagy and apoptosis inducers are sensitive to variation in mTOR activity. These results confirm that autophagy has cytoprotective role and is activated in mutually exclusive manner with respect to ER stress levels.

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

    Science.gov (United States)

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

    2013-08-28

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

  2. Antimicrobial peptide LL-37 promotes the viability and invasion of skin squamous cell carcinoma by upregulating YB-1

    Science.gov (United States)

    Wang, Wei; Zheng, Yan; Jia, Jinjing; Li, Changji; Duan, Qiqi; Li, Ruilian; Wang, Xin; Shao, Yongping; Chen, Caifeng; Yan, Huling

    2017-01-01

    Antimicrobial peptide LL-37 serves a function in the host defense against microbial invasion, and also regulates cell proliferation, immune activity and angiogenesis. Previous studies have reported that LL-37 participates in the development of numerous tumour types, such as ovarian cancer, lung cancer, melanoma and breast cancer. However, the function of LL-37 in the development of skin squamous cell carcinoma (SCC) has not yet been fully elucidated. The aim of the current study was to investigate how LL-37 promotes the expression of Y-box binding protein 1 (YB-1) in SCC. Short interfering RNA (siRNA) was used to inhibit the expression of YB-1, and in vitro MTT and Transwell migration assays were used to evaluate the effect of reduced YB-1 on the viability and invasion of A431 cells. A431 cells were stimulated with LL-37, and quantitative polymerase chain reaction, immunofluorescence and western blot analyses were used to detect changes in YB-1 expression. Mitogen-activated protein kinase kinase, mitogen-activated protein kinase and nuclear factor (NF)-κB signaling pathway inhibitors were also used to evaluate the mechanism of LL-37-induced YB-1 protein expression. It was found that YB-1 expression was increased in SCC tissue compared with normal tissue. Inhibiting YB-1 expression using siRNA significantly reduced the viability and suppressed the invasion of tumour cells (P1 protein expression (P1, LL-37 can promote the occurrence and development of SCC, and this process involves the NF-κB signaling pathway. PMID:28672959

  3. Effects of voluntary exercise on the viability, proliferation and BDNF levels of bone marrow stromal cells in rat pups born from morphine- dependent mothers during pregnancy.

    Science.gov (United States)

    Haydari, Sakineh; Safari, Manouchehr; Zarbakhsh, Sam; Bandegi, Ahmad Reza; Miladi-Gorji, Hossein

    2016-11-10

    This study was designed to investigate whether free access to a running wheel during pregnancy in morphine-dependent mothers would influence the viability, proliferation and BDNF levels of bone marrow stromal cells in rat pups. Pregnant rats were made dependent by chronic administration of morphine in drinking water simultaneously with free access to a running wheel. Male pups are weaned at 21days of birth and their bones marrows were aspirated from the femurs and tibias and also the bone marrow stromal cells (BMSCs) cultured. MTT assay was used to determine cell viability and proliferation rate. The level of BDNF was measured in the supernant of BMSCs culture by ELISA. The sedentary morphine-dependent mothers' pups showed a significant increase in the percentage cell viability and proliferation rate and also a significant decrease in the BDNF protein levels in BMSCs. The rat pups borne from exercising the control and morphine-dependent mothers exhibited an increase in the percentage viability, proliferation rate and BDNF levels of the BMSCs. This study showed that maternal exercise during pregnancy in morphine-dependent and non-dependent mothers, with increasing of BDNF levels increased the proliferation and viability of BMSCs in the rat pups. Also, chronic administration of morphine during pregnancy was able to increase the proliferation and viability of BMSCs in the rat pups. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. A Critical Review on the Effect of Docosahexaenoic Acid (DHA) on Cancer Cell Cycle Progression.

    Science.gov (United States)

    Newell, Marnie; Baker, Kristi; Postovit, Lynne M; Field, Catherine J

    2017-08-17

    Globally, there were 14.1 million new cancer diagnoses and 8.2 million cancer deaths in 2012. For many cancers, conventional therapies are limited in their successes and an improved understanding of disease progression is needed in conjunction with exploration of alternative therapies. The long chain polyunsaturated fatty acid, docosahexaenoic acid (DHA), has been shown to enhance many cellular responses that reduce cancer cell viability and decrease proliferation both in vitro and in vivo. A small number of studies suggest that DHA improves chemotherapy outcomes in cancer patients. It is readily incorporated into cancer cell membranes and, as a result there has been considerable research regarding cell membrane initiated events. For example, DHA has been shown to mediate the induction of apoptosis/reduction of proliferation in vitro and in vivo. However, there is limited research into the effect of DHA on cell cycle regulation in cancer cells and the mechanism(s) by which DHA acts are not fully understood. The purpose of the current review is to provide a critical examination of the literature investigating the ability of DHA to stall progression during different cell cycle phases in cancer cells, as well as the consequences that these changes may have on tumour growth, independently and in conjunction with chemotherapy.

  5. Dual Pressure versus Hybrid Recuperation in an Integrated Solid Oxide Fuel Cell Cycle – Steam Cycle

    DEFF Research Database (Denmark)

    Rokni, Masoud

    2014-01-01

    steam in a HRSG (heat recovery steam generator). The bottoming steam cycle was modeled with two configurations: (1) a simple single pressure level and (2) a dual pressure level with both a reheat and a pre-heater. The SOFC stacks in the present SOFC-ST hybrid cycles were not pressurized. The dual......A SOFC (solid oxide fuel cell) cycle running on natural gas was integrated with a ST (steam turbine) cycle. The fuel is desulfurized and pre-reformed before entering the SOFC. A burner was used to combust the remaining fuel after the SOFC stacks. The off-gases from the burner were used to produce...... pressure configuration steam cycle combined with SOFC cycle (SOFC-ST) was new and has not been studied previously. In each of the configuration, a hybrid recuperator was used to recovery the remaining energy of the off-gases after the HRSG. Thus, four different plants system setups were compared to each...

  6. Protein kinase C signaling and cell cycle regulation

    Science.gov (United States)

    Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about 30 years. However, despite the wealth of information on PKC-mediated control of, T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. The outcome of PKC activation is highly context-dependent, with the precise cell cycle target(s) and overall effects determined by the specific isozyme involved, the timing of PKC activation, the cell type, and the signaling environment. Although PKCs can regulate all stages of the cell cycle, they appear to predominantly affect G0/G1 and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (cdks), cdk inhibitors and cdc25 phosphatases; however, evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of PKC-regulated cell cycle-specific effects. Several PKC isozymes can target Cip/Kip proteins to control G0/G1 → S and/or G2 → M transit, while effects on D-type cyclins regulate entry into and progression through G1. Analysis of PKC signaling in T cells has largely focused on its roles in T cell activation; thus, observed cell cycle effects are mainly positive. A prominent role is emerging for PKCθ, with non-redundant functions of other isozymes also described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in these cells. As in other cell types, context-dependent effects of individual isozymes have been noted in T cells, and Cip/Kip cdk inhibitors and D-type cyclins appear to be major PKC targets. Future studies are anticipated to take advantage of the similarities between these various systems to enhance understanding of PKC-mediated cell cycle regulation in T cells. PMID

  7. Protective effect of p53 on the viability of intervertebral disc nucleus pulposus cells under low glucose condition.

    Science.gov (United States)

    Xiong, Xifeng; Dai, Libing; Liang, Weiguo; Zhang, Jinli; Qin, Shengnan; Cao, Wenjuan; Ye, Dongping; Liang, Peihong; Liu, Zhihe

    2017-09-02

    P53 is a famous cancer suppressor and plays key roles in metabolism. Intervertebral disc (IVD) is the largest avascular cartilaginous structure in humans and its degeneration is a common cause of spine diseases initiated from damaged nucleus pulposus (NP) cells. The potential cause of disc degeneration has been attributed to aging, genetic factors, mechanical factors and nutrition. In this study, we found that p53 decreased and leaked to the cytoplasm in NP cells as the glucose level decreases, in contrast to cancer cells in which p53 increases and concentrates to the nuclei. Comparing with in p53 knockdown NP cells, relative high p53 expression in normal control NP cells inhibited autophagy and the pentose phosphate pathway. Furthermore, the expression of Sox 9 and type II collagen were higher in p53 normal control than p53 knockdown NP cells. Based on these results, we believe that relative high p53 facilitates NP cell viability and integrity. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

    Science.gov (United States)

    JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

    2016-06-17

    Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.

  9. Diacerein retards cell growth of chondrosarcoma cells at the G2/M cell cycle checkpoint via cyclin B1/CDK1 and CDK2 downregulation.

    Science.gov (United States)

    Lohberger, Birgit; Leithner, Andreas; Stuendl, Nicole; Kaltenegger, Heike; Kullich, Werner; Steinecker-Frohnwieser, Bibiane

    2015-11-10

    Chondrosarcoma is characterized for its lack of response to conventional cytotoxic chemotherapy, propensity for developing lung metastases, and low rates of survival. Research within the field of development and expansion of new treatment options for unresectable or metastatic diseases is of particular priority. Diacerein, a symptomatic slow acting drug in osteoarthritis (SYSADOA), implicates a therapeutic benefit for the treatment of chondrosarcoma by an antitumor activity. After treatment with diacerein the growth behaviour of the cells was analyzed with the xCELLigence system and MTS assay. Cell cycle was examined using flow cytometric analysis, RT-PCR, and western blot analysis of specific checkpoint regulators. The status for phosophorylation of mitogen-activated protein kinases (MAPKs) was analyzed with a proteome profiler assay. In addition, the possible impact of diacerein on apoptosis was investigated using cleaved caspase 3 and Annexin V/PI flow cytometric analysis. Diacerein decreased the cell viability and the cell proliferation in two different chondrosarcoma cell lines in a dose dependent manner. Flow cytometric analysis showed a classical G2/M arrest. mRNA and protein analysis revealed that diacerein induced a down-regulation of the cyclin B1-CDK1 complex and a reduction in CDK2 expression. Furthermore, diacerein treatment increased the phosphorylation of p38α and p38β MAPKs, and Akt1, Akt2, and Akt 3 in SW-1353, whereas in Cal-78 the opposite effect has been demonstrated. These observations accordingly to our cell cycle flow cytometric analysis and protein expression data may explain the G2/M phase arrest. In addition, no apoptotic induction after diacerein treatment, neither in the Cal-78 nor in the SW-1353 cell line was observed. Our results demonstrate for the first time that the SYSADOA diacerein decreased the viability of human chondrosarcoma cells and induces G2/M cell cycle arrest by CDK1/cyclin B1 down-regulation.

  10. A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays.

    Science.gov (United States)

    Chan, Grace Ka Yan; Kleinheinz, Tracy L; Peterson, David; Moffat, John G

    2013-01-01

    In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1) significant underestimation of compound potency and efficacy, and 2) non-monotonic dose-response curves due to concentration-dependent phenotypic 'switching'. In particular, potency and efficacy of DNA synthesis-targeting agents such as gemcitabine and etoposide could be profoundly underestimated by ATP and MTS-reduction assays. In the same image-based assay we showed that drug-induced increases in ATP content were associated with increased cell size and proportionate increases in mitochondrial content and respiratory flux concomitant with cell cycle arrest. Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity.

  11. Metformin Induces Growth Inhibition and Cell Cycle Arrest by Upregulating MicroRNA34a in Renal Cancer Cells.

    Science.gov (United States)

    Xie, Wei; Wang, Lei; Sheng, Halei; Qiu, Jing; Zhang, Di; Zhang, Le; Yang, Fan; Tang, Dahai; Zhang, Kebin

    2017-01-03

    BACKGROUND Metformin is a widely used biguanide drug for the treatment of type 2 diabetes. It has been revaluated as a potential anti-cancer drug with promising activity in various tumors. However, the precise mechanisms underlying the suppression of cancer cells by metformin remain not well understood. MATERIAL AND METHODS In this study, human renal cell carcinoma cell line ACHN was used to investigate the anti-proliferation effect of metformin. A cell counting kit-8 assay was used to detect the cell viability. The cell cycle distribution and apoptosis were analyzed by flow cytometry. The expression of cyclin D1 and p27KIP1 was detected by Western blot. The underlying mechanism involving miRNA34a was further investigated by quantitative RT-PCR and transfection with miRNA inhibitor specific for miRNA34a in ACHN, 769-P, and A498 cells. RESULTS Metformin could significantly inhibit the proliferation of ACHN cells in a dose- and time-dependent manner. In addition, the results showed that metformin induced G0/G1 phase arrest and delayed entry into S phase in ACHN cells. It was shown that metformin downregulates the expression of cyclin D1 and increases the p27KIP1 level. Furthermore, metformin increased ACHN cell death. Lastly, miRNA34a was found to be upregulated by metformin in ACHN, 769-P, and A498 cells. Subsequently, it was demonstrated that inhibition of miRNA34a could partially attenuate the suppressive effect of metformin on renal cancer cell proliferation. CONCLUSIONS The study data revealed that metformin induced cell growth inhibition and cell cycle arrest partially by upregulating miRNA34a in renal cancer cells.

  12. The apoptosis linked gene ALG-2 is dysregulated in tumors of various origin and contributes to cancer cell viability

    DEFF Research Database (Denmark)

    la Cour, Jonas; Høj, Berit Rahbek; Mollerup, Jens

    2008-01-01

    The apoptosis linked gene-2 (ALG-2), discovered as a proapoptotic calcium binding protein, has recently been found upregulated in lung cancer tissue indicating that this protein may play a role in the pathology of cancer cells and/or may be a tumor marker. Using immunohistochemistry on tissue...... microarrays we analysed the expression of ALG-2 in 7371 tumor tissue samples of various origin as well as in 749 normal tissue samples. Most notably, ALG-2 was upregulated in mesenchymal tumors. No correlation was found between ALG-2 staining intensity and survival of patients with lung, breast or colon...... cancer. siRNA mediated ALG-2 downregulation led to a significant reduction in viability of HeLa cells indicating that ALG-2 may contribute to tumor development and expansion....

  13. Human Bone Marrow-Derived Mesenchymal Cell Reactions to 316L Stainless Steel: An in Vitro Study on Cell Viability and Interleukin-6 Expression.

    Science.gov (United States)

    Anwar, Iwan Budiwan; Santoso, Asep; Saputra, Eko; Ismail, Rifky; Jamari, J; Van der Heide, Emile

    2017-06-01

    Purpose: Human bone marrow-derived mesenchymal cell (hBMC) reactions to 316L stainless steel (316L-SS) have never been evaluated. The objective of this study was to assess cell viability and interleukin-6 expression of hBMC cultures upon treatment with a 316L-SS implant. Methods: A cytotoxicity analysis was conducted with a 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium (MTT) assay after a period of 24, 48 and 72 hours of incubation. Expression of interleukin-6 was measured using enzyme-linked immunosorbent assay (ELISA). Results: Cell viability measurement was performed via IC50 formula. All treatment group showed a > 50 % cell viability with a range of 56,5 - 96,9 % at 24 hours, 51,8-77,3% at 48 hours and 70,1- 120 % at 72 hours. Interleukin-6 expression was downregulated subsequent to treatment with 316L-SS compared to the control group. Conclusion: We found that 316L-SS did not exhibit toxicity towards hBMC culture.

  14. Effect of laser energy, substrate film thickness and bioink viscosity on viability of endothelial cells printed by Laser-Assisted Bioprinting

    Science.gov (United States)

    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.

  15. Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability.

    Science.gov (United States)

    Park, Jina; Jin, Sung Il; Kim, Hyung Min; Ahn, Junhyoung; Kim, Yeon-Gu; Lee, Eun Gyo; Kim, Min-Gon; Shin, Yong-Beom

    2015-02-15

    We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

    Science.gov (United States)

    Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

    2015-01-01

    It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life

  17. Krebs cycle rewired for macrophage and dendritic cell effector functions.

    Science.gov (United States)

    Ryan, Dylan Gerard; O'Neill, Luke A J

    2017-10-01

    The Krebs cycle is an amphibolic pathway operating in the mitochondrial matrix of all eukaryotic organisms. In response to proinflammatory stimuli, macrophages and dendritic cells undergo profound metabolic remodelling to support the biosynthetic and bioenergetic requirements of the cell. Recently, it has been discovered that this metabolic shift also involves the rewiring of the Krebs cycle to regulate cellular metabolic flux and the accumulation of Krebs cycle intermediates, notably, citrate, succinate and fumarate. Interestingly, a new role for Krebs cycle intermediates as signalling molecules and immunomodulators that dictate the inflammatory response has begun to emerge. This review will discuss the latest developments in Krebs cycle rewiring and immune cell effector functions, with a particular focus on the regulation of cytokine production. © 2017 Federation of European Biochemical Societies.

  18. Cell-cycle inhibition by Helicobacter pylori L-asparaginase.

    Directory of Open Access Journals (Sweden)

    Claudia Scotti

    Full Text Available Helicobacter pylori (H. pylori is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application.

  19. Influence of boron addition to Ti-13Zr-13Nb alloy on MG63 osteoblast cell viability and protein adsorption.

    Science.gov (United States)

    Majumdar, P; Singh, S B; Dhara, S; Chakraborty, M

    2015-01-01

    Cell proliferation, cell morphology and protein adsorption on near β-type Ti-13Zr-13Nb (TZN) alloy and Ti-13Zr-13Nb-0.5B (TZNB) composite have been investigated and compared to evaluate the effect of boron addition which has been added to the Ti alloy to improve their poor tribological properties by forming in situ TiB precipitates. MG63 cell proliferation on substrates with different chemistry but the same topography was compared. The MTT assay test showed that the cell viability on the TZN alloy was higher than the boron containing TZNB composite after 36 h of incubation and the difference was pronounced after 7 days. However, both the materials showed substantially higher cell attachment than the control (polystyrene). For the same period of incubation in fetal bovine serum (FBS), the amount of protein adsorbed on the surface of boron free TZN samples was higher than that in the case of boron containing TZNB composite. The presence of boron in the TZN alloy influenced protein adsorption and cell response and they are lower in TZNB than in TZN as a result of the associated difference in chemical characteristics. Copyright © 2014. Published by Elsevier B.V.

  20. Kinase activity ranking using phosphoproteomics data (KARP) quantifies the contribution of protein kinases to the regulation of cell viability.

    Science.gov (United States)

    Wilkes, Edmund H; Casado, Pedro; Rajeeve, Vinothini; Cutillas, Pedro R

    2017-09-01

    Cell survival is regulated by a signaling network driven by the activity of protein kinases; however, determining the contribution that each kinase in the network makes to such regulation remains challenging. Here, we report a computational approach that uses mass spectrometry-based phosphoproteomics data to rank protein kinases based on their contribution to cell regulation. We found that the scores returned by this algorithm, which we have termed kinase activity ranking using phosphoproteomics data (KARP), were a quantitative measure of the contribution that individual kinases make to the signaling output. Application of KARP to the analysis of eight hematological cell lines revealed that cyclin-dependent kinase (CDK) 1/2, casein kinase (CK) 2, extracellular signal-related kinase (ERK), and p21-activated kinase (PAK) were the most frequently highly ranked kinases in these cell models. The patterns of kinase activation were cell-line specific yet showed a significant association with cell viability as a function of kinase inhibitor treatment. Thus, our study exemplifies KARP as an untargeted approach to empirically and systematically identify regulatory kinases within signaling networks. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. High-Dose {sup 111}In Induces G1 Cell Cycle Arrest and Cell Death in Rat Bone Marrow Mesenchymal Stem Cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Bok Nam; Shim, Woo Young; Ahn, Young Hwan; Lee, Jae Ho; Yoon, Joon Kee [Ajou Univ. School of Medicine, Suwon (Korea, Republic of)

    2012-06-15

    This study was performed to evaluate the effect of {sup 111}In-labeling on the cell growth, cycle and viability of bone marrow mesenchymal stem cells (BMSCs). Rat BMSCs were labeled with various doses of {sup 111}In (0.4-11.1 Bq/cell). The growth curve of {sup 111}In-BMSCs was obtained up to 14th day of labeling. The cell cycle was evaluated by 5-bromo-2-deoxyuridine (BrdU) labeling or prospidium iodide (PI) staining. Senescent cells were counted under a light microscope after staining with 5-bromo-4-chloro-3-indolyl-{sup D-}galactopyranoside. Flow cytometry was performed to measure apoptotic and necrotic fractions after staining with annexin V-FITC and PI. The growth of BMSCs labeled with higher doses of {sup 111}In (4.4 or 11.1 Bq/cell) was significantly inhibited from the 3rd day of labeling. Flow cytometry revealed less BrdU-positive BMSCs at 11.1 Bq {sup 111}In/cell (9.07%/3.18%) on the 14th day (control=1.60%/0.39%). However, no cellular senescence was visualized up to the 14th day. A high dose of {sup 111}In-labeling induced cell cycle arrest and death in BMSCs; therefore, it should be used with a careful dosimetry in case of applying it to humans.

  2. Genome-wide examination of myoblast cell cycle withdrawal duringdifferentiation

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Xun; Collier, John Michael; Hlaing, Myint; Zhang, Leanne; Delshad, Elizabeth H.; Bristow, James; Bernstein, Harold S.

    2002-12-02

    Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40 percent fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly

  3. Technoeconomy of different solid oxide fuel cell based hybrid cycle

    DEFF Research Database (Denmark)

    Rokni, Masoud

    2014-01-01

    Gas turbine, steam turbine and heat engine (Stirling engine) is used as bottoming cycle for a solid oxide fuel cell plant to compare different plants efficiencies, CO2 emissionsand plants cost in terms of $/kW. Each plant is then integrated with biomass gasification and finally six plants...... configurations are compared with each other. Technoeconomy is used when calculating the cost if the plants. It is found that when a solid oxide fuel cell plant is combined with a gas turbine cycle then the plant efficiency will be the highest one while if a biomass gasification plant is integrated...... with these hybrid cycles then integrated biomass gasification with solid oxide fuel cell and steam cycle will have the highest plant efficiency. The cost of solid oxide fuel cell with steam plant is found to be the lowest one with a value of about 1030$/kW....

  4. Cellular Clocks : Coupled Circadian Dispatch and Cell Division Cycles

    NARCIS (Netherlands)

    Merrow, Martha; Roenneberg, Till

    2004-01-01

    Gating of cell division by the circadian clock is well known, yet its mechanism is little understood. Genetically tractable model systems have led to new hypotheses and questions concerning the coupling of these two cellular cycles.

  5. Kinetics of cadmium accumulation and its effects on microtubule integrity and cell viability in the seagrass Cymodocea nodosa

    Energy Technology Data Exchange (ETDEWEB)

    Malea, Paraskevi, E-mail: malea@bio.auth.gr [Department of Botany, School of Biology, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki (Greece); Adamakis, Ioannis-Dimosthenis S. [Department of Botany, School of Biology, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki (Greece); Kevrekidis, Theodoros [Laboratory of Environmental Research and Education, Democritus University of Thrace, Nea Hili, GR-68100 Alexandroupolis (Greece)

    2013-11-15

    Highlights: •Cd effect on microtubules and viability of seagrass leaf cells was assessed. •The Michaelis–Menten equation satisfactorily dercribed the kinetics of Cd uptake. •Cd depolymerized MTs after 3–9 d of exposure, cell death occurred at later time. •Toxicity appeared to depend on Cd uptake rate rather than on tissue Cd content. •MTs can be used as biomarker of Cd stress and uptake rate for predicting effects. -- Abstract: The kinetics of cadmium accumulation and its effects on microtubule cytoskeleton and cell viability in leaf blades of the seagrass Cymodocea nodosa were investigated under laboratory conditions in exposure concentrations ranging from 0.5 to 40 mg L{sup −1}. An initial rapid accumulation of cadmium was followed by a steady state. The Michaelis–Menten model adequately described metal accumulation; equilibrium concentration and uptake velocity tended to increase, whereas bioconcentration factor at equilibrium to decrease, as the exposure concentration increased. Cadmium depolymerized microtubules after 3–9 d of exposure, depending on trace metal concentration, indicating that microtubules could be used as an early biomarker of cadmium stress; cell death, occurring at later time than microtubule disturbance, was also observed. Microtubule depolymerization expressed as percentage of reduction of fluorescence intensity and cell mortality expressed as percentage of live cells increased with time. The lowest experimental tissue concentration associated with the onset of microtubule depolymerization and cell death (98.5–128.9 μg g{sup −1} dry wt, 0.5 mg L{sup −1} treatment, 7th and 9th d) was within the wide range of reported cadmium concentrations in leaves of seagrass species from various geographical areas. This lowest tissue concentration was exceeded up to the 3rd d at higher exposure concentrations, but toxic effects were generally detected at later time. The time periods required for the onset of depolymerization and

  6. PEP-1-CAT-Transduced Mesenchymal Stem Cells Acquire an Enhanced Viability and Promote Ischemia-Induced Angiogenesis

    Science.gov (United States)

    Zhang, Lei; Dong, Xiao-Wei; Wang, Jia-Ning; Tang, Jun-Ming; Yang, Jian-Ye; Guo, Ling-Yun; Zheng, Fei; Kong, Xia; Huang, Yong-Zhang; Chen, Shi-You

    2012-01-01

    Objective Poor survival of mesenchymal stem cells (MSC) compromised the efficacy of stem cell therapy for ischemic diseases. The aim of this study is to investigate the role of PEP-1-CAT transduction in MSC survival and its effect on ischemia-induced angiogenesis. Methods MSC apoptosis was evaluated by DAPI staining and quantified by Annexin V and PI double staining and Flow Cytometry. Malondialdehyde (MDA) content, lactate dehydrogenase (LDH) release, and Superoxide Dismutase (SOD) activities were simultaneously measured. MSC mitochondrial membrane potential was analyzed with JC-1 staining. MSC survival in rat muscles with gender-mismatched transplantation of the MSC after lower limb ischemia was assessed by detecting SRY expression. MSC apoptosis in ischemic area was determined by TUNEL assay. The effect of PEP-1-CAT-transduced MSC on angiogenesis in vivo was determined in the lower limb ischemia model. Results PEP-1-CAT transduction decreased MSC apoptosis rate while down-regulating MDA content and blocking LDH release as compared to the treatment with H2O2 or CAT. However, SOD activity was up-regulated in PEP-1-CAT-transduced cells. Consistent with its effect on MSC apoptosis, PEP-1-CAT restored H2O2-attenuated mitochondrial membrane potential. Mechanistically, PEP-1-CAT blocked H2O2-induced down-regulation of PI3K/Akt activity, an essential signaling pathway regulating MSC apoptosis. In vivo, the viability of MSC implanted into ischemic area in lower limb ischemia rat model was increased by four-fold when transduced with PEP-1-CAT. Importantly, PEP-1-CAT-transduced MSC significantly enhanced ischemia-induced angiogenesis by up-regulating VEGF expression. Conclusions PEP-1-CAT-transduction was able to increase MSC viability by regulating PI3K/Akt activity, which stimulated ischemia-induced angiogenesis. PMID:23285080

  7. Proanthocyanidins from the American Cranberry (Vaccinium macrocarpon Induce Cell Cycle Alterations in DU145 Human Prostate Cancer Cells in Vitro by Affecting the Expression of Cell Cycle-Associated Proteins

    Directory of Open Access Journals (Sweden)

    Joseph Kim

    2014-04-01

    Full Text Available Background: Prostate cancer is one of the most common cancers in the world. There are genetic and environmental factors that can potentially impact the development and progression of many types of cancer, including prostate cancer. As a consequence of environmental factors, such as diet having a potential effect on the development of prostate cancer, considerable interest in the possible health benefits associated with the inclusion and consumption of certain foods in the diet exists. Context and purpose of this study: This study describes the effects of a proanthocyanidinenriched fraction (PACs isolated from the American cranberry (Vaccinium macrocarpon on the behaviour of androgen-refractory (insensitive DU145 human prostate cancer cells in vitro. Results: Following treatment of DU145 human prostate cancer cells with 25 µg/mL of PACs for six hours, PACs significantly decreased the cellular viability of DU145 cells. PACs treatment (25 µg/mL for 6 hours of DU145 cells increased the proportion of cells in the G2-M phase of the cell cycle and decreased the proportion of cells in the G1 phase of the cell cycle. These alterations were associated with changes in cell cycle regulatory proteins and other cell cycle associated proteins. PACs increased the expression of cyclin E, cyclin D1, CDK2 and CDK4, and decreased the expression of cyclin A and cyclin B1. The protein expression level of p27 increased, and the protein expression levels of p16INK4a, p21, and pRBp107 decreased in response to PACs treatment. The protein expression level of pRBp130 was unchanged in Functional Foods in Health and Disease 2014; 4(2:130- 146 Page 131 of 146 response to PACs treatment. Conclusions: These findings demonstrate that proanthocyanidins from the American cranberry can affect the behaviour of human prostate cancer cells in vitro and further support the potential health benefits associated with cranberries.

  8. A machine vision system for automated non-invasive assessment of cell viability via dark field microscopy, wavelet feature selection and classification

    Directory of Open Access Journals (Sweden)

    Friehs Karl

    2008-10-01

    Full Text Available Abstract Background Cell viability is one of the basic properties indicating the physiological state of the cell, thus, it has long been one of the major considerations in biotechnological applications. Conventional methods for extracting information about cell viability usually need reagents to be applied on the targeted cells. These reagent-based techniques are reliable and versatile, however, some of them might be invasive and even toxic to the target cells. In support of automated noninvasive assessment of cell viability, a machine vision system has been developed. Results This system is based on supervised learning technique. It learns from images of certain kinds of cell populations and trains some classifiers. These trained classifiers are then employed to evaluate the images of given cell populations obtained via dark field microscopy. Wavelet decomposition is performed on the cell images. Energy and entropy are computed for each wavelet subimage as features. A feature selection algorithm is implemented to achieve better performance. Correlation between the results from the machine vision system and commonly accepted gold standards becomes stronger if wavelet features are utilized. The best performance is achieved with a selected subset of wavelet features. Conclusion The machine vision system based on dark field microscopy in conjugation with supervised machine learning and wavelet feature selection automates the cell viability assessment, and yields comparable results to commonly accepted methods. Wavelet features are found to be suitable to describe the discriminative properties of the live and dead cells in viability classification. According to the analysis, live cells exhibit morphologically more details and are intracellularly more organized than dead ones, which display more homogeneous and diffuse gray values throughout the cells. Feature selection increases the system's performance. The reason lies in the fact that feature

  9. Detection of viability of transplanted beta cells labeled with a novel contrast agent - polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles by magnetic resonance imaging.

    Science.gov (United States)

    Zhang, Bo; Jiang, Biao; Chen, Ying; Huang, Hai; Xie, Qiuping; Kang, Muxing; Zhang, Hui; Zhai, Chuanxin; Wu, Yulian

    2012-01-01

    Islets can be visualized on MRI by labeling with superparamagnetic contrast agent during the transplantation procedure. However, whether the signal intensity reflects the cell number and cellular viability has not been determined. We used a self-synthesized novel superparamagnetic contrast agent -polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles (PVP-SPIO) - to label β-TC-6 cells (a mouse insulinoma cell line) or primary islets with commercial Feridex as a control. The labeling efficiency of two agents was compared by Prussian blue staining, intracellular iron content determination and MR scanning. Cells were exposed to hypoxia, high-glucose or exogenous H₂O₂ stimulation before/after PVP-SPIO labeling. Normal and injured cells were also transplanted into renal subcapsule. A clinically used 3.0 T MR scan was performed in vitro and 24 h post-transplantation to investigate the correlation between cellular viability and signal. Our PVP-SPIO displayed superior biocompatibility and magnetic properties. All of the cells could be labeled at 100 µg/ml iron concentration after 24 h incubation. At 100 µg/ml iron concentration, 1 × 10⁵ β cells labeled with PVP-SPIO could already be visualized in vitro by MRI, less than the detection threshold of Feridex. There existed a linear correlation between the number of labeled cells and R₂ value on the T₂ -weighted images. The signal intensity and the intracellular iron content declined along with the decreased viability of labeled cells. There was also a significant difference in signal intensity between injured and normal labeled cells after transplantation. From these results, we concluded that PVP-SPIO possessed superior cell labeling efficiency, and β cells could be labeled without compromising viability and function. The signal intensity on MRI might be a useful predictor to evaluate the number and the viability of PVP-SPIO-labeled cells. Copyright © 2012 John Wiley & Sons, Ltd.

  10. Variety in intracellular diffusion during the cell cycle

    Science.gov (United States)

    Selhuber-Unkel, Christine; Yde, Pernille; Berg-Sørensen, Kirstine; Oddershede, Lene B.

    2009-06-01

    During the cell cycle, the organization of the cytoskeletal network undergoes dramatic changes. In order to reveal possible changes of the viscoelastic properties in the intracellular space during the cell cycle we investigated the diffusion of endogenous lipid granules within the fission yeast Schizosaccharomyces Pombe using optical tweezers. The cell cycle was divided into interphase and mitotic cell division, and the mitotic cell division was further subdivided in its stages. During all stages of the cell cycle, the granules predominantly underwent subdiffusive motion, characterized by an exponent α that is also linked to the viscoelastic moduli of the cytoplasm. The exponent α was significantly smaller during interphase than during any stage of the mitotic cell division, signifying that the cytoplasm was more elastic during interphase than during division. We found no significant differences in the subdiffusive exponents from granules measured in different stages of cell division. Also, our results for the exponent displayed no significant dependence on the position of the granule within the cell. The observation that the cytoplasm is more elastic during interphase than during mitotic cell division is consistent with the fact that elastic cytoskeletal elements such as microtubules are less abundantly present during cell division than during interphase.

  11. Impact of photodynamic inactivation (PDI) using the photosensitizer chlorin e6 on viability, apoptosis, and proliferation of human corneal endothelial cells.

    Science.gov (United States)

    Wang, Jiong; Stachon, Tanja; Eppig, Timo; Langenbucher, Achim; Seitz, Berthold; Szentmáry, Nóra

    2013-04-01

    Photodynamic inactivation (PDI) may be a potential alternative in case of therapy-resistant infectious keratitis. PDI using the photosensitizer chlorin e6 (Ce6) with high photosensitizing efficacy offers a valuable option, also for keratitis. The purpose of our study was to determine the impact of PDI with the photosensitizer Ce6 on viability, apoptosis, and proliferation of human corneal endothelial cells (HCECs), in vitro. Human corneal endothelial cell line was cultured in DMEM/Ham's F12 medium supplemented with 5 % fetal calf serum. HCECs cultures underwent illumination using red (670 nm) light for 13 min following exposure to 50-500 nM concentrations of Ce6 in the culture medium. Twenty-four hours after PDI, cell viability was evaluated by the Alamar blue assay, total DNA content of the cells and apoptosis using the APO-DIRECT Kit, and cell proliferation by the BrdU Cell Proliferation Assay Kit. Using Ce6 or illumination only, we did not detect significant changes of cell viability, apoptosis, and proliferation. Following PDI, viability and total DNA content of HCECs decreased significantly above 150 nM Ce6 concentration (P proliferation of endothelial cells decreased significantly (P proliferation, and also triggers apoptosis of HCECs in vitro. PDI using the photosensitizer Ce6 may be a potential treatment alternative in infectious keratitis. However, to avoid endothelial cell damage, the photosensitizer must not penetrate the endothelium.