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Sample records for cell viability dna

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

    Science.gov (United States)

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

    2016-04-15

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

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

    Directory of Open Access Journals (Sweden)

    Haller Thomas

    2011-02-01

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

  3. Cadmium affects viability of bone marrow mesenchymal stem cells through membrane impairment, intracellular calcium elevation and DNA breakage

    Directory of Open Access Journals (Sweden)

    Abnosi Mohammad Hussein

    2010-01-01

    Full Text Available Background: Cadmium is an important heavy metal with occupational and environmental hazard. Cadmium toxicity results mainly in bone-related complication such as itai-itai disease. Mesenchymal stem cells of the bone marrow have the ability to differentiate to osteoblasts which ensure the well-being of the bone tissue. Thus the aim was to investigate the effect of cadmium on viability of rat bone marrow mesenchymal stem cells. Materials and Methods: The rat bone marrow mesenchymal stem cells were grown to confluency in DMEM medium supplemented with 15% fetal bovine serum and penicillin-streptomycin up to third passage. Then the cells were treated with 0, 5, 15, 25, 35, and 45 of CdCl 2 at 12, 24, 36, and 48 h, and their viability was investigated using trypan blue staining. In addition, after treatment with selected dose (15 and 45 μM and time (24 and 48 h the cell morphology, DNA damage and calcium content of the cells were evaluated. Data was analyzed using one and two-way ANOVA (Tukey test and the P2+ was observed. Conclusion: Cadmium chloride is a toxic compound which might affect the well-being of bone tissue through affecting the mesenchymal stem cells.

  4. Inactivation of E. Coli cell viability and DNA Photo-breakage by Pulsed Nitrogen Laser Radiation

    International Nuclear Information System (INIS)

    The mutagenic and lethal effect of nitrogen laser radiation: 337.1 nm wave length, 1.5 millijoul pulse energy, 10 nanosecond pulse with and pulse repetition rate range from 1 to 50 Pulse/ second was evaluated on E. Coli cells. Results indicated that irradiation of E. coli JMP39 with pulse repetition of 8 , 16 , 32 pulse/sec, for 1, 5 , 10, 25 min respectively led to a significant decrease in cell count proportional to irradiation dose with significant increase in lacmutation frequency accompanied with some mutations in pattern of antibiotic resistance. The effect of nitrogen laser on the genomic content of the strain JMP39 was also studied by irradiating the total DNA with 30 pulse/second for 1 ,5, 15 , 30 min then subjected to both agarose gel electrophoresis and scanning spectrophotometry. The first technique revealed to DNA photo breakage and significant decrease in DNA absorbency was noticed by scanning spectrophotometry. This could be attributed to photo-decomposition resulted from multi-photo-excitation of UV-Laser pulses

  5. Individual and combined effects of ochratoxin A and citrinin on viability and DNA fragmentation in cultured Vero cells and on chromosome aberrations in mice bone marrow cells

    International Nuclear Information System (INIS)

    Ochratoxin A (OTA) and citrinin (CTN) are two common contaminant mycotoxins which can occur jointly in a wide range of food commodities. Both mycotoxins have several toxic effects but share a significant nephrotoxic and carcinogenic potential since OTA and CTN were reported to be responsible for naturally occurring human and animal kidney diseases and tumors. Considering the concomitant production of OTA and CTN, it is very likely that humans and animals are always exposed to the mixture rather than to individual compounds. Therefore, the aim of the present study was to investigate, in vivo and in vitro, whether DNA damage is enhanced by combination of both mycotoxins as compared to their effect separately. To this end, we have assessed their effects individually or combined on cell proliferation and DNA fragmentation in cultured Vero cells and in vivo by monitoring the induction of chromosome aberrations. Our results clearly showed that cultured renal cells respond to OTA and CTN exposure by a moderate and weak inhibition of cell proliferation, respectively. However, when combined, they exert a significant increase in inhibition of cell viability. Similar results were found for the investigated genotoxicity endpoints (DNA fragmentation and chromosome aberrations). Altogether, our study showed that OTA and CTN combination effects are clearly synergistic. The synergistic induction of DNA damage observed with OTA and CTN taken concomitantly could be relevant to explain the molecular basis of the renal diseases and tumorogenesis induced by naturally occurring mycotoxins

  6. Viability of mesenchymal stem cells during electrospinning

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    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.

  7. Cell Viability Assessment: Toward Content-Rich Platforms

    Science.gov (United States)

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

    2013-01-01

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

  8. 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

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

    Science.gov (United States)

    Shen, Shan-Wei; Song, Hou-Yan

    2009-10-25

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

  10. Viability of dielectrophoretically trapped neural cortical cells in culture

    NARCIS (Netherlands)

    Heida, T.; Vulto, P.; Rutten, W.L.C.; Marani, E.

    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

  11. >Effect of progesterone hormon on cell viability and stem cell activation in dental pulp cells

    Directory of Open Access Journals (Sweden)

    Segah Altuntaş

    2016-03-01

    Full Text Available Objective: The dental pulp is the part in the center of a tooth made up of living connective tissue and cells called odontoblasts. The vitality of the dentin structure, both during health and after injury, depends on pulp cell activity and the signaling processes that regulate the cell’s behavior. Dental pulp tissue has condensed stem cell activity. Dental pulp stem cells are multipotent stem cells that have the potential to differentiate into a variety of cell types. Several publications have stressed the importance of the expression of pluripotentiality associated markers: the transcription factors Nanog, Sox2, Oct3/4, SSEA4, CD13, Stro1 are indispensable for the stem cells to divide indefinitely without affecting their differentiation potential (self renewal capacity. Progesterone is a steroid hormone leading to menstrual cycle and gestation. There is a widespread rumor among people that pregnancy causes toothy loss. Method: So, progesterone was applied in different concentrations on human dental pulp cells in cell culture. Cell viability assay was applied 24th hour later with trypan blue. RNA isolation, cDNA synthesis and Real Time PCR analysis were applied on selected transcription factors (Nanog and Oct4 (POU5F1 genes which have role on steamness of stem cells. Gene expression analyses results were correlated with the cell viability assay results. Results: Cell viability assay results were 80% viable in control, 82% viable in 7 ml progesterone application, 81% viable in 14 ml progesterone application, 83% viable in 21 ml progesterone application. Due to our findings, progesterone in different concentrations did not chance the cell viability in dental pulpa cells. On gene expression analyses, preliminary results supported that high concentrations of progesterone enhance the gene expressions of steamness genes (Nanog, and Oct4 in dental pulp cells. Conclusions: So, progesterone did not change cell viability in high concentrations. We

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

    Science.gov (United States)

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

    2014-02-15

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

  13. Mammalian cell viability in electrospun composite nanofiber structures.

    Science.gov (United States)

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

    2011-10-10

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

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

    Science.gov (United States)

    Sharma, Meenakshi

    2016-01-01

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

  15. 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.

  16. 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.

  17. DNA-cell conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2015-10-01

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

  20. Viability Tests for Fresh and Stored Haemopoietic Cells

    International Nuclear Information System (INIS)

    This paper reviews current methods of measurement of the viability of fresh and stored haemopoietic cells. The life expectancy of granulocytes and monocytes after transfusion can be studied by in-vitro labelling with 3H-DFP and subsequent autoradiography. The evaluation of data in about 30 patients with various haemopoietic conditions indicates a wide variation of the disappearance half-time of granulocytes. 3H-cytidine labels essentially all lymphocytes in vitro, predominantly in their RNA. Transfusion of 3H-cytidine-labelled lymphocytes enables one to measure the lower limit of their life-expectancy as well as their rate of RNA metabolism. If bone-marrow cells are labelled in vitro with 3H-thymidine and subsequently transfused, their capability to circulate, to reach the haemopoietic tissue of the host, to proliferate and to mature can be demonstrated. However, the repopulating capacity of frozen and thawed marrow is independent of the ability of 3H-TDR-labelled marrow cells to circulate, proliferate and mature. It is assumed that bone-marrow cells capable of repopulating depleted haemopoietic tissue are resting under steady-state conditions and can be labelled by means of 3H-TDR only using special conditions. Thus the only viability tests for fresh and stored bone-marrow cells at present appear to be bioassay methods at the animal experimental level. The results indicate the need for the development of reliable viability tests for stem cells applicable in both experimental and clinical conditions. (author)

  1. Influence of location-dependent protuberance damage on cell viability

    Institute of Scientific and Technical Information of China (English)

    YANG HaiFeng; ZHOU Ming; DI JianKe; ZHAO EnLan; YANG PeiFang; GONG AiHua; SUN XiangLan

    2009-01-01

    The influence of femtosecond laser-induced damages on viability of olfactory ensheathing cells (OECs) is investigated. Several cytokinetic processes including cellular damage, recovery and death are dis-cussed. Using femtosecond laser with the power of 100 μW and cutting speed of 2 μm/s, we cut the cellular protuberance with smaller diameter twice in different locations, and then observe the viability of the damaged cells. Under the same conditions, the root of protuberance with larger diameter is cut six times to observe changes of cellular shape. Whether the damage is located in the end, middle or root of protuberance with smaller diameter, the cell viability can recover within 3 h. When the damage is located in the root of protuberance with larger diameter, the damaged cell will die in the way of oncoais. Cytokinetic phenomena including intracellular high Ca2+ concentration, cellular morphologic change, recovery and oncosis are discussed. Meanwhile, high Ca2+ concentration is observed after femtosec-ond laser surgery. Therefore, femtosecond laser surgery is an important tool for establishing cell damage model and studying cytokinetics.

  2. Femtosecond Optical Trapping of Cells: Efficiency and Viability

    Institute of Scientific and Technical Information of China (English)

    GONG Jixian; LI Fang; XING Qirong

    2009-01-01

    The femtosecond optical trapping capability and the effect of femtosecond laser pulses on cell viability were studied. The maximum lateral velocity at which the particles just failed to be trapped, together with the measured average trapping power, were used to calculate the lateral trapping force(Q-value). The viability of the cells after femtosecond laser trapping was ascertained by vital staining. Measurement of the Q-values shows that femtosecond optical tweezers are just as effective as continuous wave optical tweezers. The experiments demonstrate that there is a critical limit for expo-sure time at each corresponding laser power of femtosecond optical tweezers, and femtosecond laser tweezers are safe for optical trapping at low power with short exposure time.

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

    International Nuclear Information System (INIS)

    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

  4. 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.

  5. 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.

  6. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  8. New small molecules targeting apoptosis and cell viability in osteosarcoma.

    Directory of Open Access Journals (Sweden)

    Doris Maugg

    Full Text Available Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS, the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2 nor primary human osteoblasts (hOB. In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    Gabriel eVinderola

    2011-05-01

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

  11. 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

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

    Science.gov (United States)

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

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

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

    Science.gov (United States)

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

    2015-06-01

    The objectives of the study were to (1) examine and optimize the impact of sucrose during slow freezing and (2) compare the results of two freezing methods (slow freezing and vitrification) on cellular viability (germinal and stromal cells), follicle morphology, DNA integrity, and gap junction protein expression (connexin 43 [Cx 43]). Different sucrose supplementations (0, 0.1, and 0.3 M) in standard freezing medium were compared before and after slow freezing. Ovarian tissue slow frozen using 0.1- (4.0 ± 0.4) or 0.3-M sucrose (3.9 ± 0.5) yielded better follicular viability (number of positive follicles per 0.0625 mm(2)) than the group without sucrose (1.9 ± 0.2; P sucrose-treated groups (0.1 M, 47.4% and 0.3 M, 43.5%) than the group without sucrose (0 M, 33.8%; P sucrose groups (0.1 M, 1.2% and 0.3 M, 1.9%) than the group without sucrose (7.7%; P sucrose concentrations. In terms of the freezing methods used, vitrified ovarian tissues had fewer viable follicles (3.2 ± 0.6) than the slow-freezing method (4.6 ± 0.6; P sucrose supplementation and slow-freezing method on the follicular viability, follicular histologic appearances of follicles, and apoptosis of the follicles and stromal cells in cat ovarian tissues.

  14. 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.

  15. Cell viability and repair systems in mammal cells

    International Nuclear Information System (INIS)

    Synchronized cell cultures of mice are irradiated with 4,0J/m2 ultraviolet light at different times. The possible mechanisms involved in the recuperation of the cellular survival observed, are discussed. (M.A.)

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

    Science.gov (United States)

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

    2016-01-01

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

  17. 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.

  18. 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.)

  19. 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.

  20. 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

    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...... 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...

  1. Effect of Sodium Arsenite on Rat Bone Marrow Mesenchymal Stem Cells: Cells Viability and Morphological Study

    Directory of Open Access Journals (Sweden)

    M.H. Abnosi

    2010-07-01

    Full Text Available Introduction & Objective: Sodium arsenite as an environmental pollutant being found in the air, water, and earth crust threats the human beings' health. The aim of this study was to investigate the effect of sodium arsenite on viability and morphology of mesenchymal stem cells in rat bone marrow.Materials & Methods: In this exprimental study the cells were extracted in DMEM containing 15% FBS and Pen/Strep until the 3rd passage then treated with 0, 0.1, 0.5, 2.5, 12.5 and 20 µM of sodium arsenite for 12, 24, 36 and 48 hrs. Viability of the cells was carried out with trypan blue and MTT staining, then 0.1 µM and 36 hrs treatment was selected for further investigations. Morphology of the cells was studied using fluorescent dye (Hochest, propidium iodide and acridine orange as well as protein profile of the cells were studied using SDS-PAGE. Data was analyzed using one and two way ANOVA.Results: Based on the two way ANOVA, cumulative effect of treatment time and used dosage caused highly significant reduction (p<0.001 in viability of rat bone marrow mesenchymal stem cells. One way ANOVA indicated that the viability of the cells reduced significantly (p<0.05 from 0.1 µM of sodium arsenite on wards in all the treatment time. Morphological changes including condensation and deformation of the nuclei, membrane disruption, and shrinkage of cytoplasm were also observed. Conclusion: Sodium arsenite toxicity caused morphological and protein profile changes as well as dose and time dependent reduction in viability of rat bone marrow mesenchymal stem cells.

  2. Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization:

    OpenAIRE

    Hudej, Rosana; Kandušer, Maša; Miklavčič, Damijan; Trontelj, Katja; Ušaj, Marko

    2009-01-01

    Background. Various electrofusion parameters have to be adjusted to obtain theoptimal electrofusion efficiency. Based on published data, good electrofusion conditions can be achieved with the hypotonic treatment. However, the duration of the hypotonic treatment before electroporation and buffer hypoosmolarity have to be adjusted in order to cause cell swelling, to avoid regulatory volume decrease and to preserve cell viability. The aims of our study were to determine cell size dynamics and vi...

  3. Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization

    OpenAIRE

    Trontelj, Katja; Kandušer, Maša; Miklavčič, Damijan; Hudej, Rosana; Ušaj, Marko

    2015-01-01

    Background. Various electrofusion parameters have to be adjusted to obtain theoptimal electrofusion efficiency. Based on published data, good electrofusion conditions can be achieved with the hypotonic treatment. However, the duration of the hypotonic treatment before electroporation and buffer hypoosmolarity have to be adjusted in order to cause cell swelling, to avoid regulatory volume decrease and to preserve cell viability. The aims of our study were to determine cell size dynamics and vi...

  4. Pseudorabies Virus Induces Viability Changes and Oxidative Stress in Swine Testis Cell-Line

    Directory of Open Access Journals (Sweden)

    Xiao-Zhan Zhang§1, Ye Chen§1, Hong-Liang Huang§2, Dong-Lei Xu1, Chang-Bao Ren2, Bi-Tao Liu1, Shuo Su1 and Zhao-Xin Tang1, 2*

    2013-11-01

    Full Text Available In this study, we evaluated the association between pseudorabies (PRV virus-induced viability changes and oxidative stress in vitro cultivated swine testis (ST cells. The kinetic of 2, 12, 24, 36 and 48 h during the cell culture with PRV by using a multiplicity of infection (MOI of 1 TCID50 per cell were adopted. The results suggested a complex relation between cell viability and oxidative stress during PRV infection. In the early stages of PRV infection, the cell viability was higher than the control group, and the state of cellular oxidative stress remained relatively stable. After 24 h, the cell viability began to decrease, and the amount of the cellular malondialdehyde in ST cells increased significantly, and the activities of superoxide dismutase and catalase decreased significantly (P<0.05. Meanwhile, the rising concentrations of cellular hydrogen peroxide were detected prior to the changes in cell viability and oxidative stress. In conclusion, the PRV infection of ST cells leads to oxidative stress, and this stress could play a crucial role on the cell viability as the PRV infection time progresses.

  5. 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.

  6. 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

    not accompanied by concomitant changes in viability. However, changes in expression of marker genes for cell cycle inhibition or progression, such as p21 and PCNA, could not explain the changes in DNA content. Interestingly, aspecific upregulation of GAPDH activity was found, which was limited to cartilaginous......% 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......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...

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

    Science.gov (United States)

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

    2016-01-01

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

  8. 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.

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

    Science.gov (United States)

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

    2015-12-28

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

  10. Cell viability - Open TG-GATEs | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us Open... Database Description Download License Update History of This Database Site Policy | Contact Us Cell viability - Open TG-GATEs | LSDB Archive ...

  11. 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 biocompatibl...

  12. Preparation of Nanoporous Polymer Films for Real-Time Viability Monitoring of Cells

    Directory of Open Access Journals (Sweden)

    Chia-Man Chou

    2011-01-01

    Full Text Available We have demonstrated an alternative way to monitor the viability of cells adhered on a nanoporous polymer film in real time. The nanoporous polymer films were prepared by laser interference pattering. During exposure of holographic patterning, the dissolved solvents were phase separated with photocured polymer and the nanopores were created as the solvents evaporated. The diffracted spectra from the nanoporous polymer film responded to each activity of the cell cycle, from initial cell seeding, through growth, and eventual cell death. This cell-based biosensor uses a nanoporous polymer film to noninvasively monitor cell viability and may prove useful for biotechnological applications.

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

    Science.gov (United States)

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

    2016-09-15

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

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

    Science.gov (United States)

    Kwolek-Mirek, Magdalena; Zadrag-Tecza, Renata

    2014-11-01

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

  15. 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. PMID:25070929

  16. 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.

  17. 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. PMID:26952444

  18. 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...... 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...

  19. Aspiration, but not injection, decreases cultured equine mesenchymal stromal cell viability

    OpenAIRE

    Williams, Lynn B.; Russell, Keith A.; Koenig, Judith B.; Thomas G. Koch

    2016-01-01

    Background Recently, equine multipotent mesenchymal stromal cells (MSC) have received significant attention as therapy for various conditions due to their proposed regenerative and immune-modulating capacity. MSC are commonly administered to the patient through a hypodermic needle. Currently, little information is available on the effect of such injection has on equine MSC immediate and delayed viability. We hypothesize that viability of equine MSC is not correlated with needle diameter durin...

  20. Effect of amyloid peptides on serum withdrawal-induced cell differentiation and cell viability

    Institute of Scientific and Technical Information of China (English)

    Yi Peng WANG; Ze Fen WANG; Ying Chun ZHANG; Qing TIAN; Jian Zhi WANG

    2004-01-01

    Abnormal deposition of amyloid-β(Aβ) peptides and formation of neuritic plaques are recognized as pathological processes in Alzheimer's disease (AD) brain. By using amyloid precursor protein (APP) transfected cells, this study aims to investigate the effect of overproduction of Aβ on cell differentiation and cell viability. It was shown that after serum withdrawal, untransfected cell (N2a/Wt) and vector transfected cells (N2a/vector) extended long and branched cell processes, whereas no neurites was induced in wild type APP (N2a/APP695) and Swedish mutant APP (N2a/APPswe) transfected N2a cells. After differentiation by serum withdrawal, the localization of APP/Aβ and neurofilament was extended to neurites, whereas those of APP-transfected cells were still restricted within the cell body. Levels of both APP and Aβ were significantly higher in N2a/APP695 and N2a/APPswe than in N2a/Wt, as determined by Western blot and Sandwich ELISA, respectively. To further investigate the effect of Aβ on the inhibition of cell differentiation,we added exogenously the similar level or about 10-times of the Aβ level produced by N2a/APP695 and N2a/APPswe to the culture medium and co-cultured with N2a/Wt for 12 h, and we found that the inhibition of serum withdrawalinduced differentiation observed in N2a/APP695 and N2a/APPswe could not be reproduced by exogenous administration of Aβ into N2a/Wt. We also observed that neither endogenous production nor exogenous addition of Aβ1-40 or Aβ1-42, even to hundreds fold of the physiological concentration, affected obviously the cell viability. These results suggest that the overproduction of Aβ could not arrest cell differentiation induced by serum deprivation and that, at least to a certain degree and in a limited time period, is not toxic to cell viability.

  1. 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.

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

    Science.gov (United States)

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

    2015-10-01

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

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

    Science.gov (United States)

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

    1976-01-01

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

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

    Science.gov (United States)

    Petrunkina, A M; Harrison, R A P

    2011-05-31

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

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

    OpenAIRE

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

    2016-01-01

    During cancer metastasis, circulating tumor cells constantly experience hemodynamic shear stress in the circulation. Cellular responses to shear stress including cell viability and proliferation thus play critical roles in cancer metastasis. Here, we developed a microfluidic approach to establish a circulatory microenvironment and studied circulating human colon cancer HCT116 cells in response to a variety of magnitude of shear stress and circulating time. Our results showed that cell viabili...

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

    Science.gov (United States)

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

    2014-01-01

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

  7. 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.

  8. 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.

  9. 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.

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

    Science.gov (United States)

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

    2013-11-01

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

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

    Science.gov (United States)

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

    2003-01-01

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

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

    Science.gov (United States)

    Xia, Bingzhao; Krutkramelis, Kaspars; Oakey, John

    2016-07-11

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

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

    Science.gov (United States)

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

    2016-06-01

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

  14. Comparison of viability of adipose-derived Mesenchymal stem cells on agarose and fibrin glue scaffolds

    Directory of Open Access Journals (Sweden)

    Farzaneh Tafvizi

    2015-06-01

    Full Text Available Background & aim: Utilizing tissue engineering techniques and designing similar structures of the damaged tissues require the use of tools such as scaffolds, cells, and bioactive molecules in vitro. Meanwhile, appropriate cell cultures with the ability to divide and differentiate on the natural scaffolds lacking features like immunogenicity and tumorgenesis is particularly important. Adipose tissue has attracted researchers’ attention due to its abundance of mesenchymal stem cells and its availability through a liposuction. The purpose of the present study was to investigate the reproducibility and viability of the adipose-derived stem cells on natural scaffolds of fibrin glue and agarose. Methods: In the present experimental study, the isolation and identification of the mesenchymal stem cells was performed on tissue obtained from liposuction. The tissues were extensively washed with PBS and were digested with collagenase I, then the mesenchymal stem cells were isolated. The cells were cultured in RPMI medium supplemented with antibiotic. Subsequently, the expression of cell surface markers including CD34, CD44, CD90, and CD105 were analyzed by flow cytometry to confirm the mesenchymal cells. After preparing fibrin glue and agarose scaffolds, the viability and proliferation of the adipose tissue-derived mesenchymal stem cells were examined at the period of 24, 48, and 72 hours by MTT and ELISA assays. The obtained results were analyzed by SPSS ver.19. Results: The results of adipose tissue-derived mesenchymal stem cells culture on the fibrin glue and agarose scaffolds indicated that cell viability on fibrin glue and agarose scaffold were 68.22% and 89.75% in 24 hrs, 64.04% and 66.97% in 48 hours, 222.87% and 1089.68% in 72 hours respectively. Significant proliferation and viability cells on a synthesized agarose scaffold were seen compared to the fibrin glue scaffold after 72 hrs. The viability of the cells significantly increased on the

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    Ingrid Garzón

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

  17. Enhanced reduction in cell viability by hyperthermia induced by magnetic nanoparticles

    Directory of Open Access Journals (Sweden)

    Héctor L Rodríguez-Luccioni

    2011-02-01

    Full Text Available Héctor L Rodríguez-Luccioni, Magda Latorre-Esteves, Janet Méndez-Vega, Orlando Soto, Ana R Rodríguez, Carlos Rinaldi, Madeline Torres-LugoDepartment of Chemical Engineering, University of Puerto Rico, Mayagüez 00681, Puerto RicoAbstract: Colloidal suspensions of iron oxide magnetic nanoparticles are known to dissipate energy when exposed to an oscillating magnetic field. Such energy dissipation can be employed to locally raise temperature inside a tumor between 41°C and 45°C (hyperthermia to promote cell death, a treatment known as magnetic fluid hyperthermia (MFH. This work seeks to quantify differences between MFH and hot-water hyperthermia (HWH in terms of reduction in cell viability using two cancer cell culture models, Caco-2 (human epithelial colorectal adenocarcinoma and MCF-7 (human breast cancer. Magnetite nanoparticles were synthesized via the co-precipitation method and functionalized with adsorbed carboxymethyl dextran. Cytotoxicity studies indicated that in the absence of an oscillating magnetic field, cell viability was not affected at concentrations of up to 0.6 mg iron oxide/mL. MFH resulted in a significant decrease in cell viability when exposed to a magnetic field for 120 minutes and allowed to rest for 48 hours, compared with similar field applications, but with shorter resting time. The results presented here suggest that MFH most likely induces apoptosis in both cell types. When compared with HWH, MFH produced a significant reduction in cell viability, and these effects appear to be cell-type related.Keywords: magnetic fluid hyperthermia, carboxymethyl dextran magnetite, cell death, apoptosis

  18. 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.

  19. 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.

  20. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  6. 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)

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

    Science.gov (United States)

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

    2015-11-01

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

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

  9. The effect of tuning cold plasma composition on glioblastoma cell viability.

    Directory of Open Access Journals (Sweden)

    Xiaoqian Cheng

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

  10. Role of Cell-Cell bond for the viability and the function of vascular smooth muscle cells

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    M. Mura

    2010-01-01

    Full Text Available Vascular smooth muscle cell (VSMC viability and homeostasis is regulated by cell-matrix and cell-cell contact: disruption of these interactions are responsible of a switch from a mature to a high proliferative phenotype. VSMCs migration, rate of growth and apoptosis, and the extent of their extracellular matrix (ECM deposition can be also modulated by proatherogenic peptides. Among them, ATII induces the transactivation of IGF I R, which, together with the binding protein IGFBP3, represents a determinant of cell survival, growth and proliferation. Aim of our in vitro study was to verify the role of elective cell-cell bond in moulating the response to ATII. Thus, we evaluated viability, proliferation, IGFIR, IGFBP3 expression and the long term survival and production of ECM in a provisional tissue. A7r5 cell-line was used in adherent cultures or incubated in agarose-coated culture plates to inhibit cell-matrix interactions. Cells, treated or not with ATII 100 nM, were evaluated for apoptosis rate, cell cycle, IGFIR and IGFBP3 protei expression. Fibrin provisional tissue was developed polymerizing a fibrin solution. cantaining A7r5 cells with thrombin. Histological stainings for ECM components were performed on sections of prvisional tissue. An exclusive cell-cell contact resulted to monolayer cell cultures. ATII did not affect the cell survival in both culture conditions, but promoted a 10% decrease in "S" phase and an increases IGFIR expression only in adherent cells. while suspended cell aggregates were resistant to ATII administration; IGFBP3 was reduced both in ATII treated adherent cells and in floating clustered cells, irrespective of the treatmentn. VSMC conditioning in agarose-coated plates before seeding in fibrin provisional matrix reduced, but not abolished, the cell ability to colonize the clot and to produce ECM. This study demonstrates that the elective cell-cell contact induces a quiescent status in cells lacking of cell

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-01-01

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

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

    OpenAIRE

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

    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 re...

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

    Science.gov (United States)

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

    2016-06-01

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

  14. Biochanin A Modulates Cell Viability, Invasion, and Growth Promoting Signaling Pathways in HER-2-Positive Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Vikas Sehdev

    2009-01-01

    Full Text Available Overexpression of HER-2 receptor is associated with poor prognosis and aggressive forms of breast cancer. Scientific literature indicates a preventive role of isoflavones in cancer. Since activation of HER-2 receptor initiates growth-promoting events in cancer cells, we studied the effect of biochanin A (an isoflavone on associated signaling events like receptor activation, downstream signaling, and invasive pathways. HER-2-positive SK-BR-3 breast cancer cells, MCF-10A normal breast epithelial cells, and NIH-3T3 normal fibroblast cells were treated with biochanin A (2–100 μM for 72 hours. Subsequently cell viability assay, western blotting and zymography were carried out. The data indicate that biochanin A inhibits cell viability, signaling pathways, and invasive enzyme expression and activity in SK-BR-3 cancer cells. Biochanin A did not inhibit MCF-10A and NIH-3T3 cell viability. Therefore, biochanin A could be a unique natural anticancer agent which can selectively target cancer cells and inhibit multiple signaling pathways in HER-2-positive breast cancer cells.

  15. 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.

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

    Institute of Scientific and Technical Information of China (English)

    Tebekeme Okoko; Diepreye Ere

    2012-01-01

    Objective: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. Methods:The macrophage cell line U937 was treated with cadmium (0.1μmol/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 (10μmol/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. Results:It revealed that the anthocynanin-rich extract significantly (P <0.05) increased the viability of the cells which was suppressed by cadmium when compared to quercetin dihydrate. The extract also reduced the cadmium-mediated production of the markers of macrophage-activation when compared to quercetin dihydrate. In both experiments, the activity of the extract was concentration-dependent (P <0.05). Conclusion:The findings show that H. 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.

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

    Science.gov (United States)

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

    2014-12-01

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

  18. >Effect of progesterone hormon on cell viability and stem cell activation in dental pulp cells

    OpenAIRE

    Segah Altuntaş; Muhammed Ali Kara; Deniz Selin Aksoy; Zehra Dilşad Çoban; Şefik Güran

    2016-01-01

    Objective: The dental pulp is the part in the center of a tooth made up of living connective tissue and cells called odontoblasts. The vitality of the dentin structure, both during health and after injury, depends on pulp cell activity and the signaling processes that regulate the cell’s behavior. Dental pulp tissue has condensed stem cell activity. Dental pulp stem cells are multipotent stem cells that have the potential to differentiate into a variety of cell types. Several publications hav...

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

    Science.gov (United States)

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

    2012-10-01

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

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

    Science.gov (United States)

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

    2012-01-01

    To be effective, probiotic bacteria must exhibit a number of functional characteristics, including the resistance to gastric acidity and the ability to adhere to the intestinal epithelium. In this study, we examined in vitro the viability of lactic acid bacteria (LAB) combination after exposure to low pH, and the adhesion of LAB to Caco-2 cells during coincubation of 9 bacterial strains. To test bacterial viability, 6 commercially available products were incubated in 0.1 N HCl at pH 1.2 for 60 min. The greatest growth inhibition was noted for the non-capsulated product containing the Lactobacillus rhamnosus strain (log reduction of CFU = 6.4), and the best survival observed for the product containing 9 bacterial strains, equipped with a modern capsule made according to the Multi-Resistant Encapsulation technology (log reduction of CFU = 0.1). In the adhesion experiment, the combination of 9 bacterial strains was added to 17-day-old Caco-2 cell culture for 90 min. The greatest efficiency of adhesion was observed for the inoculum containing 5.5x10(8) CFU/mL/9.6 cm(2) of Caco-2 and the dose of probiotic bacteria of 190 cells per one Caco-2 cell. As a result, approximately 157 bacterial cells adhered to one Caco-2 cell. The results indicate that the combination of 9 bacterial strains in the examined product is characterized as highly adhesive. PMID:22462453

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

    Science.gov (United States)

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

    2008-05-01

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

  2. Cytotoxicity and genotoxicity of a monazite component: lanthanum effects on the viability and induction of breaks in the DNA of human lymphocytes

    International Nuclear Information System (INIS)

    The Monazite is a mineral extracted from open mines. It is constituted by lanthanum element aggregated with cerium, yttrium and thorium [(Ce, La, Y, Th)PO4]. Lanthanum (La) is a rare-earth metal with applications in agriculture, industry and medicine. Since lanthanides and their compounds show a broad spectrum of applications there is an increased risk of incorporation in human. Inhalation of aerosols containing La is the main route of incorporation in workers exposed to several chemical forms of La. Herein, we examined the effect of lanthanum nitrate - La(NO3)3 in human lymphocytes. JURKAT cells and human peripheral lymphocytes (HPL) were used to evaluate the effect of La(NO3)3 on viability (apoptosis or necrosis) and DNA strand breaks induction or/and alkali-labile sites (ALS). We demonstrate that La has a cytotoxic and genotoxic effect on both cell lines. The results indicate that necrosis is the pathway by which La(NO3)3 induces cytotoxicity. The vitamin E is able to diminish DNA strand breaks induced by La(NO3)3 suggesting that reactive oxygen species (ROS) may be involved in the genotoxic process. (author)

  3. Ca2+-induced changes in energy metabolism and viability of melanoma cells

    OpenAIRE

    Glass-Marmor, L; Penso, J; Beitner, R

    1999-01-01

    Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. We show here that a rise in intracellular-free calcium ion (Ca2+), induced by Ca2+-ionophore A23187, exerted a deleterious effect on glycolysis and viability of B16 melanoma cells. Ca2+-ionophore caused a dose-dependent detachment of phosphofructokinase (EC 2.7.1.11), one of the key enzymes of glycolysis, from cytoskeleton. It also induced a decrease in the levels of glucose 1,6-bisphosphate and...

  4. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2016-04-01

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

  8. 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.

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

    Science.gov (United States)

    Lestard, Nathalia R.

    2016-01-01

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

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

    Science.gov (United States)

    Lestard, Nathalia R; Capella, Marcia A M

    2016-01-01

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

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

    Science.gov (United States)

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

    2010-08-01

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

  12. 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

    ), and a nonadherent mouse B-lymphoid cell line (HB-94). Analyses of variance (ANOVAs) revealed no significant main or dose-related effects of spiritual healing compared to controls for either of the two cell lines or any of the assays (P-values between 0.09 and 0.96). When comparing healing and control across all......Alternative treatments such as spiritual healing and prayer are increasingly popular, especially among patients with life-threatening diseases such as cancer. According to theories of spiritual healing, this intervention is thought to influence living cells and organisms independently...... 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...

  13. 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...... oxidation, an essential energy supply, in HT-29 and DLD-1 cells. TNF-alpha+IFN-gamma induced a parallel profound decline in cell viability in HT-29 cells, but not in DLD-1 cells, where impairment of butyrate oxidation seemed to precede later occurrence of cell damage. TNF-alpha+INF-gamma induced CEC damage...

  14. 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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    International Nuclear Information System (INIS)

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

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

    Directory of Open Access Journals (Sweden)

    Arias Covadonga R

    2012-11-01

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

  18. Mechanisms of DNA uptake by cells

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1977-01-01

    Three categories of cellular uptake of DNA can be distinguished. First, in the highly transformable bacteria, such as Diplococcus pneumoniae, Haemophilus influenzae and Bacillus subtilis, elaborate mechanisms of DNA transport have evolved, presumably for the purpose of genetic exchange. These mechanisms can introduce substantial amounts of DNA into the cell. Second, methods have been devised for the forced introduction of DNA by manipulation of bacterial cells under nonphysiological conditions. By such means small but significant amounts of DNA have been introduced into various bacteria, including Escherichia coli. Third, mammalian cells are able to take up biologically active DNA. This has been most clearly demonstrated with viral DNA, although the mechanism of uptake is not well understood. The intention, here, is to survey current understanding of the various mechanisms of DNA uptake. A review of experience with the bacterial systems may throw some light on the mammalian system and lead to suggestions for enhancing DNA uptake by mammalian cells.

  19. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

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

    2016-07-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    International Nuclear Information System (INIS)

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

  6. The effect of silver nanoparticles on Staphylococcus epidermidis biofilm biomass and cell viability

    Directory of Open Access Journals (Sweden)

    Bahman Khameneh

    2014-10-01

    Full Text Available Objective(s: Bacterial biofilm has been considered responsible for many deaths and high health costs worldwide. Their better protection against antibacterial agents compared to free living cells leads to poor treatment efficiency. Nanotechnology is promising approach to combat biofilm infections. The aim of the present study was to eradicate Staphylococcus epidermidis biofilm with silver nanoparticles (SNPs. Materials and Methods: SNPs were used at different concentrations (two fold dilutions and incubation times (24, 48, 72 h. The crystal violet staining and pour plate assays were used to assess biofilm biomass and bacterial viability, respectively. The ability of SNPs on biofilm matrix eradication was assessed through optical density ratio (ODr. Positive control was defined as an ODr =1.0. Results: The crystal violet assay indicated that the biofilm matrixes were intact at different concentrations of SNOs and incubation times. There were no significant differences between these parameters (P >0.05. Bacterial enumeration studies revealed that higher concentrations of SNPs were more effective in killing bacteria than lower ones. Although, longer incubation times  led to enhancement of anti-biofilm activity of SNPs. Conclusion: The anti-biofilm activity of SNPs was concentration- and time-dependent. The results of this study highlighted that SNPs were effective against cell viability; however they were ineffective against biomass.

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

    Science.gov (United States)

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

    2016-08-01

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

  8. DNA repair in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

  9. Biased DNA Segregation during Stem Cell Division

    OpenAIRE

    Anversa, Piero; Leri, Annarosa; Kajstura, Jan

    2012-01-01

    Adult skeletal muscle stem cells are a heterogeneous cell population characterized by a small subset of undifferentiated cells that express at high level the paired/homeodomain gene Pax7. This category of satellite cells divides predominantly by asymmetric chromatid segregation generating a daughter cell that carries the mother DNA and retains stem cell property, and a daughter cell that inherits the newly-synthesized DNA and acquires the myocyte lineage.1

  10. Conservation science in a terrorist age: the impact of airport security screening on the viability and DNA integrity of frozen felid spermatozoa.

    Science.gov (United States)

    Gloor, Kayleen T; Winget, Doug; Swanson, William F

    2006-09-01

    In response to growing terrorism concerns, the Transportation Security Administration now requires that all checked baggage at U.S. airports be scanned through a cabinet x-ray system, which may increase risk of radiation damage to transported biologic samples and other sensitive genetic material. The objective of this study was to investigate the effect of these new airport security regulations on the viability and DNA integrity of frozen felid spermatozoa. Semen was collected from two domestic cats (Felis silvestris catus) and one fishing cat (Prionailurus viverrinus), cryopreserved in plastic freezing straws, and transferred into liquid nitrogen dry shippers for security screening. Treatment groups included frozen samples from each male scanned once or three times using a Transportation Security Administration-operated cabinet x-ray system, in addition to non-scanned samples (i.e., negative control) and samples previously scanned three times and exposed to five additional high-intensity x-ray bursts (i.e., positive control). Dosimeters placed in empty dry shippers were used to quantify radiation exposure. Following treatment, straws were thawed and spermatozoa analyzed for post-thaw motility (percentage motile and rate of progressive movement), acrosome status, and DNA integrity using single-cell gel electrophoresis (i.e., the comet assay). Dosimeter measurements determined that each airport screening procedure produced approximately 16 mrem of radiation exposure. Our results indicated that all levels of radiation exposure adversely affected (P 0.05) among treatment groups. Results also showed that the amount of double-stranded DNA damage was greater (P < 0.05) in sperm samples from both cat species scanned three times compared to samples scanned once or negative controls. Findings suggest that new airport security measures may cause radiation-induced damage to frozen spermatozoa and other valuable biologic samples transported on passenger aircraft and that

  11. 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.

  12. Pulmonary surfactant preserves viability of alveolar type II cells exposed to polymyxin B in vitro.

    Directory of Open Access Journals (Sweden)

    Guido Stichtenoth

    Full Text Available BACKGROUND: Exogenous surfactant derived from animal lungs is applied for treatment of surfactant deficiency. By means of its rapid spreading properties, it could transport pharmaceutical agents to the terminal air spaces. The antimicrobial peptide Polymyxin B (PxB is used as a topical antibiotic for inhalation therapy. Whereas it has been shown that PxB mixed with surfactant is not inhibiting surface activity while antimicrobiotic activity is preserved, little is known concerning the effects on synthesis of endogenous surfactant in alveolar type II cells (ATIIC. OBJECTIVE: To investigate ATIIC viability and surfactant-exocytosis depending on PxB and/or surfactant exposure. METHODS: ATIIC were isolated from rat lungs as previously described and were cultivated for 48 h. After incubation for a period of 1-5 h with either PxB (0.05 or 0.1 mg/ml, modified porcine surfactant (5 or 10 mg/ml or mixtures of both, viability and exocytosis (spontanously and after stimulation were determined by fluorescence staining of intracellular surfactant. RESULTS: PxB 0.1 mg/ml, but not porcine surfactant or porcine surfactant plus PxB reduces ATIIC-viability. Only PxB alone, but not in combination with porcine surfactant, rapidly reduces fluorescence in ATIIC at maximum within 3 h, indicating stimulation of exocytosis. Subsequent ionomycin-stimulation does not further increase exocytosis of PxB incubated ATIIC. In presence of surfactant, stimulating effects of PxB and ionomycin on exocytosis are reduced. CONCLUSION: PxB alone shows negative effects on ATIIC, which are counterbalanced in mixtures with surfactant. So far, our studies found no results discouraging the concept of a combined treatment with PxB and surfactant mixtures.

  13. Quercetin Stimulates Insulin Secretion and Reduces the Viability of Rat INS-1 Beta-Cells

    Directory of Open Access Journals (Sweden)

    Michael Kittl

    2016-06-01

    Full Text Available Background/Aims: Previously we described insulinotropic effects of Leonurus sibiricus L. plant extracts used for diabetes mellitus treatment in Traditional Mongolian Medicine. The flavonoid quercetin and its glycoside rutin, which exert anti-diabetic properties in vivo by interfering with insulin signaling in peripheral target tissues, are constituents of these extracts. This study was performed to better understand short- and long-term effects of quercetin and rutin on beta-cells. Methods: Cell viability, apoptosis, phospho-protein abundance and insulin release were determined using resazurin, annexin-V binding assays, Western blot and ELISA, respectively. Membrane potentials (Vmem, whole-cell Ca2+ (ICa- and ATP-sensitive K+ (IKATP currents were measured by patch clamp. Intracellular Ca2+ (Cai levels were measured by time-lapse imaging using the ratiometric Ca2+ indicator Fura-2. Results: Rutin, quercetin and the phosphoinositide-3-kinase (PI3K inhibitor LY294002 caused a dose-dependent reduction in cell viability with IC50 values of ∼75 µM, ∼25 µM and ∼3.5 µM, respectively. Quercetin (50 µM significantly increased the percentage of Annexin-V+ cells within 48 hrs. The mean cell volume (MCV of quercetin-treated cells was significantly lower. Within 2 hrs, quercetin significantly decreased basal- and insulin-stimulated Akt(T308 phosphorylation and increased Erk1/2 phosphorylation, without affecting P-Akt(S473 abundance. Basal- and glucose-stimulated insulin release were significantly stimulated by quercetin. Quercetin significantly depolarized Vmem by ∼25 mV which was prevented by the KATP-channel opener diazoxide, but not by the L-type ICa inhibitor nifedipine. Quercetin significantly stimulated ICa and caused a 50% inhibition of IKATP. The effects on Vmem, ICa and IKATP rapidly reached peak values and then gradually diminished to control values within ∼1 minute. With a similar time-response quercetin induced an elevation in Cai

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

    Science.gov (United States)

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

    2016-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Mohammad Kazemi Pilehrood

    2014-01-01

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

  16. The Effect of Sericin from Various Extraction Methods on Cell Viability and Collagen Production

    Directory of Open Access Journals (Sweden)

    Pornanong Aramwit

    2010-05-01

    Full Text Available Silk sericin (SS can accelerate cell proliferation and attachment; however, SS can be extracted by various methods, which result in SS exhibiting different physical and biological properties. We found that SS produced from various extraction methods has different molecular weights, zeta potential, particle size and amino acid content. The MTT assay indicated that SS from all extraction methods had no toxicity to mouse fibroblast cells at concentrations up to 40 μg/mL after 24 h incubation, but SS obtained from some extraction methods can be toxic at higher concentrations. Heat-degraded SS was the least toxic to cells and activated the highest collagen production, while urea-extracted SS showed the lowest cell viability and collagen production. SS from urea extraction was severely harmful to cells at concentrations higher than 100 μg/mL. SS from all extraction methods could still promote collagen production in a concentration-dependent manner, even at high concentrations that are toxic to cells.

  17. Cell Attachment and Viability Study of PCL Nano-fiber Modified by Cold Atmospheric Plasma.

    Science.gov (United States)

    Atyabi, Seyed Mohammad; Sharifi, Fereshteh; Irani, Shiva; Zandi, Mojgan; Mivehchi, Houri; Nagheh, Zahra

    2016-06-01

    The field of tissue engineering is an emerging discipline which applies the basic principles of life sciences and engineering to repair and restore living tissues and organs. The purpose of this study was to investigate the effect of cold and non-thermal plasma surface modification of poly (ϵ-caprolactone) (PCL) scaffolds on fibroblast cell behavior. Nano-fiber PCL was fabricated through electrospinning technique, and some fibers were then treated by cold and non-thermal plasma. The cell-biomaterial interactions were studied by culturing the fibroblast cells on nano-fiber PCL. Scaffold biocompatibility test was assessed using an inverted microscope. The growth and proliferation of fibroblast cells on nano-fiber PCL were analyzed by MTT viability assay. Cellular attachment on the nano-fiber and their morphology were evaluated using scanning electron microscope. The result of cell culture showed that nano-fiber could support the cellular growth and proliferation by developing three-dimensional topography. The present study demonstrated that the nano-fiber surface modification with cold plasma sharply enhanced the fibroblast cell attachment. Thus, cold plasma surface modification greatly raised the bioactivity of scaffolds.

  18. 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

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

    Science.gov (United States)

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

    2012-01-01

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

  20. 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

  1. Effect of Ichnocarpus frutescens (L.) R.Br. hexane extract on preadipocytes viability and lipid accumulation in 3T3-L1 cells

    Institute of Scientific and Technical Information of China (English)

    M Saravanan; S Ignacimuthu

    2013-01-01

    Objective: To investigate the crude extracts of Ichnocarpus frutescens (I. frutescens) for antiobesity effect. Methods: Leaves of I. frutescens were sequentially extracted with hexane, ethyl acetate, and methanol and their effect on viability of 3T3-L1 preadipocytes were evaluated. Based on this the apoptosis on preadipocytes was confirmed by DNA fragmentation and LDH (Lactate dehydrogenase) leakage assays. Anti-adipogenesis was performed by oil red O (ORO) staining and free glycerol release in the medium of differentiated adipocytes. Results: The hexane extract of I. frutescens (IFHE) inhibited cell viability in a time- and dose-related manner. An increased release of LDH, as a marker of membrane integrity, was observed at a dose of 200 μg/mL. The discontinuous DNA fragments on agarose gel electrophoresis showed the apoptotic effect of the IFHE. Morphological observations of cells stained with ORO showed a decrease in cellular lipid content at the concentrations tested compared to the induced control cells. In the experiment of lipolytic activity, treatment with IFHE enhanced glycerol secretion with the rates of approximately 28%, 55%, and 46% at the concentrations of 100, 200 and 300 μg/mL, respectively. Conclusions:The observed properties clearly revealed the medicinal property of I. frutescens in the treatment of obesity.

  2. 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.

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

    Science.gov (United States)

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

    2008-09-01

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

  4. Antioxidant potential of herbs extracts and impact on HepG2 cells viability

    Directory of Open Access Journals (Sweden)

    Anna Gramza-Michałowska

    2008-12-01

    Full Text Available Mercury poisoning is responsible for inducing serious adverse effects in living organisms. One of protection factors could be substances proven to possess high antioxidant and metal chelating activity – plant polyphenols. There are many sources of polyphenols in plant kingdom but the most interesting for food industry could be widely consumed herbs. Aim of the research was to evaluate antioxidative potential of selected plant extracts and its influence on HepG2 cells in different conditions. Ethanolic herbs extracts were characterised by total polyphenol content. Antioxidant activity was estimated with use of DPPH• and ABTS+• radicals scavenging methods and FRAP. Research included cells viability estimation by the MTT assay and cells exposition to HgCl2, chemical agent inducing cell death. Analysis of herbs extracts antioxidative activity showed best potential represented thyme and marjoram, highest FRAP was evaluated in samples with mint and marjoram extracts. On the basis of received results it was found that examined plant extracts showed weak protection against Hg presence in examined cells environment.

  5. DNA charge transport within the cell.

    Science.gov (United States)

    Grodick, Michael A; Muren, Natalie B; Barton, Jacqueline K

    2015-02-01

    The unique characteristics of DNA charge transport (CT) have prompted an examination of roles for this chemistry within a biological context. Not only can DNA CT facilitate long-range oxidative damage of DNA, but redox-active proteins can couple to the DNA base stack and participate in long-range redox reactions using DNA CT. DNA transcription factors with redox-active moieties such as SoxR and p53 can use DNA CT as a form of redox sensing. DNA CT chemistry also provides a means to monitor the integrity of the DNA, given the sensitivity of DNA CT to perturbations in base stacking as arise with mismatches and lesions. Enzymes that utilize this chemistry include an interesting and ever-growing class of DNA-processing enzymes involved in DNA repair, replication, and transcription that have been found to contain 4Fe-4S clusters. DNA repair enzymes containing 4Fe-4S clusters, that include endonuclease III (EndoIII), MutY, and DinG from bacteria, as well as XPD from archaea, have been shown to be redox-active when bound to DNA, share a DNA-bound redox potential, and can be reduced and oxidized at long-range via DNA CT. Interactions between DNA and these proteins in solution, in addition to genetics experiments within Escherichia coli, suggest that DNA-mediated CT can be used as a means of cooperative signaling among DNA repair proteins that contain 4Fe-4S clusters as a first step in finding DNA damage, even within cells. On the basis of these data, we can consider also how DNA-mediated CT may be used as a means of signaling to coordinate DNA processing across the genome. PMID:25606780

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

    Science.gov (United States)

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

    2013-08-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  9. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2008-04-01

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

  12. Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields

    Directory of Open Access Journals (Sweden)

    Jian Wang

    2016-01-01

    Full Text Available After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF, Adipose-derived Stem Cells (ASCs and those labeled by superparamagnetic iron oxide (SPIO nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT assay, proliferation by cell counting and bromodeoxyuridine (BrdU incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF, Insulin-like Growth Factor-1 (IGF-1, Transforming Growth Factor Beta 1 (TGF-β1, genetic markers comprising Stem Cell Antigen-1 (Sca1, Octamer-4 (Oct-4, ATP-binding Cassette Subfamily B Member 1 (ABCB1, adipogenic marker genes containing Lipoprotein Lipase (LPL, Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ, and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1 and Osterix (OSX. Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling.

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

    Science.gov (United States)

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

    2016-06-01

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

  14. 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

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

    Science.gov (United States)

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

    2014-04-01

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

  16. Tracing Conidial Fate and Measuring Host Cell Antifungal Activity Using a Reporter of Microbial Viability in the Lung

    OpenAIRE

    Jhingran, Anupam; Mar, Katrina B.; Kumasaka, Debra K.; Sue E Knoblaugh; Ngo, Lisa Y.; Segal, Brahm H; Iwakura, Yoichiro; Lowell, Clifford A.; Hamerman, Jessica A.; Lin, Xin; Tobias M Hohl

    2012-01-01

    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-spe...

  17. 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

  18. 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.

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

    Science.gov (United States)

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

    2013-11-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  2. Nutritional stress enhances cell viability of odontoblast-like cells subjected to low level laser irradiation

    International Nuclear Information System (INIS)

    In spite of knowing that cells under stress are biostimulated by low level laser (LLL) irradiation, the ideal condition of stress to different cell lines has not yet been established. Consequently, the aim of the present in vitro study was to evaluate the effects of a defined parameter of LLL irradiation applied on stressed odontoblast-like pulp cells (MDPC-23). The cells were seeded (12500 cells/cm2) in wells of 24-well plates using complete culture medium (DMEM) and incubated for 24 hours. Then, the DMEM was replaced by a new medium with low concentrations (nutritional stress condition) of fetal bovine serum (FBS) giving rise to the following experimental groups: G1: 2% FBS; G2: 5% FBS; and G3: 10% FBS. The cells were irradiated three times with LLL in specific parameters (808±3 nm, 100 mW, 1.5 J/cm2) every 24 hours. No irradiation was carried out in groups G4 (2% FBS-Control), G5 (5% FBS-Control), and G6 (10% FBS-Control). For all groups, the cell metabolism (MTT assay) and morphology (SEM) was evaluated. The experimental groups showed enhanced cell metabolism and normal cell morphology regardless of FBS concentration. A slight increase in the cell metabolism was observed only in group G2. It was concluded that cell nutritional stress caused by reducing the concentration of FBS to 5% is the most suitable method to assess the biostimulation of LLL irradiated MDPC-23 cells

  3. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  5. DNA damage response in adult stem cells.

    Science.gov (United States)

    Insinga, Alessandra; Cicalese, Angelo; Pelicci, Pier Giuseppe

    2014-04-01

    This review discusses the processes of DNA-damage-response and DNA-damage repair in stem and progenitor cells of several tissues. The long life-span of stem cells suggests that they may respond differently to DNA damage than their downstream progeny and, indeed, studies have begun to elucidate the unique stem cell response mechanisms to DNA damage. Because the DNA damage responses in stem cells and progenitor cells are distinctly different, stem and progenitor cells should be considered as two different entities from this point of view. Hematopoietic and mammary stem cells display a unique DNA-damage response, which involves active inhibition of apoptosis, entry into the cell-cycle, symmetric division, partial DNA repair and maintenance of self-renewal. Each of these biological events depends on the up-regulation of the cell-cycle inhibitor p21. Moreover, inhibition of apoptosis and symmetric stem cell division are the consequence of the down-regulation of the tumor suppressor p53, as a direct result of p21 up-regulation. A deeper understanding of these processes is required before these findings can be translated into human anti-aging and anti-cancer therapies. One needs to clarify and dissect the pathways that control p21 regulation in normal and cancer stem cells and define (a) how p21 blocks p53 functions in stem cells and (b) how p21 promotes DNA repair in stem cells. Is this effect dependent on p21s ability to inhibit p53? Such molecular knowledge may pave the way to methods for maintaining short-term tissue reconstitution while retaining long-term cellular and genomic integrity.

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

    Directory of Open Access Journals (Sweden)

    Domenico Tricarico

    Full Text Available Emerging evidences suggest that Ca(2+activated-K(+-(BK channel is involved in the regulation of cell viability. The changes of the cell viability observed under hyperkalemia (15 mEq/L or hypokalemia (0.55 mEq/L conditions were investigated in HEK293 cells expressing the hslo subunit (hslo-HEK293 in the presence or absence of BK channel modulators. The BK channel openers(10(-11-10(-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(2 x 10(-7M-5 x 10(-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

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

  8. 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.

  9. Treatment of Leptothrix Cells with Ultrapure Water Poses a Threat to Their Viability

    Directory of Open Access Journals (Sweden)

    Tatsuki Kunoh

    2015-01-01

    Full Text Available The genus Leptothrix, a type of Fe/Mn-oxidizing bacteria, is characterized by its formation of an extracellular and microtubular sheath. Although almost all sheaths harvested from natural aquatic environments are hollow, a few chained bacterial cells are occasionally seen within some sheaths of young stage. We previously reported that sheaths of Leptothrix sp. strain OUMS1 cultured in artificial media became hollow with aging due to spontaneous autolysis within the sheaths. In this study, we investigated environmental conditions that lead the OUMS1 cells to die. Treatment of the cells with ultrapure water or acidic buffers (pH 6.0 caused autolysis of the cells. Under these conditions, the plasma membrane and cytoplasm of cells were drastically damaged, resulting in leakage of intracellular electrolytes and relaxation of genomic DNA. The autolysis was suppressed by the presence of Ca2+. The hydrolysis of peptidoglycan by the lysozyme treatment similarly caused autolysis of the cells and was suppressed also by the presence of Ca2+. However, it remains unclear whether the acidic pH-dependent autolysis is attributable to damage of peptidoglycan. It was observed that L. discophora strain SP-6 cells also underwent autolysis when suspended in ultrapure water; it is however, uncertain whether this phenomenon is common among other members of the genus Leptothrix.

  10. Changes in Cell Viability of Wounded Fibroblasts following Laser Irradiation in Broad-Spectrum or Infrared Light

    International Nuclear Information System (INIS)

    Objective. This study aimed to establish if broad-spectrum or infrared (IR) light in combination with laser therapy can assist phototherapy to improve the cell function of wounded cells. Background. The effect of laser light may be partly or completely reduced by broad-spectrum light. Methods. Wounded human skin fibroblasts were irradiated with 5 J/cm2 using a helium-neon laser, a diode laser, or an Nd:YAG laser in the dark, in the light, or in IR. Changes in cell viability were evaluated by cell morphology, ATP cell viability, LDH membrane integrity, and caspase 3/7 as an early marker of apoptosis. Results. Wounded cells exposed to 5 J/cm2 using 632.8 nm in the dark or 830 nm in the light or 1064 nm in the dark showed an increase in ATP viability, an increase in cytokine expression, and a decrease in LDH cytotoxicity indicating that the metabolic activity of the wounded cells was stimulated. Conclusion. Wounded cells irradiated in IR light showed an undesirable thermal effect that was proportional to the duration of exposure.

  11. 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...... 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....

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

    Energy Technology Data Exchange (ETDEWEB)

    Jayasuriya, Ambalangodage C., E-mail: a.jayasuriya@utoledo.edu [Department of Orthopaedic Surgery, University of Toledo, Toledo, OH 43614 (United States); Aryaei, Ashkan; Jayatissa, Ahalapitiya H. [Departments of Mechanical Engineering, University of Toledo, Toledo, OH 43606 (United States)

    2013-10-15

    The aim of this paper is to develop novel chitosan–zinc oxide nanocomposite films for biomedical applications. The films were fabricated with 1, 5, 10 and 15% w/w of zinc oxide (ZnO) nanoparticles (NPs) incorporated with chitosan (CS) using a simple method. The prepared nanocomposite films were characterized using atomic force microscopy, Raman and X-ray diffraction studies. In addition, nano and micro mechanical properties were measured. It was found that the microhardness, nanohardness and its corresponding elastic modulus increased with the increase of ZnO NP percentage in the CS films. However, the ductility of films decreased as the percentage of ZnO NPs increased. Cell attachment and cytotoxicity of the prepared films at days two and five were evaluated in vitro using osteoblasts (OBs). It was observed that OB viability decreased in films with higher than 5% ZnO NPs. This result suggests that although ZnO NPs can improve the mechanical properties of pure CS films, only a low percentage of ZnO NPs can be applied for biomedical and bioengineering applications because of the cytotoxicity effects of these particles. Highlights: • Chitosan–zinc oxide nanocomposite films were fabricated using a simple method. • Material characterization methods showed that adding zinc oxide up to 15% does not change the crystal structure of chitosan. • Zinc oxide nanoparticles improve nano and micro mechanical properties of chitosan films. • Adding more than 5% w/w zinc oxide nanoparticles demonstrates cytotoxicity on osteoblast cells.

  13. Cell viability and dopamine secretion of 6-hydroxydopamine-treated PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells☆

    OpenAIRE

    Tang, Yue; Cui, Yongchun; Luo, Fuliang; Liu, Xiaopeng; Wang, XiaoJuan; Wu, Aili; Zhao, Junwei; Tian, Zhong; Wu, Like

    2012-01-01

    In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson's Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal α-synuclein accumulation in cells. Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and do...

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

    Science.gov (United States)

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

    2015-05-01

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

  15. 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.

  16. 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.

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

    Science.gov (United States)

    Janakiraman, Narayanan; Johnson, Marimuthu

    2016-06-01

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

  18. The effect of the essential oils from five different Lippia species on the viability of tumor cell lines

    Directory of Open Access Journals (Sweden)

    Mayna da S. Gomide

    2013-12-01

    Full Text Available Several Lippia species have been used in folk medicine mainly for gastrointestinal and respiratory diseases. Their biological properties have been partially associated to the terpenoids found in their essential oils. According to the World Health Organization, cancer is the leading cause of death worldwide and is described as a complex group of diseases with several hallmarks. One of its acceptable defining features is the cell proliferation beyond their boundaries forming the tumors. Importantly, some drugs currently available were discovered by the investigation of plant secondary metabolites. Thus, this study aimed to evaluate in vitro cytotoxic effect of the essential oils extracted from five Lippia species against tumor cell lines. The results indicated that mouse colon carcinoma CT26.WT cell line viability was significantly reduced showing an IC50 of 19.05, 30.20 and 36.30 µg/ml when treated with the essential oils of L. sidoides, L. salviifolia and L. rotundifolia, respectively. Human lung carcinoma A549 cell line also had a compromised viability to the action of L. alba carvone chemotype essential oil. The tested essential oils did not compromise viability of the normal cell line CHO. These finds suggest that the studied Lippia essential oils might be good candidates for further in-depth studies.

  19. 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.

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

    Science.gov (United States)

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

    2015-11-30

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

    Missan, Sergey; Hrytsenko, Olga

    2015-03-01

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

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

    Science.gov (United States)

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

    2014-07-01

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

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

    Science.gov (United States)

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

    2007-06-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

  6. Changes in Cell Viability of Wounded Fibroblasts following Laser Irradiation in Broad-Spectrum or Infrared Light

    OpenAIRE

    Hawkins, Denise; Abrahamse, Heidi

    2007-01-01

    Objective. This study aimed to establish if broad-spectrum or infrared (IR) light in combination with laser therapy can assist phototherapy to improve the cell function of wounded cells. Background. The effect of laser light may be partly or completely reduced by broad-spectrum light. Methods. Wounded human skin fibroblasts were irradiated with 5 J/cm2 using a helium-neon laser, a diode laser, or an Nd:YAG laser in the dark, in the light, or in IR. Changes in cell viability were evaluated by ...

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Johanna Mäkelä

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

  10. Differences in DNA Repair Capacity, Cell Death and Transcriptional Response after Irradiation between a Radiosensitive and a Radioresistant Cell Line.

    Science.gov (United States)

    Borràs-Fresneda, Mireia; Barquinero, Joan-Francesc; Gomolka, Maria; Hornhardt, Sabine; Rössler, Ute; Armengol, Gemma; Barrios, Leonardo

    2016-01-01

    Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in "DNA damage response", "direct p53 effectors" and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols. PMID:27245205

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

    OpenAIRE

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

    2015-01-01

    Background: Activation of bone morphogenetic protein 4 (BMP4) signaling pathway in embryonic stem (ES) cells plays an important role in controlling cell proliferation, differentiation, and apoptosis. Adverse effects of BMP4 occur in a time dependent manner; however, little is known about the effect of different time exposure of this growth factor on cell number in culture media. In this study, we investigated the role of two different exposure times to BMP4 in cell viability, embryoid body (E...

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

    Science.gov (United States)

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

    2016-09-01

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

  13. 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.

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

    International Nuclear Information System (INIS)

    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 (Tf) 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 Tf 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

  15. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  17. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    OpenAIRE

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

    2014-01-01

    Extracellular acidification has been observed in allergic inflammatory diseases. Recently, we demonstrated that the proton-sensing receptor G protein-coupled receptor 65 (GPR65) regulates eosinophil survival in an acidic environment in vitro and eosinophil accumulation in an allergic lung inflammation model. For mast cells, another inflammatory cell type critical for allergic responses, it remains unknown whether GPR65 is expressed and/or regulates mast cell viability. Thus, in the present st...

  20. 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

  1. Cell viability and dopamine secretion of 6-hydroxydopamine-treated PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Yue Tang; Yongchun Cui; Fuliang Luo; Xiaopeng Liu; Xiaojuan Wang; Aili Wu; Junwei Zhao; Zhong Tian; Like Wu

    2012-01-01

    In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson's Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal α-synuclein accumulation in cells. Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and dopamine secretion in a cell dose-dependent manner. MitoLight staining was used to confirm that PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells demonstrate reduced levels of cell apoptosis. Immunocytochemistry and western blot analysis found the quantity of α-synuclein accumulation was significantly reduced in PC12 cell and bone marrow-derived mesenchymal stem cell co-cultures. These results indicate that bone marrow-derived mesenchymal stem cells can attenuate 6-hydroxydopamine-induced cytotoxicity by reducing abnormal α-synuclein accumulation in PC12 cells.

  2. Estrogen Enhances the Cell Viability and Motility of Breast Cancer Cells through the ERα-ΔNp63-Integrin β4 Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Jar-Yi Ho

    Full Text Available Estrogen induces ERα-positive breast cancer aggressiveness via the promotion of cell proliferation and survival, the epithelial-mesenchymal transition, and stem-like properties. Integrin β4 signaling has been implicated in estrogen/ERα-induced tumorigenicity and anti-apoptosis; however, this signaling cascade poorly understood. ΔNp63, an N-terminally truncated isoform of the p63 transcription factor, functions as a transcription factor of integrinβ4 and therefore regulates cellular adhesion and survival. Therefore, the aim of the present study was to investigate the estrogen-induced interaction between ERα, ΔNp63 and integrin β4 in breast cancer cells. In ERα-positive MCF-7 cells, estrogen activated ERα transcription, which induced ΔNp63 expression. And ΔNp63 subsequently induced integrin β4 expression, which resulted in AKT phosphorylation and enhanced cell viability and motility. Conversely, there was no inductive effect of estrogen on ΔNp63-integrinβ4-AKT signaling or on cell viability and motility in ERα-negative MDA-MB-231 cells. ΔNp63 knockdown abolishes these estrogen-induced effects and reduces cell viability and motility in MCF-7 cells. Nevertheless, ΔNp63 knockdown also inhibited cell migration in MDA-MB-231 cells through reducing integrin β4 expression and AKT phosphorylation. In conclusion, estrogen enhances ERα-positive breast cancer cell viability and motility through activating the ERα-ΔNp63-integrin β4 signaling pathway to induce AKT phosphorylated activation. Those findings should be useful to elucidate the crosstalk between estrogen/ER signaling and ΔNp63 signaling and provide novel insights into the effects of estrogen on breast cancer progression.

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

    Science.gov (United States)

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

    2015-08-01

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

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

    Science.gov (United States)

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

    2014-06-01

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

  5. Pancreatic islet-cell viability, functionality and oxidative status remain unaffected at pharmacological concentrations of commonly used antibiotics in vitro

    Indian Academy of Sciences (India)

    Yogita Shewade; Suraj Tirth; R R Bhonde

    2001-09-01

    Environmental factors such as diet, physical activity, drugs, pollution and life style play an important role in the progression and/or precipitation of diseases like diabetes, hypertension, obesity and cardiovascular disorders. Indiscriminate use of antibiotics to combat infectious diseases is one of the commonest forms of misuse of drugs. Antibiotics seem to have a correlation with diabetes and pancreatic function. There are controversial reports about the effect of antibiotics on the pancreatic islets; some suggesting their harmless action, some depicting a beneficial role and others indicating deleterious effect. Moreover, use of antibiotics is mandatory during islet isolation and cultivation to reduce incidences of microbial contamination. It is likely that antibiotic treatment may adversely affect islet viability and its functioning leading to failure of islet transplantation. The present in vitro study was undertaken to examine the effect of commonly used antibiotics such as gentamycin, penicillin, streptomycin, tetracycline, neomycin, erythromycin and chloramphenicol on islet viability, its functioning and induction of oxidative stress if any. The viability and insulin production data showed that none of the antibiotics used in the present study affect the viability and the functioning of the islets at their pharmacological concentrations. Free radical levels measured in terms of melonyldialdehyde (MDA), nitric oxide (NO) and reduced glutathione (GSH) reveal that except for a marginal increase in lipid peroxidation with tetracycline and slight increase in NO levels with streptomycin, none of these antibiotics affect the oxidative status of the cells. Antioxidant enzymes such as superoxide dismutase and catalase remain unaffected after this treatment. Our results reveal the innocuous nature of the antibiotics used at pharmacological concentrations, suggesting their safety whenever prescribed to combat infections and also during islet isolation procedures.

  6. Impact of UV-radiation on viability, photosynthetic characteristics and DNA of brown algal zoospores : implications for depth zonation

    NARCIS (Netherlands)

    Wiencke, C; Gomez, [No Value; Pakker, H; Flores-Moya, A; Altamirano, M; Hanelt, D; Bischof, K; Figueroa, FL

    2000-01-01

    Measurements of photosynthesis, germination capacity and assessment of DNA damage were carried out in the laboratory to determine the effect of different conditions of ultraviolet (UV) and photosynthetically active radiation (PAR) on zoospores of various large brown algae collected on Spitsbergen (S

  7. 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

    ), and a nonadherent mouse B-lymphoid cell line (HB-94). Analyses of variance (ANOVAs) revealed no significant main or dose-related effects of spiritual healing compared to controls for either of the two cell lines or any of the assays (P-values between 0.09 and 0.96). When comparing healing and control across all......Alternative treatments such as spiritual healing and prayer are increasingly popular, especially among patients with life-threatening diseases such as cancer. According to theories of spiritual healing, this intervention is thought to influence living cells and organisms independently...... 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...

  8. 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.

  9. Synergistic effect of radon and sodium arsenite on DNA damage in HBE cells.

    Science.gov (United States)

    Liu, Xing; Sun, Bin; Wang, Xiaojuan; Nie, Jihua; Chen, Zhihai; An, Yan; Tong, Jian

    2016-01-01

    Human epidemiological studies showed that radon and arsenic exposures are major risk factors for lung cancer in Yunnan tin miners. However, biological evidence for this phenomenon is absent. In this study, HBE cells were exposed to different concentrations of sodium arsenite, different radon exposure times, or a combination of these two factors. The results showed a synergistic effect of radon and sodium arsenite in cell cytotoxicity as determined by cell viability. Elevated intracellular ROS levels and increased DNA damage indexed by comet assay and γ-H2AX were detected. Moreover, DNA HR repair in terms of Rad51 declined when the cells were exposed to both radon and sodium arsenite. The synergistic effect of radon and sodium arsenite in HBE cells may be attributed to the enhanced DSBs and inhibited HR pathway upon co-exposure.

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

    Science.gov (United States)

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

    2016-01-01

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

  11. DNA analysis of epithelial cell suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, J.S.; Johnson, N.F.; Holland, L.M.

    1985-01-01

    Cell suspensions of skin were obtained by animals exposed by skin painting of several crude oils. DNA analysis of these cell suspensions labeled with mithramycin provide determination of percentages of cells in the G/sub 1/, S and G/sub 2/M phases of the cell cycle. Data acquired showed differences from control animals occurring as early as 7 days after treatment and persisting through 21 days afterwards. There was histological evidence of erythema and hyperplasia in shale oil-exposed skins. Flow cytometric analysis of DNA content in shale-oil-exposed skin cells showed an increased percentage of cycling cells plus evidence of aneuploidy. Similar data from simply abraded skin showed increased percentages of cycling cells, but no aneuploidy. The shale-oil-exposed group, when compared to a standard petroleum-exposed group, had significantly increased percentages of cycling cells. This early indication of differing response to different complex mixtures was also seen in long-term skin exposures to these compounds. Similar analytical techniques were applied to tracheal cell suspensions from ozone-exposed rats. 12 refs., 4 figs., 4 tabs. (DT)

  12. Lipid emulsions differentially affect LPS-induced acute monocytes inflammation: in vitro effects on membrane remodeling and cell viability.

    Science.gov (United States)

    Boisramé-Helms, Julie; Delabranche, Xavier; Klymchenko, Andrey; Drai, Jocelyne; Blond, Emilie; Zobairi, Fatiha; Mely, Yves; Hasselmann, Michel; Toti, Florence; Meziani, Ferhat

    2014-11-01

    The aim of this study was to assess how lipid emulsions for parenteral nutrition affect lipopolysaccharide (LPS)-induced acute monocyte inflammation in vitro. An 18 h long LPS induced human monocyte leukemia cell stimulation was performed and the cell-growth medium was supplemented with three different industrial lipid emulsions: Intralipid(®), containing long-chain triglycerides (LCT--soybean oil); Medialipid(®), containing LCT (soybean oil) and medium-chain triglycerides (MCT--coconut oil); and SMOFlipid(®), containing LCT, MCT, omega-9 and -3 (soybean, coconut, olive and fish oils). Cell viability and apoptosis were assessed by Trypan blue exclusion and flow cytometry respectively. Monocyte composition and membrane remodeling were studied using gas chromatography and NR12S staining. Microparticles released in supernatant were measured by prothrombinase assay. After LPS challenge, both cellular necrosis and apoptosis were increased (threefold and twofold respectively) and microparticle release was enhanced (sevenfold) after supplementation with Medialipid(®) compared to Intralipid(®), SMOFlipid(®) and monocytes in the standard medium. The monocytes differentially incorporated fatty acids after lipid emulsion challenge. Finally, lipid-treated cells displayed microparticles characterized by disrupted membrane lipid order, reflecting lipid remodeling of the parental cell plasma membrane. Our data suggest that lipid emulsions differentially alter cell viability, monocyte composition and thereby microparticle release. While MCT have deleterious effects, we have shown that parenteral nutrition emulsion containing LCT or LCT and MCT associated to n-3 and n-9 fatty acids have no effect on endotoxin-induced cell death and inflammation. PMID:25038627

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

    OpenAIRE

    Mezghani Sana; Hammami Amira; Amri Mohamed

    2015-01-01

    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 radiatio...

  14. Cell-Free Fetal DNA and Cell-Free Total DNA Levels in Spontaneous Abortion with Fetal Chromosomal Aneuploidy

    OpenAIRE

    Ji Hyae Lim; Min Hyoung Kim; You Jung Han; Da Eun Lee; So Yeon Park; Jung Yeol Han; Moon Young Kim; Hyun Mee Ryu

    2013-01-01

    BACKGROUND: Cell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA) with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy....

  15. Cells by design: a mini-review of targeting cell engineering using DNA microarrays.

    Science.gov (United States)

    Jaluria, Pratik; Chu, Chia; Betenbaugh, Michael; Shiloach, Joseph

    2008-06-01

    Recent studies have demonstrated the utility of DNA microarray technology in engineering cellular properties. For instance, cellular adhesion, the necessity of cells to attach to a surface in order to to proliferate, was examined by comparing two distinct HeLa cell lines. Two genes, one encoding a type II membrane glycosylating sialyltransferase (siat7e) and the other encoding a secreted glycoprotein (lama4), were found to influence adhesion. The expression of siat7e correlated with reduced adhesion, whereas expression of lama4 correlated with increased adhesion, as shown by various assays. In a separate example, a gene encoding a mitochondrial assembly protein (cox15) and a gene encoding a kinase (cdkl3), were found to influence cellular growth. Enhanced expression of either gene resulted in slightly higher specific growth rates and higher maximum cell densities for HeLa, HEK-293, and CHO cell lines. Another investigated property was the adaptation of HEK-293 cells to serum-free media. The genes egr1 and gas6, both with anti-apoptotic properties, were identified as potentially improving adaptability by impacting viability at low serum levels. In trying to control apoptosis, researchers found that by altering the expression levels of four genes faim, fadd, alg-2, and requiem, apoptotic response could be altered. In the present work, these and related studies in microorganisms (prokaryote and eukaryote) are examined in greater detail focusing on the approach of using DNA microarrays to direct cellular behavior by targeting select genes. PMID:18327555

  16. AKT2-knockdown suppressed viability with enhanced apoptosis, and attenuated chemoresistance to temozolomide of human glioblastoma cells in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Cui Y

    2015-07-01

    Full Text Available Yong Cui,1,* Jing Lin,1,* Jianling Zuo,2 Lei Zhang,1 Yan Dong,1 Guohan Hu,1 Chun Luo,1 Juxiang Chen,1 Yicheng Lu1 1Department of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China; 2Department of Neurosurgery, First Affiliated Hospital of Soochow University, Suzhou, People’s Republic of China *These authors contributed equally to this work Abstract: The AKT2 kinase (protein kinase Bβ is overexpressed in high-grade gliomas. Upregulation of the AKT2 gene has been previously observed in glioblastoma patients suffering from chemotherapy failure and tumor progress. In this study, we aimed to evaluate the effect of AKT2 on viability and chemoresistance in the human glioblastoma cell line U251. The U251 cell line was stably transfected with short hairpin RNA (shRNA targeting AKT2. U251 cells underexpressing AKT2 were then examined for viability with temozolomide (TMZ treatment, and tested for cell apoptosis both in vitro and in tumor-implanted mice. Next, expressions of several chemoresistance-related molecules were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR and western blot analysis. The results showed that the 50% inhibitory concentration (IC50 of AKT2 shRNA-transfected cells was significantly lower compared with Lenti-GFP-transfected and nontransfected controls and that the tumor growth of the AKT2-shRNA and TMZ combined-treated mice was obviously suppressed in either mass or volume. Concomitantly, the apoptosis of TMZ-treated tumor cells was significantly enhanced after knockdown of AKT2, as measured by flow cytometry and in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL analysis. Furthermore, AKT2-inhibition in TMZ-treated glioblastoma U251 cells upregulated apoptotic effector caspase-3, whereas it downregulated antiapoptotic protein Bcl-2, DNA repairing protein MGMT, and drug efflux pump protein MRP1. Our study

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

    Directory of Open Access Journals (Sweden)

    Shen QL

    2016-05-01

    Full Text Available Qinglin Shen1,2,*, Caixia Peng2,3,*, Yan Zhan1, Liang Fan1, Mengyi Wang1, Qing Zhou4, Jue Liu2,4, Xiaojuan Lv1, Qiu Tang1, Jun Li1,2, Xiaodong Huang2, Jiahong Xia2 1Department of Oncology, 2Key Laboratory for Molecular Diagnosis of Hubei Province, 3Central Laboratory, 4Department of Pharmacy, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China *These authors contributed equally to this work Abstract: Selection of the optimal chemotherapy regimen for an individual cancer patient is challenging. The existing chemosensitivity tests are costly, time-consuming, and not amenable to wide utilization within a clinic. This limitation might be addressed by the recently proposed use of circulating tumor cells (CTCs, which provide an opportunity to noninvasively monitor response to therapy. Over the past few decades, various techniques were developed to capture and recover CTCs, but these techniques were often limited by a capture and recovery performance tradeoff between high viability and high efficiency. In this work, we used anti-epithelial cell adhesion molecule coated aptamer–poly (N-isopropylacrylamide functionalized silicon nanowire substrates to capture and release epithelial cell adhesion molecule-positive CTCs at 32°C and 4°C, respectively. Then, we applied the nuclease to digest the aptamer to release the captured CTCs (near or at the end of the polymer brush, which cannot be released by heating/cooling process. High viability and purity CTCs could be achieved by decreasing the heating/cooling cycles and enzymatic treatment rounds. Furthermore, the time-saving process is helpful to maintain the morphology and enhance vitality of the recovered CTCs and is beneficial to the subsequent cell culture in vitro. We validated the feasibility of chemosensitivity testing based on the recovered HCC827 cells using an adenosine triphosphate–tumor chemosensitivity

  18. RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability

    Energy Technology Data Exchange (ETDEWEB)

    Park, E.; Prakash, L. (Univ. of Rochester School of Medicine, NY (United States)); Guzder, S.N.; Prakash, S. (Univ. of Rochester, NY (United States)); Koken, M.H.M.; Jaspers-Dekker, I.; Weeda, G.; Hoeijmakers, H.J. (Erasmus Univ., Rotterdam (Netherlands))

    1992-12-01

    Xeroderma pigmentosum (XP) patients are extremely sensitive to ultraviolet (UV) light and suffer from a high incidence of skin cancers, due to a defect in nucleotide excision repair. The disease is genetically heterogeneous, and seven complementation groups, A-G, have been identified. Homologs of human excision repair genes ERCC1, XPDC/ERCC2, and XPAC have been identified in the yeast Saccharomyces cerevisiae. Since no homolog of human XPBC/ERCC3 existed among the known yeast genes, we cloned the yeast homolog by using XPBC cDNA as a hybridization probe. The yeast homolog, RAD25 (SSL2), encodes a protein of 843 amino acids (M[sub r] 95,356). The RAD25 (SSL2)- and XPCX-encoded proteins share 55% identical and 72% conserved amino acid residues, and the two proteins resemble one another in containing the conserved DNA helicase sequence motifs. A nonsense mutation at codon 799 that deletes the 45 C-terminal amino acid residues in RAD25 (SSL2) confers UV sensitivity. This mutation shows epistasis with genes in the excision repair group, whereas a synergistic increase in UN sensitivity occurs when it is combined with mutations in genes in other DNA repair pathways, indicating that RAD25 (SSL2) functions in excision repair but not in other repair pathways. We also show that RAD25 (SSL2) is an essential gene. A mutation of the Lys[sup 392] residue to arginine in the conserved Walker type A nucleotide-binding motif is lethal, suggesting an essential role of the putative RAD 25 (SSL2) ATPase/DNA helicase activity in viability. 40 refs., 3 figs., 1 tab.

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

    Science.gov (United States)

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

    2011-12-13

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

  20. 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. PMID:26802493

  1. Chromatin condensation and differential sensitivity of mammalian and insect cells to DNA strand breaks induced by bleomycin

    Energy Technology Data Exchange (ETDEWEB)

    Lopez-Larraza, Daniel M. [IMBICE, C.C. 403, 1900 La Plata (Argentina)]. E-mail: danielop@imbice.org.ar; Padron, Juan [IMBICE, C.C. 403, 1900 La Plata (Argentina); Ronci, Natalia E. [IMBICE, C.C. 403, 1900 La Plata (Argentina); Vidal Rioja, Lidia A. [IMBICE, C.C. 403, 1900 La Plata (Argentina)

    2006-08-30

    Bleomycin (BLM) induces DNA damage in living cells. In this report we analyzed the role of chromatin compactness in the differential response of mosquito (ATC-15) and mammalian (CHO) cells to DNA strand breaks induced by BLM. We used cells unexposed and exposed to sodium butyrate (NaB), which induces chromatin decondensation. By nucleoid sedimentation assay and digestions of nuclei with DNAse I, untreated mosquito cells (no BLM; no NaB) were shown to have more chromatin condensation than untreated CHO cells. By alkaline unwinding ATC-15 cells treated with NaB showed more BLM-induced DNA strand breaks than NaB-untreated CHO cells. The time-course of BLM-induced DNA damage to nuclear DNA was similar for NaB-untreated mammalian and insect cells, but with mosquito cells showing less DNA strand breaks, both at physiological temperatures and at 4 {sup o}C. However, when DNA repair was inhibited by low temperatures and chromatin was decondensed by NaB treatments, differences in BLM-induced DNA damage between these cells lines were no longer observed. In both cell lines, NaB did not affect BLM action on cell growth and viability. On the other hand, the low sensitivity of ATC-15 cells to BLM was reflected in their better growth efficiency. These cells exhibited a satisfactory growth at BLM doses that produced a permanent arrest of growth in CHO cells. The data suggest that mosquito cells might have linker DNAs shorter than those of mammalian cells, which would result in the observed both greater chromatin condensation and greater resistance to DNA damage induced by BLM as compared to CHO cells.

  2. Engineered cell-cell communication via DNA messaging

    Directory of Open Access Journals (Sweden)

    Ortiz Monica E

    2012-09-01

    Full Text Available Abstract Background Evolution has selected for organisms that benefit from genetically encoded cell-cell communication. Engineers have begun to repurpose elements of natural communication systems to realize programmed pattern formation and coordinate other population-level behaviors. However, existing engineered systems rely on system-specific small molecules to send molecular messages among cells. Thus, the information transmission capacity of current engineered biological communication systems is physically limited by specific biomolecules that are capable of sending only a single message, typically “regulate transcription.” Results We have engineered a cell-cell communication platform using bacteriophage M13 gene products to autonomously package and deliver heterologous DNA messages of varying lengths and encoded functions. We demonstrate the decoupling of messages from a common communication channel via the autonomous transmission of various arbitrary genetic messages. Further, we increase the range of engineered DNA messaging across semisolid media by linking message transmission or receipt to active cellular chemotaxis. Conclusions We demonstrate decoupling of a communication channel from message transmission within engineered biological systems via the autonomous targeted transduction of user-specified heterologous DNA messages. We also demonstrate that bacteriophage M13 particle production and message transduction occurs among chemotactic bacteria. We use chemotaxis to improve the range of DNA messaging, increasing both transmission distance and communication bit rates relative to existing small molecule-based communication systems. We postulate that integration of different engineered cell-cell communication platforms will allow for more complex spatial programming of dynamic cellular consortia.

  3. 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.

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

    International Nuclear Information System (INIS)

    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

  5. Influence of boron addition to Ti–13Zr–13Nb alloy on MG63 osteoblast cell viability and protein adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Majumdar, P., E-mail: m.pallab@gmail.com [School of Mechanical Science, Indian Institute of Technology, Bhubaneswar (India); Singh, S.B. [Department of Metallurgical and Materials Engineering, Indian Institute of Technology, Kharagpur (India); Dhara, S. [School Medical Science and Technology, Indian Institute of Technology, Kharagpur (India); Chakraborty, M. [School of Mechanical Science, Indian Institute of Technology, Bhubaneswar (India)

    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. - Highlights: • The influence of boron addition on biocompatibility of Ti–13Zr–13Nb • Boron forms in situ TiB in TZN matrix and decreases cell proliferation on TZN surfaces. • Protein adsorption is lower in TZNB than in TZN. • Compared to TZNB composite, TZN alloy is more suitable for bone grafting applications.

  6. DNA typing of epithelial cells after strangulation.

    Science.gov (United States)

    Wiegand, P; Kleiber, M

    1997-01-01

    DNA typing was carried out on epithelial cells which were transferred from the hands of the suspect onto the neck of the victim. In an experimental study 16 suspect-victim combinations were investigated for estimating the typing success. Alternatively to an attack against the neck, the upper arm was used for "strangulation". PCR typing was carried out using the short tandem repeat systems (STRs) HumCD4, HumVWF31A (VWA) and Hum-FIBRA (FGA) and the success rate was > 70% for all 3 systems. In most of the cases mixed patterns containing the phenotype of the suspect and the victim were obtained. In a case where strangulation was the cause of death, epithelial cells could be removed from the neck of the victim. The DNA pattern of the suspect could be successfully amplified using four STRs, demonstrating the applicability of this approach for practical casework. PMID:9274940

  7. X-ray radiation effect of C-arm on adipose tissue-mesenchymal stem cell viability and population doubling time

    Directory of Open Access Journals (Sweden)

    Ahmad J. Rahyussalim

    2016-04-01

    Full Text Available Background: Adipose tissue derived mesenchymal stem cells (AT-MSCs are relatively easy in isolation procedure compared to bone marrow-derived. Minimally invasive MSC injections need C-arm as guidance that potentially influence the cell viability and doubling time. This study aimsed to determine the effect of C-arm X-ray exposure on AT-MSC viability and population doubling time (PDT.Methods: This experimental study used cryopreserved adipose tissue derived MSCs stored in Stem Cell Medical Technology Integrated Service Unit Cipto Mangunkusumo Hospital. Cells were thawed, propagated, and exposed to varying doses of C-arm X-ray radiation. Stem cell viability was measured, and then the cells were cultured to assess their PDT. Generalized linear models test was used to compare cell viability between post-thaw, post-propagation, post-radiation, post-culture post-radiation, and control and between radiation dose groups. Kruskal-Wallis test assessed PDT between various radiation doses in post-radiation groups. Wilcoxon test was used to assess PDT between pre-radiation and post-radiation groups.Results: Mean confluence period of adipose MSCs post- irradiation was 4.33 days. There was no statistically significant difference in MSC viability after X-ray exposure between pre- and post-irradiation groups (p=0.831. There was no correlation between post-irradiation viability and radiation dose (p=0.138, r=0.503. There were no significant differences in PDT between pre- and post-culture post-irradiation groups and between various radiation doses in post-irradiation groups (p=0.792.  Conclusion: MSC viability and PDT were not influenced by radiation exposure up to 32.34 mgray.

  8. Effect of single-walled carbon nanotubes on tumor cells viability and formation of multicellular tumor spheroids

    Science.gov (United States)

    Yakymchuk, Olena M.; Perepelytsina, Olena M.; Dobrydnev, Alexey V.; Sydorenko, Mychailo V.

    2015-03-01

    This paper describes the impact of different concentrations of single-walled carbon nanotubes (SWCNTs) on cell viability of breast adenocarcinoma, MCF-7 line, and formation of multicellular tumor spheroids (MTS). Chemical composition and purity of nanotubes is controlled by Fourier transform infrared spectroscopy. The strength and direction of the influence of SWCNTs on the tumor cell population was assessed by cell counting and measurement of the volume of multicellular tumor spheroids. Effect of SWCNTs on the formation of multicellular spheroids was compared with the results obtained by culturing tumor cells with ultra dispersed diamonds (UDDs). Our results demonstrated that SWCNTs at concentrations ranging from 12.5 to 50 μg/ml did not have cytotoxic influence on tumor cells; instead, they had weak cytostatic effect. The increasing of SWCNTs concentration to 100 to 200 μg/ml stimulated proliferation of tumor cells, especially in suspension fractions. The result of this influence was in formation of more MTS in cell culture with SWCNTs compared with UDDs and control samples. In result, the median volume of MTS after cultivation with SWCNTs at 100 to 200 μg/ml concentrations is 3 to 5 times greater than that in samples which were incubated with the UDDs and is 2.5 times greater than that in control cultures. So, if SWCNTs reduced cell adhesion to substrate and stimulated formation of tumor cell aggregates volume near 7 · 10-3 mm3, at the same time, UDDs reduced adhesion and cohesive ability of cells and stimulated generation of cell spheroids volume no more than 4 · 10-3 mm3. Our results could be useful for the control of cell growth in three-dimensional culture.

  9. Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation.

    Science.gov (United States)

    Wang, Zhihao; Wang, Yun; Zhang, Dongxu; Li, Jianghua; Hua, Zhaozhe; Du, Guocheng; Chen, Jian

    2010-02-01

    Alkaline polygalacturonate lyase (PGL) production by Pichia pastoris GS115 was used as a model to study the mechanism and strategy for enhancing heterologous protein production. In order to enhance cell viability and volumetric recombinant protein productivity, sorbitol, which had been confirmed to be a non-repressive carbon source, was added together with methanol during the induction phase. The resultant PGL activity was up to 1593 U mL(-1), which was enhanced 1.85-fold compared to the control (863 U mL(-1)) cultured with sorbitol added at a constant rate of 3.6 g h(-1)L(-1) after an induction period of 100 h. Further results revealed that an appropriate sorbitol co-feeding strategy not only decreased the cell mortality to 8.8% (the control is about 23.1%) in the end of fermentation, but also reduced the proteolytic degradation of PGL.

  10. Mitochondrial DNA mutations in single human blood cells.

    Science.gov (United States)

    Yao, Yong-Gang; Kajigaya, Sachiko; Young, Neal S

    2015-09-01

    Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34(+) cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34(+) cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34(+) cells obtained from the same donor over time suggests stability of some somatic mutations. CD34(+) cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification. PMID:26149767

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

    Science.gov (United States)

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

    2016-05-01

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

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

    Science.gov (United States)

    Librizzi, Mariangela; Tobiasch, Edda; Luparello, Claudio

    2016-01-01

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

  13. Effect of(12)C (+5) ion beam irradiation on cell viability and plant regeneration in callus, protoplasts and cell suspensions ofLavateva thuringiaca.

    Science.gov (United States)

    Vazquez-Tello, A; Uozumi, T; Hidaka, M; Kobayashi, Y; Watanabe, H

    1996-11-01

    The biological effects of irradiation with(12)C(+5) ion beam on plant cells have been analyzed. Protoplasts and cell suspensions ofLavatera thuringiaca, and a somatic hybrid callus (Hibiscus rosa-sinensis +Lavatera thuringiaca), were irradiated with doses from 0.05 to 50 Gy, and the effects on cell growth, cell division, cell viability and embryogenesis rates were analyzed. Irradiation with(12)C(+5) ion beam at relatively very low doses (5.0 Gy) significantly inhibited cell division, yet the survival rate and regeneration capability of the cells through somatic embryogenesis were conserved in more than 70 and 50 %, respectively. These results indicate that cell division is the most sensitive parameter to irradiation, accounting for the inhibition of colony formation and callus growth. The potential use of the(12)C(+5) ion beam in asymmetric protoplast fusion experiments is discussed. PMID:24178652

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

    Directory of Open Access Journals (Sweden)

    Aneta Hill-Bator

    2014-01-01

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

  15. Mitochondrial aldehyde dehydrogenase 2 protects gastric mucosa cells against DNA damage caused by oxidative stress.

    Science.gov (United States)

    Duan, Yantao; Gao, Yaohui; Zhang, Jun; Chen, Yinan; Jiang, Yannan; Ji, Jun; Zhang, Jianian; Chen, Xuehua; Yang, Qiumeng; Su, Liping; Zhang, Jun; Liu, Bingya; Zhu, Zhenggang; Wang, Lishun; Yu, Yingyan

    2016-04-01

    Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a member of the aldehyde dehydrogenase superfamily and is involved with the metabolic processing of aldehydes. ALDH2 plays a cytoprotective role by removing aldehydes produced during normal metabolism. We examined the cytoprotective role of ALDH2 specifically in gastric mucosa cells. Overexpression of ALDH2 increased the viability of gastric mucosa cells treated with H2O2, while knockdown of ALDH2 had an opposite effect. Moreover, overexpression of ALDH2 protected gastric mucosa cells against oxidative stress-induced apoptosis as determined by flow cytometry, Hoechst 33342, and TUNEL assays. Consistently, ALDH2 knockdown had an opposite effect. Additionally, DNA damage was ameliorated in ALDH2-overexpressing gastric mucosa cells treated with H2O2. We further identified that this cytoprotective role of ALDH2 was mediated by metabolism of 4-hydroxynonenal (4-HNE). Consistently, 4-HNE mimicked the oxidative stress induced by H2O2 in gastric mucosa cells. Treatment with 4-HNE increased levels of DNA damage in ALDH2-knockdown GES-1 cells, while overexpression of ALDH2 decreased 4-HNE-induced DNA damage. These findings suggest that ALDH2 can protect gastric mucosa cells against DNA damage caused by oxidative stress by reducing levels of 4-HNE.

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

    OpenAIRE

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

    2013-01-01

    Osteosarcoma is the most common primary bone tumor in children and adolescents. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have antitumor effects in several cancers. Here, we report that a novel synthetic berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of G292, KHOS, and MG-63 human os...

  17. 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

  18. 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.

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

    Science.gov (United States)

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

    2016-10-31

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

  20. Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies.

    Science.gov (United States)

    Lu, Yujie; Darne, Chinmay D; Tan, I-Chih; Zhu, Banghe; Hall, Mary A; Lazard, Zawaunyka W; Davis, Alan R; Simpson, Lashan; Sevick-Muraca, Eva M; Olmsted-Davis, Elizabeth A

    2013-10-01

    Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models.

  1. 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

  2. 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...

  3. Influence of conductive polymer doping on the viability of cardiac progenitor cells

    OpenAIRE

    Gelmi, Amy; Kozak Ljunggren, Monika; Rafat, Mehrdad; Jager, Edwin

    2014-01-01

    Cardiac tissue engineering via the use of stem cells is the future for repairing impaired heart function that results from a myocardial infarction. Developing an optimised platform to support the stem cells is vital to realising this, and through utilising new smart materials such as conductive polymers we can provide a multi-pronged approach to supporting and stimulating the stem cells via engineered surface properties, electrical, and electromechanical stimulation. Here we present a fundame...

  4. Detection of G-quadruplex DNA in mammalian cells

    NARCIS (Netherlands)

    Henderson, Alexander; Wu, Yuliang; Huang, Yu Chuan; Chavez, Elizabeth A.; Platt, Jesse; Johnson, F. Brad; Brosh, Robert M.; Sen, Dipankar; Lansdorp, Peter M.

    2014-01-01

    It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal antibodi

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

    NARCIS (Netherlands)

    Łopacińska, Joanna M; Grǎdinaru, Cristian; Wierzbicki, Rafal; Købler, Carsten; Schmidt, Michael S; Madsen, Martin T; Skolimowski, Maciej; Dufva, Martin; Flyvbjerg, Henrik; Mølhave, Kristian

    2012-01-01

    Knowledge of cells' interactions with nanostructured materials is fundamental for bio-nanotechnology. We present results for how individual mouse fibroblasts from cell line NIH3T3 respond to highly spiked surfaces of silicon black that were fabricated by maskless reactive ion etching (RIE). We did s

  6. Ochratoxin A: induction of (oxidative) DNA damage, cytotoxicity and apoptosis in mammalian cell lines and primary cells

    International Nuclear Information System (INIS)

    Ochratoxin A (OTA) is a nephrotoxic/-carcinogenic mycotoxin, produced by several Aspergillus- and Penicillium-strains. Humans are exposed to OTA via food contamination, a causal relationship of OTA to human endemic Balkan nephropathy is still under debate. Since DNA-adducts of OTA or its metabolites could not be identified unambiguously, its carcinogenic effectiveness might be related to secondary effects, such as oxidative cell damage or cell proliferation. In this study, OTA mediated induction of (oxidative) DNA damage, cytotoxicity (necrosis, growth inhibition, apoptosis) and modulation of glutathione were investigated in cell lines (V79, CV-1) and primary rat kidney cells. After 24 h incubation, viability of V79 cells was strongly decreased by OTA concentrations >2.5 μmol/L, whereas CV-1 cells were clearly less sensitive. Strong growth inhibition occurred in both cell lines (IC50 ∼2 μmol/L). Apoptosis, detected with an immunochemical test and with flow cytometry, was induced by >1 μmol/L OTA. Oxidative DNA damage, detected by comet assay after additional treatment with repair enzymes, was induced in all cell systems already at five-fold lower concentrations. Glutathione in CV-1 cells was depleted after 1 h incubation (>100 μmol/L). In contrast, an increase was measured after 24 h incubation (>0.5 μmol/L). In conclusion, OTA induces oxidative DNA damage at low, not yet cytotoxic concentrations. Oxidative DNA damage might initiate cell transformation eventually in connection with proliferative response following cytotoxic cell death. Both events might represent pivotal factors in the chain of cellular events leading into nephro-carcinogenicity of OTA

  7. Using amorphous silicon solar cells to boost the viability of luminescent solar concentrators

    Energy Technology Data Exchange (ETDEWEB)

    Farrell, Daniel J. [Physics Department, Imperial College London, South Kensington campus, SW7 2AZ, London (United Kingdom); Sark, Wilfried G.J.H.M. van [Utrecht University, Faculty of Science, Debye Institute for Nanomaterials Science, Nanophotonics - Physics of Devices, P.O. Box 80000, 3508 TA Utrecht (Netherlands); Utrecht University, Copernicus Institute for Sustainable Development and Innovation, Science, Technology and Society, Heidelberglaan 2, 3584 CS Utrecht (Netherlands); Velthuijsen, Steven T.; Schropp, Ruud E.I. [Utrecht University, Faculty of Science, Debye Institute for Nanomaterials Science, Nanophotonics - Physics of Devices, P.O. Box 80000, 3508 TA Utrecht (Netherlands)

    2010-04-15

    We have, for the first time, designed and fabricated hydrogenated amorphous silicon solar cells to be used in conjunction with Luminescent Solar Concentrators (LSCs). LSCs are planar plastic sheets doped with organic dyes that absorb solar illumination and down shift the energy to narrowband luminescence which is collected by solar cells attached to the sheet edge. We fabricated an LSC module with two bonded solar cells and performed characterisation with the cells connected in series and parallel configurations. We find that the LSC module has an optical collection efficiency of 9.5% and an optimum power conversion efficiency of approaching 1% when the cells are in a parallel connection. (copyright 2010 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  8. Effects of hydroxyapatite nanostructure on channel surface of porcine acellular dermal matrix scaffold on cell viability and osteogenic differentiation of human periodontal ligament stem cells

    Directory of Open Access Journals (Sweden)

    Ge S

    2013-05-01

    Full Text Available Shaohua Ge,1 Ning Zhao,1 Lu Wang,1 Hong Liu,2 Pishan Yang11Shandong Provincial Key Laboratory of Oral Biomedicine, Department of Periodontology, Shandong University; 2State Key Laboratory of Crystal Materials, Center of Bio and Micro/Nano Functional Materials, Shandong University, Jinan, People's Republic of ChinaAbstract: A new nanostructured hydroxyapatite-coated porcine acellular dermal matrix (HAp-PADM was fabricated by a biomimetic mineralization method. Human periodontal ligament stem cells were seeded on HAp-PADM and the effects of this scaffold on cell shape, cytoskeleton organization, cell viability, and osteogenic differentiation were examined. Periodontal ligament stem cells cultured on HAp-PADM exhibited different cell shape when compared with those on pure PADM. Moreover, HAp-PADM promoted cell viability and alkaline phosphatase activity significantly. Based on quantitative real-time polymerase chain reaction, the expression of bone-related markers runt-related transcription factor 2 (Runx2, osteopontin (OPN, and osteocalcin (OCN upregulated in the HAp-PADM scaffold. The enhancement of osteogenic differentiation of periodontal ligament stem cells on the HAp-PADM scaffold was proposed based on the research results. The results of this study highlight the micro-nano, two-level, three-dimensional HAp-PADM composite as a promising scaffold for periodontal tissue engineering.Keywords: hydroxyapatite, scaffold, nanostructure, proliferation, differentiation, tissue engineering

  9. Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex

    OpenAIRE

    Mayanagi, Kouta; Kiyonari, Shinichi; Nishida, Hirokazu; Saito, Mihoko; Kohda, Daisuke; Ishino, Yoshizumi; Shirai, Tsuyoshi; Morikawa, Kosuke

    2011-01-01

    DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally ...

  10. A Viability Approach for Robustness Measurement, Organizational Autopoiesis, and Cell Turnover in a Multicellular System.

    Science.gov (United States)

    Sarr, Abdoulaye; Désilles, Anya; Fronville, Alexandra; Rodin, Vincent

    2016-04-01

    In this article, we use the potential of computational biology to highlight the key role of cell apoptosis for studying some tissue's properties through in silico experiments of morphogenesis. Our morphogenesis model is a new approach focusing on the deterministic program within cells that controls their placement and their differentiation at the beginning of the embryogenesis. Indeed, when the tissue is made by just a few pair of cells, we consider that cellular mechanisms are related neither to the influence of mechanical forces nor to the spread of chemicals. Dynamics are based on spatial and logical choices, the other factors being involved when the tissue contains a large number of cells. We had established a mathematical formulation of such a model and had enlightened the link between phenotype (cell placement and cell differentiation) and genotype (cell program) at the early embryogenesis. Indeed, that work allowed for generating any early tissue and the associated program that designs it. We propose now to study and assess some properties of these tissues for further selection and classification purposes. More precisely, we present in this article novel methods to measure tissue robustness based on the backward morphogenesis of our model. We also show some implementations of their self-maintenance properties, on the one hand to deal with environment disturbances through autopoiesis and on the other hand to achieve a dynamical steady state which ensures tissue renewal. PMID:26958901

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

    Science.gov (United States)

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

    2010-07-01

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

  12. Detection on emamectin benzoate-induced apoptosis and DNA damage in Spodoptera frugiperda Sf-9 cell line.

    Science.gov (United States)

    Wu, Xiwei; Zhang, Lei; Yang, Chao; Zong, Mimi; Huang, Qingchun; Tao, Liming

    2016-01-01

    Emamectin benzoate (EMB), an important macrocyclic lactone insecticide that belongs to the avermectin family and possesses excellent potency in controlling pests, is non-carcinogenic and non-mutagenic conducted in rats and mice, but EMB-induced cytotoxicity and genotoxicity in arthropod insect have been seldom reported yet. In the present paper, we quantified the cytotoxicity of EMB through the detections on cell viability, DNA damage, and cell apoptosis in Spodoptera frugiperda Sf-9 cells in vitro. The results showed that EMB caused a concentration- and time-dependent reduction on the viability of Sf-9 cells, and the median inhibitory concentrations (IC50) were 3.34μM at 72h of exposure. The dual acridine orange/ethidium bromide staining showed that exposure to EMB induced a significant time- and concentration-dependent increase on cell apoptosis. The alkaline comet assay revealed that EMB induced significant increases on single-strand DNA breaks, and the percentage of γH2AX-positive cells represented a time- and concentration-dependent formation of DNA double-strand breaks in Sf-9 cells. Interestingly, the similar cytotoxic actions of EMB also went for the human cancerous HeLa cells as a control cell group. Data demonstrated the potential cytotoxic effect of EMB on Sf-9 cells that was significantly greater than the effect of hydrogen peroxide at the same concentrations.

  13. Relationship between reactive oxygen species and sodium-selenite-induced DNA damage in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    ZOU Yunfeng; NIU Piye; GONG Zhiyong; YANG Jin; YUAN Jing; WU Tangchun; CHEN Xuemin

    2007-01-01

    Selenium compounds,as an effective chemopreventive agent,can induce apoptosis in tumor cells.Reactive oxygen species(ROS)are important mediators in apoptosis induced by various stimuli,which include chemopreventive agents.In this study,we investigated the relationship between ROS and the levels of DNA damage induced by selenite in HepG2 cells.After HepG2 cells were treated with selenite,there was a dose-dependent decrease in cell viability.The levels of ROS induced by selenite were measured by 2',7'-dichlorofluorescein diacetate(DCFH-DA)fluorescence,which shows a dose-and time-dependent increase in HepG2 cells.The levels of DNA damage in HepG2 increased in all cells treated with an increasing dose of selenite at 0,2.5,5,10,and 20 μmol/L.N-acetylcysteine(NAC),a known antioxidant,increased cell viability and decreased ROS generation.Moreover,NAC effectively blocked DNA damage induced by selenite.These results revealed that ROS might play an important role in selenite-induced DNA damage that can be reduced by NAC treatment.

  14. Viability and proliferation of L929, tumour and hybridoma cells in the culture media containing sericin protein as a supplement or serum substitute.

    Science.gov (United States)

    Cao, Ting-Ting; Zhang, Yu-Qing

    2015-09-01

    Cell cultures often require the addition of animal serum and other supplements. In this study, silk sericin, a bioactive protein, recovered from the waste of silk floss production was hydrolysed into three pepsin-degraded sericin peptides with different ranges of molecular mass. Normal animal cells, tumour cells and hybridoma cells were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute. The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture. The cell culture results showed that with the degradation of sericin, for normal mouse fibroblast L929 cells, addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone. When all serum was replaced by sericin, cell viability in the sericin medium could reach about one half of that in FBS medium. When in a medium containing a mixture of FBS: sericin (6:4, v/v), the cell culture effect is about 80 %. For the cultures of four tumour and one hybridoma cells, regardless of the molecular weight range, these degraded sericin peptides could substitute all serum in FBS media. The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium. In other words, cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media. The mechanism of the sericin protein to promote cell growth and proliferation will be further investigated later. PMID:25895088

  15. Role of surface-electrical properties on the cell-viability of carbon thin films grown in nanodomain morphology

    Science.gov (United States)

    Javid, Amjed; Kumar, Manish; Yoon, Seokyoung; Lee, Jung Heon; Tajima, Satomi; Hori, Masaru; Geon Han, Jeon

    2016-07-01

    Carbon thin films, having a combination of unique physical and chemical properties, exhibit an interesting biocompatibility and biological response to living entities. Here, the carbon films are developed in the morphology form of nano-domains with nanoscale inter-domain separations, tuned by plasma conditions in the facing target magnetron sputtering process. The wettability and surface energy are found to have a close relation to the inter-domain separations. The chemical structure of carbon films exhibited the relative enhancement of sp3 in comparison to sp2 with the increase of domain separations. The cell-viability of these films shows promising results for L929 mouse fibroblast and Saos-2 bone cells, when inter-domain separation is increased. Electrical conductivity and surface energy are identified to play the key role in different time-scales during the cell-proliferation process. The contribution from electrical conductivity is dominant in the beginning of the cultivation, whereas with the passage of time (~3–5 d) the surface energy takes control over conductivity to enhance the cell proliferation.

  16. 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

    2016-01-01

    Arctigenin (ARC) has been shown to have an anti-cancer effect in various cell types and tissues. However, there have been no studies concerning metastatic colorectal cancer (CRC). In this study, we investigated the anti-metastatic properties of ARC on colorectal metastasis and present a potential candidate drug. ARC induced cell cycle arrest and apoptosis in CT26 cells through the intrinsic apoptotic pathway via MAPKs signaling. In several metastatic phenotypes, ARC controlled epithelial-mesenchymal transition (EMT) through increasing the expression of epithelial marker E-cadherin and decreasing the expressions of mesenchymal markers; N-cadherin, vimentin, β-catenin, and Snail. Moreover, ARC inhibited migration and invasion through reducing of matrix metalloproteinase-2 (MMP-2) and MMP-9 expressions. In an experimental metastasis model, ARC significantly inhibited lung metastasis of CT26 cells. Taken together, our study demonstrates the inhibitory effects of ARC on colorectal metastasis. PMID:27618887

  17. 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.

  18. Delivery, Effect on Cell Viability, and Plasticity of Modified Aptamer Constructs

    DEFF Research Database (Denmark)

    Gissberg, Olof; Zaghloul, Eman M; Lundin, Karin E;

    2016-01-01

    AS1411 is a g-quadruplex-forming aptamer capable of selectively entering cancer cells by nucleolin receptor-mediated uptake. In this study, we investigated the cell internalization properties and plasticity of AS1411 carrying different locked nucleic acid-containing cargo oligonucleotides (ONs......) for delivery into A549 and U2OS cells. We found that internalization efficiency is highly governed by ON cargo chemistry and composition since the inherent antitumor properties of AS1411 were lost when attached to a nontoxic ON, noTox. However, a toxic ON, Tox, demonstrated potent cytotoxicity after aptamer......-mediated uptake in A549 cells. We also examined the effect of unlocked nucleic acid (UNA) modifications in the loop region of the aptamer, and how the cargo ONs and UNA incorporation affect the secondary structure of AS1411, in the presence or absence of two novel ellipticine derivatives. These findings add new...

  19. Effect of low-dimensional alumina structures on viability of L 929 cells

    Energy Technology Data Exchange (ETDEWEB)

    Fomenko, Alla N., E-mail: alserova@ispms.tsc.ru; Korovin, Matvey S., E-mail: msk@ispms.tsc.ru; Bakina, Olga V., E-mail: ovbakina@ispms.tsc.ru; Kazantsev, Sergey O., E-mail: kzso@ispms.tsc.ru; Glazkova, Elena A., E-mail: eagl@ispms.tsc.ru; Svarovskaya, Natalia V., E-mail: nvsv@ispms.tsc.ru; Lozhkomoev, Aleksandr S., E-mail: asl@ispms.tsc.ru [National Research Tomsk Polytechnic University, Tomsk, 634050 (Russian Federation)

    2015-10-27

    In the study, we estimated the cytotoxicity of alumina nanoparticles differing in shape (nanofibers, nanoplates, nanosheets, agglomerates of nanosheets) and close in physicochemical properties (particle size, specific surface area, phase composition, and zeta potential). The alumina structures were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD) data, low-temperature nitrogen adsorption, and dynamic light scattering (DLS). The cytotoxicity was estimated on fibroblast cells of the L929 line. It was found that a more adverse effect on the cells was exerted by alumina nanofibers and nanosheets. The action of nanosheets on the cells was inhibitory and was of about the same level, irrespective of the observation period. The effect of alumina nanosheet agglomerates and nanoplates on the cell proliferation was weak even at an exposure time of 72 h.

  20. 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.

  1. The Effect of Sericin from Various Extraction Methods on Cell Viability and Collagen Production

    OpenAIRE

    Pornanong Aramwit; Sorada Kanokpanont; Titpawan Nakpheng; Teerapol Srichana

    2010-01-01

    Silk sericin (SS) can accelerate cell proliferation and attachment; however, SS can be extracted by various methods, which result in SS exhibiting different physical and biological properties. We found that SS produced from various extraction methods has different molecular weights, zeta potential, particle size and amino acid content. The MTT assay indicated that SS from all extraction methods had no toxicity to mouse fibroblast cells at concentrations up to 40 μg/mL after 24 h incubation, b...

  2. Validation and reproducibility of computerised cell-viability analysis of tissue slices

    Directory of Open Access Journals (Sweden)

    Elliott Janet AW

    2003-03-01

    Full Text Available Abstract Background The identification of live cells using membrane integrity dyes has become a frequently used technique, especially with articular cartilage and chondrocytes in situ where tissue slices are used to assess cell recovery as a function of location. The development of a reproducible computerised method of cell evaluation would eliminate many variables associated with manual counting and significantly reduce the amount of time required to evaluate experimental results. Methods To validate a custom computerised counting program, intra-person and inter-person cell counts of nine human evaluators (three groups – unskilled, novice, and experienced were compared with repeated pixel counts of the custom program on 15 digitised images (in triplicate of chondrocytes in situ stained with fluorescent dyes. Results Results indicated increased reproducibility with increased experience within evaluators [Intraclass Correlation Coefficient (ICC range = 0.67 (unskilled to 0.99 (experienced] and between evaluators [ICC = 0.47 (unskilled, 0.85 (novice, 0.93 (experienced]. The computer program had perfect reproducibility (ICC = 1.0. There was a significant relationship between the average of the experienced evaluators results and the custom program results (ICC = 0.77. Conclusions This study demonstrated that increased experience in cell counting resulted in increased reproducibility both within and between human evaluators but confirmed that the computer program was the most reproducible. There was a good correlation between the intact cell recovery determined by the computer program and the experienced human evaluators. The results of this study showed that the computer counting program was a reproducible tool to evaluate intact cell recovery after use of membrane integrity dyes on chondrocytes in situ. This and the significant decrease in the time used to count the cells by the computer program advocate its use in future studies because it has

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

    OpenAIRE

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

    2013-01-01

    Focal adhesion kinase (FAK), hyaluronan (HA), and hyaluronan synthase-3 (HAS3) have been implicated in cancer growth and progression. FAK inhibition with the small molecule inhibitor Y15 decreases colon cancer cell growth in vitro and in vivo. HAS3 inhibition in colon cancer cells decreases FAK expression and activation, and exogenous HA increases FAK activation. We sought to determine the genes affected by HAS and FAK inhibition and hypothesized that dual inhibition would synergistically inh...

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

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yoon Shin; Lim, Goh-Woon [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung [Department of Microbiology, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Yoo, Eun-Sun [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Chan Ra, Jeong [Stem Cell Research Center, RNL BIO, Seoul 153-768 (Korea, Republic of); Ryu, Kyung-Ha, E-mail: ykh@ewha.ac.kr [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of)

    2012-06-22

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

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2011-06-01

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

  7. In vitro organotin administration alters guinea pig cochlear outer hair cell shape and viability.

    Science.gov (United States)

    Clerici, W J; Chertoff, M E; Brownell, W E; Fechter, L D

    1993-06-01

    Trimethyltin (TMT) and triethyltin (TET) disrupt auditory function at doses far below those shown to be neurotoxic. In vivo studies suggest that the initial effect of TMT on hearing occurs at the inner hair cell/spiral ganglion cell synapse, while later, the outer hair cell (OHC) undergoes structural and functional damage. TET produces acute effects upon afferent neurotransmission similar to those observed following TMT, but TET's effects on OHC structure and function have not been examined. OHCs are motile elements within the cochlea, believed to modulate the sensitivity and tuning within the inner ear. Changes in OHC length may alter hearing function, and length changes have been reported following exposure to various ototoxic agents in vitro. In the present study, 77 OHCs from 45 pigmented male guinea pigs were isolated in primary culture and exposed for 90 min to concentrations between 30 microM and 1.0 mM of TMT or TET and then to bathing medium for 30 min to remove the toxicant. Significant shortening of the OHC cell body occurred at all doses to both organotins, with a mean reduction in length of 15.1 and 20.2% for 1.0 mM TMT and TET, respectively, at the end of testing; control cells were only 3.4% shorter at the end of 90 min of perfusion with bathing medium. The effect of organotin exposure on OHC volume was not consistently related to either TMT or TET concentration or altered cell length. In addition, disruption of the plasma membrane characterized by bleb formation, the forceful ejection of cytoplasm, or bursting was seen in 80% of cells exposed to 1.0 mM TET, although not TMT; lower concentrations of both organotins disrupted the cell membrane in 10-30% of cells. Membrane rupture was not reliably associated with either increased cell volume or decreased length, implicating a weakening of the plasma membrane or cortical lattice as the basis for this effect. Consistent with the irreversible structural weakening of the lateral wall, resorption of

  8. Synthesis, interaction with DNA, cytotoxicity, cell cycle arrest and apoptotic inducing properties of ruthenium(II) molecular "light switch" complexes.

    Science.gov (United States)

    Shobha Devi, C; Anil Kumar, D; Singh, Surya S; Gabra, Nazar; Deepika, N; Kumar, Y Praveen; Satyanarayana, S

    2013-06-01

    In an endeavor toward the development of metal-based anticancer drugs, we present here the design, synthesis and characterization of three ruthenium(II) functionalized phenanthroline complexes with extended π-conjugation. These complexes have been shown to act as promising CT-DNA intercalators as evidenced by UV-visible, luminescence, emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide and salt dependent studies. All three complexes [Ru(Hdpa)2PPIP](2+) (1), [Ru(Hdpa)2PIP](2+) (2), [Ru(Hdpa)24HEPIP](2+) (3) clearly demonstrated that they can bind to DNA through the intercalation mode. Cell viability experiments indicated that all complexes showed significant dose dependent cytotoxicity in selected cell lines. The apoptosis and cell cycle arrest were also investigated. The complexes were docked into DNA-base-pairs using the 'GOLD' (Genetic Optimization for Ligand Docking), docking program. PMID:23665797

  9. The role of ribosylated-BSA in regulating PC12 cell viability.

    Science.gov (United States)

    Kuo, Tsun-Yung; Huang, Chuen-Lin; Yang, Jung-Mou; Huang, Wei-Jung; Huang, Nai-Kuei; Chen, Yue-Wen; Lin, Ren-Jye; Yang, Ying-Chen

    2012-08-01

    Glycation, one of the post-translational modifications, is known to influence protein structure and biological function. Advanced glycation end products (AGEs) have been shown to cause pathologies of diabetes. Glycation levels in patients with Alzheimer's disease (AD) are higher than in normal people. However, whether the glycation of susceptible proteins is a triggering event for cell damage or simply a result remains to be elucidated. In this study, we demonstrated that ribose-conjugated BSA (Rib-BSA) directly induces PC12 cell death in a dose- and time-dependent manner. The IC(50) is 4.6 μM. Unlike glucose-incubated BSA, Rib-BSA rapidly forms cytotoxic AGEs. PC12 is vulnerable to Rib-BSA. However, fructose can induce AGE formation, although no effect on cell survival was observed. This effect of Rib-BSA is reversed by pretreatment of pioglitazone and rosiglitazone, which belongs to thiazolidinediones (TZDs) and are peroxisome proliferator-activated receptor (PPAR-γ) ligands. Moreover, Rib-BSA upregulates inducible nitric oxide synthase (iNOS), cycloxygenase 2 (COX-2) expression, and p-38 phosphorylation and leaves extracellular regulated protein1/2 (ERK1/2) phosphorylation unchanged. The Rib-BSA-induced signaling changes are blocked by rosiglitazone and confirmed by PPAR-γ small-interfering RNA transfection. The reduction of cell survival by Rib-BSA is blocked by the iNOS inhibitor and p38 inhibitor. No effect on cell survival was observed using the COX-2 inhibitor. Consequently, these results show that Rib-BSA directly inducing PC12 cell death is a triggering event and TZDs protect PC12 cell from Rib-BSA damage. Signaling molecules, such as PPAR-γ, P38, and iNOS, are involved in Rib-BSA-mediated cytotoxicity.

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

    Energy Technology Data Exchange (ETDEWEB)

    Teng, Chih-Chuan [Institute of Nursing and Department of Nursing, Chang Gung University of Science and Technology, Taiwan (China); Chronic Diseases and Health Promotion Research Center, CGUST, Taiwan (China); Institute of Basic Medicine Science, National Cheng Kung University, Tainan, Taiwan (China); Kuo, Hsing-Chun [Institute of Nursing and Department of Nursing, Chang Gung University of Science and Technology, Taiwan (China); Chronic Diseases and Health Promotion Research Center, CGUST, Taiwan (China); Department of Medical Research China Medical University Hospital, Taichung, Taiwan (China); Sze, Chun-I, E-mail: szec@mail.ncku.edu.tw [Institute of Basic Medicine Science, Department of Cell Biology and Anatomy and Pathology, National Cheng Kung University, Tainan, Taiwan (China)

    2013-11-01

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

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

    Science.gov (United States)

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

    2011-08-01

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

  12. Delivery, Effect on Cell Viability, and Plasticity of Modified Aptamer Constructs.

    Science.gov (United States)

    Gissberg, Olof; Zaghloul, Eman M; Lundin, Karin E; Nguyen, Chi-Hung; Landras-Guetta, Corinne; Wengel, Jesper; Zain, Rula; Smith, C I Edvard

    2016-06-01

    AS1411 is a g-quadruplex-forming aptamer capable of selectively entering cancer cells by nucleolin receptor-mediated uptake. In this study, we investigated the cell internalization properties and plasticity of AS1411 carrying different locked nucleic acid-containing cargo oligonucleotides (ONs) for delivery into A549 and U2OS cells. We found that internalization efficiency is highly governed by ON cargo chemistry and composition since the inherent antitumor properties of AS1411 were lost when attached to a nontoxic ON, noTox. However, a toxic ON, Tox, demonstrated potent cytotoxicity after aptamer-mediated uptake in A549 cells. We also examined the effect of unlocked nucleic acid (UNA) modifications in the loop region of the aptamer, and how the cargo ONs and UNA incorporation affect the secondary structure of AS1411, in the presence or absence of two novel ellipticine derivatives. These findings add new insights to the design and future applications of aptamer-guided delivery of ON cargo to cancer cells. PMID:26859550

  13. DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by Ruta graveolens in human colon cancer cells.

    Science.gov (United States)

    Arora, Shagun; Tandon, Simran

    2015-01-01

    In the present study, we investigated the anti-cancer effect of various potencies of Ruta graveolens (Ruta) on COLO-205 cell line, as evidenced by cytotoxicity, migration, clonogenecity, morphological and biochemical changes and modification in the levels of genes associated with apoptosis and cell cycle. On treatment of COLO-205 cells maximal effects were seen with mother tincture (MT) and 30C potencies, wherein decrease in cell viability along with reduced clonogenecity and migration capabilities were noted. In addition morphological and biochemical alterations such as nuclear changes (fragmented nuclei with condensed chromatin) and DNA ladder-like pattern (increased amount of fragmented DNA) in COLO-205 cells indicating apoptotic related cell death were seen. The expression of apoptosis and cell-cycle related regulatory genes assessed by reverse transcriptase-PCR revealed an up-regulation of caspase 9, caspase-3, Bax, p21 and p27 expression and down-regulation of Bcl-2 expression in treated cells. The mode of cell death was suggestive of intrinsic apoptotic pathway along with cell cycle arrest at the G2/M of the cell cycle. Our findings indicate that phytochemicals present in Ruta showed potential for natural therapeutic product development for colon carcinoma.

  14. Ultraviolet Radiation-Induced Skin Aging: The Role of DNA Damage and Oxidative Stress in Epidermal Stem Cell Damage Mediated Skin Aging

    OpenAIRE

    Uraiwan Panich; Gunya Sittithumcharee; Natwarath Rathviboon; Siwanon Jirawatnotai

    2016-01-01

    Skin is the largest human organ. Skin continually reconstructs itself to ensure its viability, integrity, and ability to provide protection for the body. Some areas of skin are continuously exposed to a variety of environmental stressors that can inflict direct and indirect damage to skin cell DNA. Skin homeostasis is maintained by mesenchymal stem cells in inner layer dermis and epidermal stem cells (ESCs) in the outer layer epidermis. Reduction of skin stem cell number and function has been...

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

    Science.gov (United States)

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

    2014-01-01

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

  16. The effect of CO2 laser beam welded AISI 316L austenitic stainless steel on the viability of fibroblast cells, in vitro.

    Science.gov (United States)

    Köse, Ceyhun; Kaçar, Ramazan; Zorba, Aslı Pınar; Bağırova, Melahat; Allahverdiyev, Adil M

    2016-03-01

    It has been determined by the literature research that there is no clinical study on the in vivo and in vitro interaction of the cells with the laser beam welded joints of AISI 316L biomaterial. It is used as a prosthesis and implant material and that has adequate mechanical properties and corrosion resistance characteristics. Therefore, the interaction of the CO2 laser beam welded samples and samples of the base metal of AISI 316L austenitic stainless steel with L929 fibroblast cells as an element of connective tissue under in vitro conditions has been studied. To study the effect of the base metal and the laser welded test specimens on the viability of the fibroblast cells that act as an element of connective tissues in the body, they were kept in DMEMF-12 medium for 7, 14, 28 days and 18 months. The viability study was experimentally studied using the MTT method for 7, 14, 28 days. In addition, the direct interaction of the fibroblast cells seeded on 6 different plates with the samples was examined with an inverted microscope. The MTT cell viability experiment was repeated on the cells that were in contact with the samples. The statistical relationship was analyzed using a Tukey test for the variance with the GraphPad statistics software. The data regarding metallic ion release were identified with the ICP-MS method after the laser welded and main material samples were kept in cell culture medium for 18 months. The cell viability of the laser welded sample has been detected to be higher than that of the base metal and the control based on 7th day data. However, the laser welded sample's viability of the fibroblast cells has diminished by time during the test period of 14 and 28 days and base metal shows better viability when compared to the laser welded samples. On the other hand, the base metal and the laser welded sample show better cell viability effect when compared to the control group. According to the ICP-MS results of the main material and laser welded

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

    Directory of Open Access Journals (Sweden)

    Xueping Xie

    2012-11-01

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

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

    Science.gov (United States)

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

    2015-02-01

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

  19. Non-thermal DNA damage of cancer cells using near-infrared irradiation.

    Science.gov (United States)

    Tanaka, Yohei; Tatewaki, Naoto; Nishida, Hiroshi; Eitsuka, Takahiro; Ikekawa, Nobuo; Nakayama, Jun

    2012-08-01

    Previously, we reported that near-infrared irradiation that simulates solar near-infrared irradiation with pre- and parallel-irradiational cooling can non-thermally induce cytocidal effects in cancer cells. To explore these effects, we assessed cell viability, DNA damage response pathways, and the percentage of mitotic cancer cells after near-infrared treatment. Further, we evaluated the anti-cancer effects of near-infrared irradiation compared with doxorubicin in xenografts in nude mice by measuring tumor volume and assessing protein phosphorylation by immunoblot analysis. The cell viability of A549 lung adenocarcinoma cells was significantly decreased after three rounds of near-infrared irradiation at 20 J/cm(2). Apoptotic cells were observed in near-infrared treated cells. Moreover, near-infrared treatment increased the phosphorylation of ataxia-telangiectasia mutated (ATM) at Ser(1981), H2AX at Ser(139), Chk1 at Ser(317), structural maintenance of chromosome (SMC) 1 at Ser(966), and p53 at Ser(15) in A549 cells compared with control. Notably, near-infrared treatment induced the formation of nucleic foci of γH2AX. The percentage of mitotic A549 cells, as measured by histone H3 phosphorylation, decreased significantly after three rounds of near-infrared irradiation at 20 J/cm(2). Both near-infrared and doxorubicin inhibited the tumor growth of MDA-MB435 melanoma cell xenografts in nude mice and increased the phosphorylation of p53 at Ser(15), Chk1 at Ser(317), SMC1 at Ser(966), and H2AX at Ser(139) compared with control mice. These results indicate that near-infrared irradiation can non-thermally induce cytocidal effects in cancer cells as a result of activation of the DNA damage response pathway. The near-infrared irradiation schedule used here reduces discomfort and side effects. Therefore, this strategy may have potential application in the treatment of cancer.

  20. Increased viability and resilience of haemolymph cells in blue mussels following pre-treatment with acute high-dose gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Jaeschke, B. [Stockholm University (Sweden)

    2014-07-01

    In an initial experiment, blue mussels (Mytilus edulis) were exposed to a range of acute high doses of gamma radiation in the laboratory. Haemolymph was extracted and the haemocytes (blood cells) were scored for cell viability (% living cells) under a microscope, directly after irradiation (0.04, 0.4 or 4 Gy) and again after a subsequent treatment with hydrogen peroxide in vitro (final H{sub 2}O{sub 2} conc.: 0.2 μM). Cell viability in controls (0 Gy) was approximately 100% and no cell death was observable from radiation exposure alone. When treated with H{sub 2}O{sub 2} a decrease in cell viability was seen across all treatments, however this decrease in viability was reduced with increasing radiation pre-treatment (0 Gy = 53%; 0.04 Gy = 66%; 0.4 Gy = 75%; 4 Gy = 83%). To investigate the mechanism for this therapeutic effect observed, the experiment was repeated. Using mussels from a different location, the same, but more extensive method of irradiation (0[control], 0.04, 0.4 Gy, 5 or 40 Gy) and H{sub 2}O{sub 2} treatment was used. Additional haemolymph sub-samples were taken for analysis of catalase concentration. In this second experiment, viability of cells from controls was only 62%, indicating the mussels were in a poorer condition than those of the previous experiment. The lowest level of radiation exposure (0.04 Gy) further decreased the viability (56%). However, at higher doses the viability was increased compared to control, which then gradually declined with increasing dose (0.4 Gy = 75%; 5 Gy = 72%; 40 Gy = 65%). Catalase analysis demonstrated a complimentary pattern of activity of the antioxidant in the haemolymph, directly correlating with radiation dose (0 Gy = 0.2 U; 0.04 Gy = 0.1 U; 0.4 Gy = 1.3 U; 5 Gy = 0.9 U; 40 Gy = 0.1 Gy). Treatment with H{sub 2}O{sub 2} decreased cell viability across all treatments, but no pattern between radiation treatments was discernable. The results indicate that an acute dose of radiation not only has negligible

  1. Poly-I:C Decreases Dendritic Cell Viability Independent of PKR Activation

    DEFF Research Database (Denmark)

    Larsen, Hjalte List; Pedersen, Anders Elm

    2012-01-01

    Vaccination with tumor-antigen pulsed, monocyte-derived dendritic cells (DCs) has emerged as a promising strategy in cancer immunotherapy. The standard DC maturation cocktail consists of a combination of tumor necrosis factor-α (TNF-α)/interleukin (IL)-1β/IL-6 and prostaglandin E2 (PGE2...

  2. Cry1Ab treatment has no effects on viability of cultured porcine intestinal cells, but triggers Hsp70 expression.

    Directory of Open Access Journals (Sweden)

    Angelika Bondzio

    Full Text Available In vitro testing can contribute to reduce the risk that the use of genetically modified (GM crops and their proteins show unintended toxic effects. Here we introduce a porcine intestinal cell culture (IPEC-J2 as appropriate in vitro model and tested the possible toxic potential of Cry1Ab protein, commonly expressed in GM-maize. For comprehensive risk assessment we used WST-1 conversion and ATP content as metabolic markers for proliferation, lactate dehydrogenase release as indicator for cells with compromised membrane and transepithelial electrical resistance as parameter indicating membrane barrier function. The results were compared to the effects of valinomycin, a potassium ionophore, known to induce cytotoxic effects in most mammalian cell types. Whereas no toxicity was observed after Cry1Ab treatment, valinomycin induced a decrease in IPEC-J2 viability. This was confirmed by dynamic monitoring of cellular responses. Additionally, two dimensional differential in-gel electrophoresis was performed. Only three proteins were differentially expressed. The functions of these proteins were associated with responses to stress. The up-regulation of heat shock protein Hsp70 was verified by Western blotting as well as by enzyme-linked immunosorbent assay and may be related to a protective function. These findings suggest that the combination of in vitro testing and proteomic analysis may serve as a promising tool for mechanism based safety assessment.

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

    Science.gov (United States)

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

    2011-03-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Wojcieszak, Damian, E-mail: damian.wojcieszak@pwr.edu.pl [Faculty of Microsystem Electronics and Photonics, Wroclaw University of Technology, Janiszewskiego 11/17, 50-372 Wroclaw (Poland); Kaczmarek, Danuta [Faculty of Microsystem Electronics and Photonics, Wroclaw University of Technology, Janiszewskiego 11/17, 50-372 Wroclaw (Poland); Antosiak, Aleksandra [Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocław (Poland); Mazur, Michal [Faculty of Microsystem Electronics and Photonics, Wroclaw University of Technology, Janiszewskiego 11/17, 50-372 Wroclaw (Poland); Rybak, Zbigniew; Rusak, Agnieszka; Osekowska, Malgorzata [Department for Experimental Surgery and Biomaterials Research, Wroclaw Medical University, Poniatowskiego 2, 50-326 Wroclaw (Poland); Poniedzialek, Agata [Faculty of Microsystem Electronics and Photonics, Wroclaw University of Technology, Janiszewskiego 11/17, 50-372 Wroclaw (Poland); Gamian, Andrzej; Szponar, Bogumila [Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocław (Poland)

    2015-11-01

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

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

    Science.gov (United States)

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

    2011-05-01

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

  6. The effect of silver nanoparticles on Staphylococcus epidermidis biofilm biomass and cell viability

    OpenAIRE

    Bahman Khameneh; Hamed Zarei; Bibi Sedigheh Fazly Bazzaz

    2014-01-01

    Objective(s): Bacterial biofilm has been considered responsible for many deaths and high health costs worldwide. Their better protection against antibacterial agents compared to free living cells leads to poor treatment efficiency. Nanotechnology is promising approach to combat biofilm infections. The aim of the present study was to eradicate Staphylococcus epidermidis biofilm with silver nanoparticles (SNPs). Materials and Methods: SNPs were used at different concentrations (two fold dilutio...

  7. Cell-free fetal DNA and cell-free total DNA levels in spontaneous abortion with fetal chromosomal aneuploidy.

    Directory of Open Access Journals (Sweden)

    Ji Hyae Lim

    Full Text Available BACKGROUND: Cell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy. METHODOLOGY/PRINCIPAL FINDINGS: A nested case-control study was conducted with maternal plasma collected from 268 women in their first trimester of pregnancy. Subjects included 41 SA with normal fetal karyotype, 26 SA with fetal chromosomal aneuploidy, and 201 normal controls. The unmethylated PDE9A gene was used to measure the maternal plasma levels of cell-free fetal DNA. The GAPDH gene was used to measure the maternal plasma levels of cell-free total DNA. The diagnostic accuracy was measured using receiver-operating characteristic (ROC curves. Levels of cell-free fetal DNA and cell-free total DNA were significantly higher in both SA women with normal fetal karyotype and SA women with fetal chromosomal aneuploidy in comparison with the normal controls (P<0.001 in both. The correlation between cell-free fetal DNA and cell-free total DNA levels was stronger in the normal controls (r = 0.843, P<0.001 than in SA women with normal karyotype (r = 0.465, P = 0.002 and SA women with fetal chromosomal aneuploidy (r = 0.412, P = 0.037. The area under the ROC curve for cell-free fetal DNA and cell-free total DNA was 0.898 (95% CI, 0.852-0.945 and 0.939 (95% CI, 0.903-0.975, respectively. CONCLUSIONS: Significantly high levels of cell-free fetal DNA and cell-free total DNA were found in SA women with fetal chromosomal aneuploidy. Our findings suggest that cell-free fetal DNA and cell-free total DNA may be useful biomarkers for the prediction of SA

  8. Rv3351c, a Mycobacterium tuberculosis gene that affects bacterial growth and alveolar epithelial cell viability.

    Science.gov (United States)

    Pavlicek, Rebecca L; Fine-Coulson, Kari; Gupta, Tuhina; Quinn, Frederick D; Posey, James E; Willby, Melisa; Castro-Garza, Jorge; Karls, Russell K

    2015-12-01

    Despite the interactions known to occur between various lower respiratory tract pathogens and alveolar epithelial cells (AECs), few reports examine factors influencing the interplay between Mycobacterium tuberculosis bacilli and AECs during infection. Importantly, in vitro studies have demonstrated that the M. tuberculosis hbha and esxA gene products HBHA and ESAT6 directly or indirectly influence AEC survival. In this report, we identify Rv3351c as another M. tuberculosis gene that impacts the fate of both the pathogen and AEC host. Intracellular replication of an Rv3351c mutant in the human AEC type II pneumocyte cell line A549 was markedly reduced relative to the complemented mutant and parent strain. Deletion of Rv3351c diminished the release of lactate dehydrogenase and decreased uptake of trypan blue vital stain by host cells infected with M. tuberculosis bacilli, suggesting attenuated cytotoxic effects. Interestingly, an isogenic hbha mutant displayed reductions in AEC killing similar to those observed for the Rv3351c mutant. This opens the possibility that multiple M. tuberculosis gene products interact with AECs. We also observed that Rv3351c aids intracellular replication and survival of M. tuberculosis in macrophages. This places Rv3351c in the same standing as HBHA and ESAT6, which are important factors in AECs and macrophages. Defining the mechanism(s) by which Rv3351c functions to aid pathogen survival within the host may lead to new drug or vaccine targets.

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

    Science.gov (United States)

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

    2014-01-01

    The anti-proliferative activitiy of Hypogymnia physodes methanol extracts (ME) and its main constituents, physodalic acid (P1), physodic acid (P2), and 3-hydroxy physodic acid (P3), was tested on human cancer HeLa cell lines. Three lichen depsidones, P1, P2 and P3, were isolated from H. physodes ME using column chromatography and their structures were determined by UV, ESI TOF MS, 1H and 13C NMR. The content of P1, P2 and P3 in ME was determined using reversed-phase highperformance liquid chromatography with photodiode array detection. P1-3 represented even 70 % of the studied extract. The HeLa cells were incubated during 24 and 72 h in the presence of ME and depsidones P1, P2 and P3, at concentrations of 10-1000 μg/ml. Compounds P2 and P3 showed higher activity than compound P1. Half maximal inhibitory concentrations (IC50, μg/ml) of P1, P2, P3 and ME for 24-h incubation were 964, 171, 97 and 254 μg/ml, respectively, while for 72-h incubation they were 283, 66, 63 and 68 μg/ml. As far as we know, this is the first report on the effect of H. physodes ME and their depsidones on HeLa cells. PMID:24785112

  10. Effects of Stratospheric Conditions on the Viability, Metabolism and Proteome of Prokaryotic Cells

    Directory of Open Access Journals (Sweden)

    Dagmar Chudobova

    2015-08-01

    Full Text Available The application of ultraviolet (UV radiation to inhibit bacterial growth is based on the principle that the exposure of DNA to UV radiation results in the formation of cytotoxic lesions, leading to inactivation of microorganisms. Herein, we present the impacts of UV radiation on bacterial cultures’ properties from the biological, biochemical and molecular biological perspective. For experiments, commercial bacterial cultures (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli and Salmonella typhimurium and isolates from patients with bacterial infections (Proteus mirabilis and Pseudomonas aeruginosa were employed. The above-mentioned strains were exposed to UV using a laboratory source and to stratospheric UV using a 3D printed probe carried by a stratospheric balloon. The length of flight was approximately two hours, and the probe was enriched by sensors for the external environment (temperature, pressure and relative humidity. After the landing, bacterial cultures were cultivated immediately. Experimental results showed a significant effect of UV radiation (both laboratory UV and UV from the stratosphere on the growth, reproduction, behavior and structure of bacterial cultures. In all parts of the experiment, UV from the stratosphere showed stronger effects when compared to the effects of laboratory UV. The growth of bacteria was inhibited by more than 50% in all cases; moreover, in the case of P. aeruginosa, the growth was even totally inhibited. Due to the effect of UV radiation, an increased susceptibility of bacterial strains to environmental influences was also observed. By using commercial tests for biochemical markers of Gram-positive and Gram-negative strains, significant disparities in exposed and non-exposed strains were found. Protein patterns obtained using MALDI-TOF mass spectrometry revealed that UV exposure is able to affect the proteins’ expression, leading to their downregulation, observed

  11. DNA conformational behavior and compaction in biomimetic systems: Toward better understanding of DNA packaging in cell.

    Science.gov (United States)

    Zinchenko, Anatoly

    2016-06-01

    In a living cell, long genomic DNA is strongly compacted and exists in the environment characterized by a dense macromolecular crowding, high concentrations of mono- and divalent cations, and confinement of ca. 10μm size surrounded by a phospholipid membrane. Experimental modelling of such complex biological system is challenging but important to understand spatiotemporal dynamics and functions of the DNA in cell. The accumulated knowledge about DNA condensation/compaction in conditions resembling those in the real cell can be eventually used to design and construct partly functional "artificial cells" having potential applications in drug delivery systems, gene therapy, and production of synthetic cells. In this review, I would like to overview the past progress in our understanding of the DNA conformational behavior and, in particular, DNA condensation/compaction phenomenon and its relation to the DNA biological activity. This understanding was gained by designing relevant experimental models mimicking DNA behavior in the environment of living cell. Starting with a brief summary of classic experimental systems to study DNA condensation/compaction, in later parts, I highlight recent experimental methodologies to address the effects of macromolecular crowding and nanoscale and microscale confinements on DNA conformation dynamics. All the studies are discussed in the light of their relevance to DNA behavior in living cells, and future prospects of the field are outlined. PMID:26976700

  12. In Vitro Study of the Effect of Vitamin E on Viability, Morphological Changes and Induction of Osteogenic Differentiation in Adult Rat Bone Marrow Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    M Soleimani Mehranjani

    2014-10-01

    Full Text Available Introduction: Vitamin E as a strong antioxidant plays an important role in inhibiting free radicals. Therefore, this study aimed to investigate the effect of vitamin E on the viability, morphology and osteogenic differentiation in bone marrow mesenchymal stem cells of an adult rat. Methods: The bone marrow mesenchymal stem cells were extracted using the flashing-out method. At the end of the third passage, cells were divided into groups of control and experimental. Experimental cells were treated withVitamin E (5,10,15,25,50,100,150μM for a period of 21 days in the osteogenic media containing 10% of fetal bovine serum. The cell viability, bone matrix mineralization, intercellular and extracellular calcium deposition, alkaline phosphatase activity, expression of genes and synthesis of proteins of osteopontin and osteocalcin as well as morphological changes of the cells were investigated. The study data was analyzed using one-way ANOVA and T-Test setting the significant P value at P<0.05. Results: Within vitamin- E treated cells, the mean viability, mean bone matrix mineralization, calcium deposition, alkaline phosphatase activity, expression and synthesis of osteopontin and osteocalcin of the mesenchymal stem cells treated with vitamin E significantly increased in a dose dependent manner. Also cytoplasm extensions were observed in the cells treated with vitamin E. Conclusion: Since vitamin E caused a significant increase in cell viability and osteogenic differentiation in the mesenchymal stem cells, therefore it can be utilized in order to increase cell differentiation and cell survival.

  13. DNA Charge Transport within the Cell

    OpenAIRE

    Grodick, Michael A.; Muren, Natalie B.; Barton, Jacqueline K.

    2015-01-01

    The unique characteristics of DNA charge transport (CT) have prompted an examination of roles for this chemistry within a biological context. Not only can DNA CT facilitate long range oxidative damage of DNA, but redox-active proteins can couple to the DNA base stack and participate in long range redox reactions using DNA CT. DNA transcription factors with redox-active moieties such as SoxR and p53 can use DNA CT as a form of redox sensing. DNA CT chemistry also provides a means to monitor th...

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

    International Nuclear Information System (INIS)

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

  15. The C-terminal subunit of artificially truncated human cathepsin B mediates its nuclear targeting and contributes to cell viability

    Directory of Open Access Journals (Sweden)

    Dallner Claudia

    2005-04-01

    Full Text Available Abstract Background Splicing variants of human cathepsinB primary transcripts (CB(-2,3 result in an expression product product which lacks the signal peptide and parts of the propeptide. This naturally truncated Δ51CB is thus unable to follow the regular CB processing and sorting pathway. It is addressed to the mitochondria through an activated N-terminal mitochondrial targeting signal instead. Although Δ51CB is supposed to be devoid of the typical CB enzymatic activity, it might play a role in malignancies and trigger cell death/apoptosis independent from the function of the regular enzyme. Cytoplasmic presence of the mature CB might occur as a result of lysosomal damage. Results We investigated such "aberrant" proteins by artificial CB-GFP chimeras covering various sequence parts in respect to their enzymatic activity, their localization in different cell types, and the effects on the cell viability. Unlike the entire full length CB form, the artificial single chain form was not processed and did not reveal typical enzymatic CB activity during transient overexpression in large cell lung carcinoma cells. Δ51CB was found predominantly in mitochondria. In contrast, the shorter artificial CB constructs localized in the cytoplasm, inside the cell nucleus, and in the midbodies of dividing cells. Bleaching experiments revealed both mobile and immobile fractions of these constructs in the nucleus. Nuclear accumulation of artificially truncated CB variants led to disintegration of nuclei, followed by cell death. Conclusion We propose that cell death associated with CB is not necessarily triggered by its regular enzymatic activity but alternatively by a yet unknown activity profile of truncated CB. Cytoplasmic CB might be able to enter the cell nucleus. According to a mutational analysis, the part of CB that mediates its nuclear import is a signal patch within its heavy chain domain. The results suggest that besides the N-terminal signal peptide also

  16. Labeling Adipose-Derived Stem Cells with Hoechst 33342: Usability and Effects on Differentiation Potential and DNA Damage

    Science.gov (United States)

    Schendzielorz, P.; Froelich, K.; Rak, K.; Gehrke, T.; Scherzad, A.; Hagen, R.; Radeloff, A.

    2016-01-01

    Adipose-derived stem cells (ASCs) have been extensively studied in the field of stem cell research and possess numerous clinical applications. Cell labeling is an essential component of various experimental protocols and Hoechst 33342 (H33342) represents a cost-effective and easy methodology for live staining. The purpose of this study was to evaluate the labeling of rat ASCs with two different concentrations of H33342 (0.5 μg/mL and 5 μg/mL), with particular regard to usability, interference with cell properties, and potential DNA damage. Hoechst 33342 used at a low concentration of 0.5 μg/mL did not significantly affect cell proliferation, viability, or differentiation potential of the ASCs, nor did it cause any significant DNA damage as measured by the olive tail moment. High concentrations of 5 μg/mL H33342, however, impaired the proliferation and viability of the ASCs, and considerable DNA damage was observed. Undesirable colabeling of unlabeled cocultivated cells was seen in particular with higher concentrations of H33342, independent of varying washing procedures. Hence, H33342 labeling with lower concentrations represents a usable method, which does not affect the tested cell properties. However, the colabeling of adjacent cells is a drawback of the technique. PMID:27375746

  17. Labeling Adipose-Derived Stem Cells with Hoechst 33342: Usability and Effects on Differentiation Potential and DNA Damage

    Directory of Open Access Journals (Sweden)

    P. Schendzielorz

    2016-01-01

    Full Text Available Adipose-derived stem cells (ASCs have been extensively studied in the field of stem cell research and possess numerous clinical applications. Cell labeling is an essential component of various experimental protocols and Hoechst 33342 (H33342 represents a cost-effective and easy methodology for live staining. The purpose of this study was to evaluate the labeling of rat ASCs with two different concentrations of H33342 (0.5 μg/mL and 5 μg/mL, with particular regard to usability, interference with cell properties, and potential DNA damage. Hoechst 33342 used at a low concentration of 0.5 μg/mL did not significantly affect cell proliferation, viability, or differentiation potential of the ASCs, nor did it cause any significant DNA damage as measured by the olive tail moment. High concentrations of 5 μg/mL H33342, however, impaired the proliferation and viability of the ASCs, and considerable DNA damage was observed. Undesirable colabeling of unlabeled cocultivated cells was seen in particular with higher concentrations of H33342, independent of varying washing procedures. Hence, H33342 labeling with lower concentrations represents a usable method, which does not affect the tested cell properties. However, the colabeling of adjacent cells is a drawback of the technique.

  18. Cytosolic DNA Sensor Upregulation Accompanies DNA Electrotransfer in B16.F10 Melanoma Cells.

    Science.gov (United States)

    Znidar, Katarina; Bosnjak, Masa; Cemazar, Maja; Heller, Loree C

    2016-06-07

    In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFNβ mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60), and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16.F10 cells, inducing effects in vitro and potentially in vivo.

  19. Cytosolic DNA Sensor Upregulation Accompanies DNA Electrotransfer in B16.F10 Melanoma Cells

    Science.gov (United States)

    Znidar, Katarina; Bosnjak, Masa; Cemazar, Maja; Heller, Loree C.

    2016-01-01

    In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFNβ mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60), and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16.F10 cells, inducing effects in vitro and potentially in vivo. PMID:27271988

  20. Phytochemicals isolated from leaves of Chromolaena odorata: impact on viability and clonogenicity of cancer cell lines.

    Science.gov (United States)

    Kouamé, Prevost Bi-Koffi; Jacques, Camille; Bedi, Gustave; Silvestre, Virginie; Loquet, Denis; Barillé-Nion, Sophie; Robins, Richard J; Tea, Illa

    2013-06-01

    The leaves of Chromolaena odorata (Asteraceae) are exploited extensively in West and Central African ethnopharmacy for the treatment of a wide range of conditions, despite this being a non-native species established in the last 50 years. With the objective of seeking bioactive principles, the nonvolatile compounds, an ethanolic (80% v/v) extract was made and fractionated. From the hexane-soluble fraction, three compounds were isolated. Two of these, 5-hydroxy-7,4'-dimethoxyflavanone and 2'-hydroxy-4,4',5',6'-tetramethoxychalcone, have previously been identified in C. odorata leaves. The third was fully characterised spectroscopically and found to be 1,6-dimethyl-4-(1-methylethyl)naphthalene (cadalene), not previously isolated from the Asteraceae. All three compounds were tested for their cytotoxicity and anticancer properties. 2'-Hydroxy-4,4',5',6'-tetramethoxychalcone was found to be both cytotoxic and anticlonogenic at 20 µm in cell lines Cal51, MCF7 and MDAMB-468, and to act synergistically with the Bcl2 inhibitor ABT737 to enhance apoptosis in Cal51 breast cancer cells. PMID:22899281

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

    Energy Technology Data Exchange (ETDEWEB)

    Chan, C.W., E-mail: c.w.chan@qub.ac.uk [School of Mechanical and Aerospace Engineering, Queen' s University, Belfast, Northern Ireland (United Kingdom); Hussain, I. [School of Life Sciences, University of Lincoln, Brayford Pool, Lincoln, Lincolnshire LN6 7TU (United Kingdom); Waugh, D.G.; Lawrence, J. [Laser Engineering and Manufacturing Research Group, Faculty of Science and Engineering, University of Chester, Parkgate Road, Chester, CH1 4BJ (United Kingdom); Man, H.C. [Department of Industrial and Systems Engineering, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong (China)

    2014-09-01

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

  2. Increase in cell viability by polyamines through stimulation of the synthesis of ppGpp regulatory protein and ω protein of RNA polymerase in Escherichia coli.

    Science.gov (United States)

    Terui, Yusuke; Akiyama, Mariko; Sakamoto, Akihiko; Tomitori, Hideyuki; Yamamoto, Kaneyoshi; Ishihama, Akira; Igarashi, Kazuei; Kashiwagi, Keiko

    2012-02-01

    It is known that polyamines increase cell growth through stimulation of the synthesis of several kinds of proteins encoded by the so-called "polyamine modulon". We recently reported that polyamines also increase cell viability at the stationary phase of cell growth through stimulation of the synthesis of ribosome modulation factor, a component of the polyamine modulon. Accordingly, we looked for other proteins involved in cell viability whose synthesis is stimulated by polyamines. It was found that the synthesis of ppGpp regulatory protein (SpoT) and ω protein of RNA polymerase (RpoZ) was stimulated by polyamines at the level of translation. Stimulation of the synthesis of SpoT and RpoZ by polyamines was due to an inefficient initiation codon UUG in spoT mRNA and an unusual location of a Shine-Dalgarno (SD) sequence in rpoZ mRNA. Accordingly, the spoT and rpoZ genes are components of the polyamine modulon involved in cell viability. Reduced cell viability caused by polyamine deficiency was prevented by modified spoT and rpoZ genes whose synthesis was not influenced by polyamines. Under these conditions, the level of ppGpp increased in parallel with increase of SpoT protein. The results indicate that polyamine stimulation of synthesis of SpoT and RpoZ plays important roles for cell viability through stimulation of ppGpp synthesis by SpoT and modulation of RNA synthesis by ppGpp-RpoZ complex.

  3. DNA damage and repair in human cells exposed to sunlight

    International Nuclear Information System (INIS)

    Cultured human cells were treated with direct sunlight under conditions which minimised the hypertonic, hyperthermic and fixative effects of solar radiation. Sunlight produced similar levels of DNA strand breaks as equitoxic 254 nm UV in two fibroblast strains and a melanoma cell line, but DNA repair synthesis and inhibition of semiconservative DNA synthesis and of DNA chain elongation were significantly less for sunlight-exposed cells. DNA breaks induced by sunlight were removed more rapidly. Thus, the repair of solar damage differs considerably from 254 nm UV repair. Glass-filtered sunlight (>320 nm) was not toxic to cells and did not induce repair synthesis but gave a low level of short-lived DNA breaks and some inhibition of DNA chain elongation; thymidine uptake was enhanced. Filtered sunlight slightly enhanced UV-induced repair synthesis and UV toxicity; photoreactivation of UV damage was not found. Attempts to transform human fibroblasts using sunlight, with or without phorbol ester, were unsuccessful. (author)

  4. Fabrication, bioactivity, in vitro cytotoxicity and cell viability of cryo-treated nanohydroxyapatite–gelatin–polyvinyl alcohol macroporous scaffold

    Directory of Open Access Journals (Sweden)

    Sanjaya Kumar Swain

    2014-09-01

    Full Text Available Freeze casting and cryogenic treatment both low temperature process have been employed to fabricate nanobiocomposite hydroxyapatite (HA–gelatin–polyvinyl alcohol (PVA macroporous scaffolds from synthesized three different spherical, rod and fibrous HA nanoparticles and composition optimized vis-á-vis porosity architecture, content and compressive strength. A critical HA morphology, solid loading and liquid nitrogen interaction time have a significant effect to enhance the mechanical response of developed scaffolds. Cryo-treated 40 wt.% nanorod HA–gelatin–PVA scaffold posses interconnected pore structure with 80 vol.% porosity, average pore diameter 50–200 μm and highest 5.8 MPa compressive strength. Different degree of the apatite deposition phenomenon in simulated body fluid solution at 37 °C and pH ∼ 7.4 varies with respect to time. In vitro cytotoxicity and L929 mouse fibroblast cell culture in the presence of Dulbecco's Modified Eagle Medium and 10% Fetal Bovine Serum at 37 °C and 5% CO2 atmosphere exhibit excellent cytocompatibility and cell viability at low extract concentration up to 25%.

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

    OpenAIRE

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

    2010-01-01

    Overactivation of certain K+ channels can mediate excessive K+ efflux and intracellular K+ depletion, which are early ionic events in apoptotic cascade. The present investigation examined a possible role of the KCNQ2/3 channel or M-channel (also named Kv7.2/7.3 channels) in the pro-apoptotic process. Whole-cell recordings detected much larger M-currents (212±31 pA or 10.5±1.5 pA/pF) in cultured hippocampal neurons than that in cultured cortical neurons (47±21 pA or 2.4±0.8 pA/pF). KCNQ2/3 cha...

  6. Viability of fibroblasts in cell culture after treatment with different chemical retraction agents.

    Science.gov (United States)

    Kopac, I; Batista, U; Cvetko, E; Marion, L

    2002-01-01

    Prior to fixed prosthodontic impression procedures, temporary horizontal retraction of the free gingival tissue should be accomplished apically to the preparation finishing line. The mechanical-chemical method using cotton retraction cords of various sizes impregnated with various retraction chemicals is the most commonly employed retraction technique. Most retraction agents have pH values from 0.8 to 0.3, and are therefore hazardous to the cut dentine and periodontal tissues. Sympathomimetic vasoconstrictors introduced recently have a pH of 5.6, and are free of systemic side-effects. The present study using the dye exclusion test, colony forming ability test and colorimetric assay was undertaken to evaluate cytotoxic effects of four chemical retraction agents on cultured V-79 fibroblasts, and the dependence of cytotoxicity on the agent concentration and time of exposure. Original concentrations of retraction agents produced stronger cytotoxic effects than dilutions of 1:1 and 1:10. The most aggressive agent, 25% aluminium chloride, took only 1 min to damage all cell cultures. The proportion of cells damaged after 10 min of exposure to tetrahydrozoline was 60%, which was significantly less compared with other chemicals tested. With the colony forming ability test using retraction agents diluted to 1:10 the greatest number of colonies emerged in samples treated with tetrahydrozoline (statistical significance: P < 0.01). The colorimetric assay showed equal cytotoxic effects for 25% aluminium sulphate and tetrahydrozoline. The colorimetric test used in the study has proved an ergonomic, accurate and reliable test for cytotoxicity determination.

  7. Viability of fibroblasts in cell culture after treatment with different chemical retraction agents.

    Science.gov (United States)

    Kopac, I; Batista, U; Cvetko, E; Marion, L

    2002-01-01

    Prior to fixed prosthodontic impression procedures, temporary horizontal retraction of the free gingival tissue should be accomplished apically to the preparation finishing line. The mechanical-chemical method using cotton retraction cords of various sizes impregnated with various retraction chemicals is the most commonly employed retraction technique. Most retraction agents have pH values from 0.8 to 0.3, and are therefore hazardous to the cut dentine and periodontal tissues. Sympathomimetic vasoconstrictors introduced recently have a pH of 5.6, and are free of systemic side-effects. The present study using the dye exclusion test, colony forming ability test and colorimetric assay was undertaken to evaluate cytotoxic effects of four chemical retraction agents on cultured V-79 fibroblasts, and the dependence of cytotoxicity on the agent concentration and time of exposure. Original concentrations of retraction agents produced stronger cytotoxic effects than dilutions of 1:1 and 1:10. The most aggressive agent, 25% aluminium chloride, took only 1 min to damage all cell cultures. The proportion of cells damaged after 10 min of exposure to tetrahydrozoline was 60%, which was significantly less compared with other chemicals tested. With the colony forming ability test using retraction agents diluted to 1:10 the greatest number of colonies emerged in samples treated with tetrahydrozoline (statistical significance: P < 0.01). The colorimetric assay showed equal cytotoxic effects for 25% aluminium sulphate and tetrahydrozoline. The colorimetric test used in the study has proved an ergonomic, accurate and reliable test for cytotoxicity determination. PMID:11844038

  8. Biocorrosion behavior and cell viability of adhesive polymer coated magnesium based alloys for medical implants

    Energy Technology Data Exchange (ETDEWEB)

    Abdal-hay, Abdalla [Departmentt of Bionano System Engineering, College of Engineering, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Department of Mechanical Design Engineering, Advanced wind power system research institute, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Dewidar, Montasser [Department of Materials and Mechanical Design, Faculty of Energy Engineering, South Valley University, Aswan (Egypt); Lim, Jae Kyoo, E-mail: jklim@jbnu.ac.kr [Department of Mechanical Design Engineering, Advanced wind power system research institute, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)

    2012-11-15

    Highlights: Black-Right-Pointing-Pointer The corrosion behavior of magnesium for orthopedic applications is extremely poor. Black-Right-Pointing-Pointer The solvent (DCM, THF and DMF) had a strong effect on the coatings performance. Black-Right-Pointing-Pointer Mg bar alloy coated with PVAc/DCM layers provided an excellent bonding strength. Black-Right-Pointing-Pointer Treated samples indicated significant damping for the degradation rate. Black-Right-Pointing-Pointer Cytocompatibility on MC3T3 cells of the PVAc/DCM samples revealed a good behavior. - Abstract: The present study was ultimately aimed to design novel adhesive biodegradable polymer, poly(vinyl acetate) (PVAc), coatings onto Mg based alloys by the dip-coating technique in order to control the degradation rate and enhance the biocompatibility of magnesium alloys. The influence of various solvents on PVAc surface topography and their protection of Mg alloys were dramatically studied in vitro. Electrochemical polarization, degradation, and PVAc film cytocompatibility were also tested. Our results showed that the solvent had a significant effect on coating quality. PVAc/dichloromethane solution showed a porous structure and solution concentration could control the porous size. The coatings prepared using tetrahydrofuran and dimethylformamide solvents are exceptional in their ability to generate porous morphology even at low polymer concentration. In general, the corrosion performance appears to be different on different PVAc-solvent system. Immersion tests illustrated that the porous morphology on PVAc stabilized corrosion rates. A uniform corrosion attack in artificial simulation body fluid was also exhibited. The cytocompatibility of osteoblast cells (MC3T3) revealed high adherence, proliferation, and survival on the porous structure of PVAc coated Mg alloy, which was not observed for the uncoated samples. This novel PVAc coating is a promising candidate for biodegradable implant materials, which might

  9. Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions.

    Directory of Open Access Journals (Sweden)

    Nan Li

    Full Text Available Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG. In this study we used mouse embryonic stem (MES and mouse embryonic fibroblast (MEF cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS. Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR defects.

  10. Cosegregation of cell wall and DNA in Bacillus subtilis.

    OpenAIRE

    Schlaeppi, J M; Karamata, D

    1982-01-01

    Cosegregation of cell wall and DNA of a lysis-negative mutant of Bacillus subtilis was examined by continuously labeling (i) cell wall, (ii) DNA, and (iii) both cell wall and DNA. After four to five generations of chase in liquid media it was found by light microscope autoradiography that the numbers of wall segregation units per cell are 29 and 9 in rich and minimal medium, respectively. Under the same conditions the numbers of segregation units of DNA were almost 50% lower: 15 and 5, respec...

  11. n Vitro Immunomodulatory Effect of R10 Fraction of Garlic on Viability and Production of TNF-? in CD8+ T Cells

    Directory of Open Access Journals (Sweden)

    T. Ghazanfari

    2014-01-01

    Full Text Available Introduction & Objective: -cells, especially CD8+ T lymphocytes are the most important cells in anti-tumor response. Previously R10 fraction of garlic extract was reported as an immuno-modulator which induced an effective cellular immunity and Th1 responses. In this study the in vitro immunomodulatory effect of R10 on CD8+ T cells viability and production of TNF-? were evaluated. Materials & Methods: In this experimental study, using monoclonal antibodies attached to magnetic beads with isolating columns by magnetic bead method, CD8+ T cells from spleen cells of Balb/C mice were isolated. R10 fraction based on molecular weight was prepared using Ultra filtration. MTT assay was used to evaluate cell viability. TNF-? level was meas-ured in the supernatant of culture of CD8+ T cells by ELISA. Obtained data was compared and analyzed using Nonparametric Test and Keraskel & Wanny's Test tests.. Results: The findings indicate that all dilutions of R10 fraction increased cell viability of CD8+ T cells in comparison with the negative control group and in the presence of ConA with dilution of 1:50 of R10 fraction significantly increased cell viability of CD8+ T Cells com-pared to ConA alone. Secretion of TNF-? significantly increased by all dilutions of R10 frac-tion. Conclusion: These findings suggest that R10 fraction of garlic can be used as an Immuno-modulator drug candidate for induction of cellular Immunity in tumor therapy. Sci J Hamadan Univ Med Sci 2014; 20 (4:273-279

  12. Biocorrosion behavior and cell viability of adhesive polymer coated magnesium based alloys for medical implants

    Science.gov (United States)

    Abdal-hay, Abdalla; Dewidar, Montasser; Lim, Jae Kyoo

    2012-11-01

    The present study was ultimately aimed to design novel adhesive biodegradable polymer, poly(vinyl acetate) (PVAc), coatings onto Mg based alloys by the dip-coating technique in order to control the degradation rate and enhance the biocompatibility of magnesium alloys. The influence of various solvents on PVAc surface topography and their protection of Mg alloys were dramatically studied in vitro. Electrochemical polarization, degradation, and PVAc film cytocompatibility were also tested. Our results showed that the solvent had a significant effect on coating quality. PVAc/dichloromethane solution showed a porous structure and solution concentration could control the porous size. The coatings prepared using tetrahydrofuran and dimethylformamide solvents are exceptional in their ability to generate porous morphology even at low polymer concentration. In general, the corrosion performance appears to be different on different PVAc-solvent system. Immersion tests illustrated that the porous morphology on PVAc stabilized corrosion rates. A uniform corrosion attack in artificial simulation body fluid was also exhibited. The cytocompatibility of osteoblast cells (MC3T3) revealed high adherence, proliferation, and survival on the porous structure of PVAc coated Mg alloy, which was not observed for the uncoated samples. This novel PVAc coating is a promising candidate for biodegradable implant materials, which might widen the use of Mg based implants.

  13. Rabbit model of intervertebral disc degeneration by external compression device characterized by X-ray, MRI, histology, and cell viability

    Directory of Open Access Journals (Sweden)

    Ismail Ismail

    2006-12-01

    Full Text Available Appropriate experimental animal models, which mimic the degenerative process occurring in human intervertebral disc (IVD breakdown and can be used for new treatment studies such as tissue engineering or disc distraction are lacking. We studied the external compression device that used by Kroeber et al to create intervertebral disc degeneration in rabbit model characterized by X-ray, MRI, Histology, and Cell Viability. Ten NZW rabbit were randomly assigned to one of five groups. Intervertebral disc VL4-L5 are compressed using an external loading device, 1.9 MPa. First group rabbit are loaded for 14 days, second loaded for 28 days, thirth group are loaded for 14 days, and unloaded for 14 days, fourth group loaded for 28 days and unloaded for 28 days. The fifth group, rabbits underwent a sham operation. Additional, rabbits were used as sample for cell viability study. In disc height : sample in group one have biggest decreasing of disc height, that is 23.9 unit. In MRI assessment, the worst grade is grade 3. In histological score, the worst group is group three (58.69, and the best is group 4 (45.69. Group one have the largest dead cell, that are 403.5, and the smallest is group four (124.75. Trypan blue staining showed that group four have better viable cell (91.1 compare than group three (86.4. The study conclude disc degeneration can be created by external axial loading for 14 days in rabbit intervertebral disc. Duration of 28 days unloading gave better result for cells to recover. (Med J Indones 2006; 15:199-207  Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 Keywords: Rabbit model –intervertebral disc degeneration- external compression device-X-ray, MRI, Histology, and Cell viabilty /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso

  14. Cell-free DNA: Comparison of Technologies.

    Science.gov (United States)

    Dar, Pe'er; Shani, Hagit; Evans, Mark I

    2016-06-01

    Cell-free fetal DNA screening for Down syndrome has gained rapid acceptance over the past few years with increasing market penetration. Three main laboratory methodologies are currently used: a massive parallel shotgun sequencing (MPSS), a targeted massive parallel sequencing (t-MPS) and a single nucleotide polymorphism (SNP) based approach. Although each of these technologies has its own advantages and disadvantages, the performance of all was shown to be comparable and superior to that of traditional first-trimester screening for the detection of trisomy 21 in a routine prenatal population. Differences in performance were predominantly shown for chromosomal anomalies other than trisomy 21. Understanding the limitations and benefits of each technology is essential for proper counseling to patients. These technologies, as well as few investigational technologies described in this review, carry a great potential beyond screening for the common aneuploidies. PMID:27235906

  15. DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation

    DEFF Research Database (Denmark)

    Shaknovich, Rita; Cerchietti, Leandro; Tsikitas, Lucas;

    2011-01-01

    cells. Among DNA methyltransferases (DNMTs), only DNMT1 was significantly up-regulated in GC B cells. Dnmt1 hypomorphic mice displayed deficient GC formation and treatment of mice with the DNA methyltransferase inhibitor decitabine resulted in failure to form GCs after immune stimulation. Notably...

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

    Science.gov (United States)

    Taparia, Shruti; Khanna, Aparna

    2016-01-01

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

  17. High modulus biodegradable polyurethanes for applications in cardiovascular stents: Evaluation of in-vitro degradation and cell viability

    Directory of Open Access Journals (Sweden)

    Melissa eSagarito

    2015-05-01

    Full Text Available We have recently reported the mechanical properties and hydrolytic degradation behaviour of a series of NovoSorb™ [1] biodegradable polyurethanes (PUs prepared by varying the hard segment (HS weight percentage from 60-100. In this study the in-vitro degradation behaviour of the PUs with and without extracellular matrix (ECM coating was investigated under accelerated hydrolytic degradation (phosphate buffer saline; PBS/70°C conditions to allow complete degradation. The mass loss at different time intervals and the effect of aqueous degradation products on the viability and growth of Human Umbilical Vein Endothelial Cells (HUVEC were examined.The results showed that for most PUs in the series the degradation medium turned acidic and the extent of this was dependent on the HS percentage. As the HS decreased, a drop in pH was observed, suggesting that the by-products of soft segment (SS degradation causing the solution to be acidic. Most of the samples were completely eroded by 18 weeks, except PU containing more than 70% HS which only showed partial degradation with no significant change in appearance. The cytotoxicity tests on HUVEC cells of the aqueous extracts obtained after 18 weeks incubation showed that toxicity was dependent on 3 factors: dose, percentage of HS and degradation time. HUVEC growth is similar to but not identical to that observed with tissue culture polystyrene (TCPS standard. The results from this in-vitro cytotoxicity study suggest that the mixture of degradation products formed during the accelerated degradation of this PU series are not toxic to cells under the experimental conditions used.

  18. DNA nanotechnology from the test tube to the cell

    Science.gov (United States)

    Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A.; Seelig, Georg

    2015-09-01

    The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology -- applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems -- lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

  19. DNA MUTAGENESIS IN PANAX GINSENG CELL CULTURES

    Directory of Open Access Journals (Sweden)

    Kiselev K.V.

    2012-08-01

    Full Text Available At the present time, it is well documented that plant tissue culture induces a number of mutations and chromosome rearrangements termed “somaclonal variations”. However, little is known about the nature and the molecular mechanisms of the tissue culture-induced mutagenesis and the effects of long-term subculturing on the rate and specific features of the mutagenesis. The aim of the present study was to investigate and compare DNA mutagenesis in different genes of Panax ginseng callus cultures of different age. It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during long-term cultivation of transgenic cell cultures of P. ginseng. In the present work, we analyzed nucleotide sequences of selected plant gene families in a 2-year-old and 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We analysed sequence variability between the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL and dammarenediol synthase (DDS genes, which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK genes, which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-year-old callus culture was markedly higher than that in cultivated plants but lower than that in the 20-year-old callus culture of P. ginseng. Most of the mutations in the 2- and 20-year-old P. ginseng calli were A↔G and T↔C transitions. The number of nonsynonymous mutations was higher in the 2- and 20-year-old callus cultures than the number of nonsynonymous mutations in the cultivated plants of P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using methylation-sensitive DNA fragmentation

  20. Mitochondrial DNA determines androgen dependence in prostate cancer cell lines

    OpenAIRE

    Higuchi, M; Kudo, T; Suzuki, S.; Evans, TT; Sasaki, R.; Wada, Y; Shirakawa, T.; Sawyer, JR; Gotoh, A

    2006-01-01

    Prostate cancer progresses from an androgen-dependent to androgen-independent stage after androgen ablation therapy. Mitochondrial DNA plays a role in cell death and metastatic competence. Further, heteroplasmic large-deletion mitochondrial DNA is verycommon in prostate cancer. To investigate the role of mitochondrial DNA in androgen dependence of prostate cancers, we tested the changes of normal and deleted mitochondrial DNA in accordance with the progression of prostate cancer. We demonstra...

  1. Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Anthony Skipper

    2016-01-01

    Full Text Available Cadmium is a heavy metal that has been shown to cause its toxicity in humans and animals. Many documented studies have shown that cadmium produces various genotoxic effects such as DNA damage and chromosomal aberrations. Ailments such as bone disease, renal damage, and several forms of cancer are attributed to overexposure to cadmium.  Although there have been numerous studies examining the effects of cadmium in animal models and a few case studies involving communities where cadmium contamination has occurred, its molecular mechanisms of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver carcinoma (HepG2 cells. To test our hypothesis, cell viability was determined by MTT assay. Lipid hydroperoxide content stress was estimated by lipid peroxidation assay. Genotoxic damage was tested by the means of alkaline single cell gel electrophoresis (Comet assay. Cell apoptosis was measured by flow cytometry assessment (Annexin-V/PI assay. The result of MTT assay indicated that cadmium chloride induces toxicity to HepG2 cells in a concentration-dependent manner, showing a 48 hr-LD50 of 3.6 µg/mL. Data generated from lipid peroxidation assay resulted in a significant (p < 0.05 increase of hydroperoxide production, specifically at the highest concentration tested. Data obtained from the Comet assay indicated that cadmium chloride causes DNA damage in HepG2 cells in a concentration-dependent manner. A strong concentration-response relationship (p < 0.05 was recorded between annexin V positive cells and cadmium chloride exposure. In summary, these in vitro studies provide clear evidence that cadmium chloride induces oxidative stress, DNA damage, and programmed cell death in human liver carcinoma (HepG2 cells.

  2. Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

    Science.gov (United States)

    Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Tamaru, Yutaka; Ogata, Shin; Takebayashi, Shin-ichiro; Ogata, Masato; Okumura, Katsuzumi

    2015-12-01

    Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

  3. Cytotoxicity and genotoxicity of a monazite component: lanthanum effects on the viability and induction of breaks in the DNA of human lymphocytes; Citotoxicidade e genotoxicidade de um componente da monazita: efeitos do lantanio na viabilidade e inducao de quebras no DNA de linfocitos humanos

    Energy Technology Data Exchange (ETDEWEB)

    Paiva, Amanda Valle de Almeida

    2007-07-01

    The Monazite is a mineral extracted from open mines. It is constituted by lanthanum element aggregated with cerium, yttrium and thorium [(Ce, La, Y, Th)PO{sub 4}]. Lanthanum (La) is a rare-earth metal with applications in agriculture, industry and medicine. Since lanthanides and their compounds show a broad spectrum of applications there is an increased risk of incorporation in human. Inhalation of aerosols containing La is the main route of incorporation in workers exposed to several chemical forms of La. Herein, we examined the effect of lanthanum nitrate - La(NO{sub 3}){sub 3} in human lymphocytes. JURKAT cells and human peripheral lymphocytes (HPL) were used to evaluate the effect of La(NO{sub 3}){sub 3} on viability (apoptosis or necrosis) and DNA strand breaks induction or/and alkali-labile sites (ALS). We demonstrate that La has a cytotoxic and genotoxic effect on both cell lines. The results indicate that necrosis is the pathway by which La(NO{sub 3}){sub 3} induces cytotoxicity. The vitamin E is able to diminish DNA strand breaks induced by La(NO{sub 3}){sub 3} suggesting that reactive oxygen species (ROS) may be involved in the genotoxic process. (author)

  4. DNA content analysis of insect cell lines by flow cytometry

    OpenAIRE

    Léry, Xavier; Charpentier, Guy; Belloncik, Serge

    1999-01-01

    The DNA content of insect cell lines (6 lepidoptera, 1 coleoptera and 1 diptera) was determined by flow cytometry. The DNA profiles of the 8 cell lines tested were different. They were characterized by the presence of several peaks (2 to 7) corresponding to different ploidy levels, by differences in the fluorescence intensity of each peak and by the proportion of cells in each peak. Two cell lines (Cf124 and BmN) were constituted of 2 distinct populations of cells. The DNA profiles of the cel...

  5. Mercuric dichloride induces DNA damage in human salivary gland tissue cells and lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Schmid, Katharina; Kroemer, Susanne [University of Regensburg, Regensburg (Germany); Sassen, Andrea [University of Regensburg, Department of Pathology, Regensburg (Germany); Staudenmaier, Rainer [Technical University of Munich, Department of Otorhinolaryngology, Head and Neck Surgery, Munich (Germany); Reichl, Franz-Xaver [University of Munich, Institute of Pharmacology and Toxicology, Munich (Germany); Harreus, Ulrich [University of Munich, Department of Otorhinolaryngology, Head and Neck Surgery, Munich (Germany); Hagen, Rudolf; Kleinsasser, Norbert [University of Wuerzburg, Department of Otorhinolaryngology, Head and Neck Surgery, Wuerzburg (Germany)

    2007-11-15

    Amalgam is still one of the most frequently used dental filling materials. However, the possible adverse effects especially that of the mercuric component have led to continued controversy. Considering that mercury may be released from amalgam fillings into the oral cavity and also reach the circulating blood after absorption and resorption, it eventually may contribute to tumorigenesis in a variety of target cells. The present investigation focuses on genotoxic effects below a cytotoxic dose level of mercuric dichloride (HgCl{sub 2}) in human samples of salivary glands and lymphocytes to elucidate a possible role in tumor initiation. DNA migration due to single strand breaks, alkali labile sites and incomplete excision repair was quantified with the aid of the single cell microgel electrophoresis (Comet) assay. The concepts of Olive Tail Moment, percentage of DNA in the Tail and Tail Length were used as measures of DNA damage. To control for cytotoxic effects, the trypan blue exclusion test was applied. Human samples of the parotid salivary gland and lymphocytes of ten donors were exposed to HgCl{sub 2} concentrations from 1 to 50 {mu}M. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and dimethyl sulfoxide (DMSO) served as controls. Increasing dose-dependent DNA migration could be demonstrated after exposure to HgCl{sub 2} in cells of the salivary glands and lymphocytes. In both cell types a significant increase in DNA migration could be shown starting from HgCl{sub 2} concentrations of 5 {mu}M in comparison to the negative control. The viability of the cell systems was not affected except at the highest concentration (50 {mu}M) tested. These data indicate genotoxic effects of mercuric dichloride in human salivary glands and lymphocytes at concentrations not leading to cytotoxic effects or cell death. Consequently, a contributory role in oral salivary gland tumor initiation warrants further investigation. (orig.)

  6. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    Science.gov (United States)

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2012-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation. PMID:22127868

  7. Assessment on Functionality and Viability of Beta Cells Following Repetitive Dosage Administration of Ethanolic Extracts of Andrographis paniculata on Streptozotocin-induced Diabetic Rats.

    OpenAIRE

    Mohd Zaini, A; A Mariam; Amirin, S; Abdul Razak, K

    2010-01-01

    The study was done at the aim to assess the functionality and viability of the beta cells of the streptozotocin-induced diabetic rats model following repetitive dosage of administration of ethanolic extracts of Andrographis paniculata. Materials and Methods: The diabetic rats were treated with the extracts for fourteen days and at the dose given was 500 mg/kg twice daily. The assessments were made on fasting blood glucose, insulin, and immunohistochemical aspect of beta cells before and after...

  8. Alterations in cell migration and cell viability of wounded human skin fibroblasts following visible red light exposure

    Science.gov (United States)

    Prabhu, Vijendra; Rao, Bola Sadashiva S.; Mahato, Krishna Kishore

    2014-02-01

    The present study intended to examine the effect of visible red light on structural and cellular parameters on wounded skin fibroblast cells. To achieve the stated objective, uniform scratch was created on confluent monolayered human skin fibroblast cells, and were exposed to single dose of He-Ne laser (15 mm spot, 6.6808 mWcm-2) at 1, 2, 3, 4, 5, 6 and 7 Jcm-2 in the presence and absence of 10% fetal bovine serum (FBS). Beam profile measurements of the expanded laser beam were conducted to ensure the beam uniformity. The influence of laser dose on the change in temperature was recorded using sensitive temperature probe. Additionally, following laser exposure cell migration and cell survival were documented at different time intervals on wounded human skin fibroblast cells grown in vitro. Beam profile measurements indicated more or less uniform power distribution over the whole beam area. Temperature monitoring of sham irradiated control and laser treatment groups displayed negligible temperature change indicating the absence of thermal effect at the tested laser doses. In the absence of 10% FBS, single exposure of different laser doses failed to produce any significant effects on cell migration or cell survival. However, in the presence of serum single exposure of 5 J/cm2 on wounded skin fibroblasts significantly enhanced the cell migration (PLLLT acts by improving cell migration and cell proliferation to produce measurable changes in wounded fibroblast cells.

  9. Mitochondrial DNA Mutations Regulate Metastasis of Human Breast Cancer Cells

    OpenAIRE

    Hirotake Imanishi; Keisuke Hattori; Reiko Wada; Kaori Ishikawa; Sayaka Fukuda; Keizo Takenaga; Kazuto Nakada; Jun-ichi Hayashi

    2011-01-01

    Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously e...

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

    Directory of Open Access Journals (Sweden)

    Nehleh Zarei Fard

    2015-03-01

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

  11. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma

    OpenAIRE

    Philip Burnham; Min Seong Kim; Sean Agbor-Enoh; Helen Luikart; Hannah A. Valantine; Kiran K Khush; Iwijn De Vlaminck

    2016-01-01

    Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in...

  12. The Impact of Glyphosate, Its Metabolites and Impurities on Viability, ATP Level and Morphological changes in Human Peripheral Blood Mononuclear Cells.

    Science.gov (United States)

    Kwiatkowska, Marta; Jarosiewicz, Paweł; Michałowicz, Jaromir; Koter-Michalak, Maria; Huras, Bogumiła; Bukowska, Bożena

    2016-01-01

    The toxicity of herbicides to animals and human is an issue of worldwide concern. The present study has been undertaken to assess toxic effect of widely used pesticide-glyphosate, its metabolites: aminomethylphosphonic acid (AMPA) and methylphosphonic acid and its impurities: N-(phosphonomethyl)iminodiacetic acid (PMIDA), N-methylglyphosate, hydroxymethylphosphonic acid and bis-(phosphonomethyl)amine on human peripheral blood mononuclear cells (PBMCs). We have evaluated the effect of those compounds on viability, ATP level, size (FSC-A parameter) and granulation (SSC-A parameter) of the cells studied. Human peripheral blood mononuclear cells were exposed to different concentrations of glyphosate, its metabolites and impurities (0.01-10 mM) for 4 and 24 h. It was found that investigated compounds caused statistically significant decrease in viability and ATP level of PBMCs. The strongest changes in cell viability and ATP level were observed after 24 h incubation of PBMCs with bis-(phosphonomethyl)amine, and particularly PMIDA. Moreover, all studied compounds changed cell granularity, while PMIDA and bis-(phosphonomethyl)amine altered PBMCs size. It may be concluded that bis-(phosphonomethyl)amine, and PMIDA caused a slightly stronger damage to PBMCs than did glyphosate. Changes in the parameters studied in PBMCs were observed only at high concentrations of the compounds examined, which clearly shows that they may occur in this cell type only as a result of acute poisoning of human organism with these substances. PMID:27280764

  13. The Impact of Glyphosate, Its Metabolites and Impurities on Viability, ATP Level and Morphological changes in Human Peripheral Blood Mononuclear Cells

    Science.gov (United States)

    Kwiatkowska, Marta; Jarosiewicz, Paweł; Michałowicz, Jaromir; Koter-Michalak, Maria; Huras, Bogumiła; Bukowska, Bożena

    2016-01-01

    The toxicity of herbicides to animals and human is an issue of worldwide concern. The present study has been undertaken to assess toxic effect of widely used pesticide—glyphosate, its metabolites: aminomethylphosphonic acid (AMPA) and methylphosphonic acid and its impurities: N-(phosphonomethyl)iminodiacetic acid (PMIDA), N-methylglyphosate, hydroxymethylphosphonic acid and bis-(phosphonomethyl)amine on human peripheral blood mononuclear cells (PBMCs). We have evaluated the effect of those compounds on viability, ATP level, size (FSC-A parameter) and granulation (SSC-A parameter) of the cells studied. Human peripheral blood mononuclear cells were exposed to different concentrations of glyphosate, its metabolites and impurities (0.01–10 mM) for 4 and 24 h. It was found that investigated compounds caused statistically significant decrease in viability and ATP level of PBMCs. The strongest changes in cell viability and ATP level were observed after 24 h incubation of PBMCs with bis-(phosphonomethyl)amine, and particularly PMIDA. Moreover, all studied compounds changed cell granularity, while PMIDA and bis-(phosphonomethyl)amine altered PBMCs size. It may be concluded that bis-(phosphonomethyl)amine, and PMIDA caused a slightly stronger damage to PBMCs than did glyphosate. Changes in the parameters studied in PBMCs were observed only at high concentrations of the compounds examined, which clearly shows that they may occur in this cell type only as a result of acute poisoning of human organism with these substances. PMID:27280764

  14. Viabilitas sel fibroblas BHK-21 pada permukaan resin akrilik rapid heat cured (Viability of fibroblast BHK-21 cells to the surface of rapid heat cured acrylic resins

    Directory of Open Access Journals (Sweden)

    Anita Yuliati

    2005-03-01

    Full Text Available Acrylic resins are widely used in the fabrication of denture bases and have been shown to be cytotoxic as a result of substances that leach from the resin. Numerous reports suggest that residual monomer may be responsible for mucosal irritation and sensitization of tissues. This information is important in eddition to the information of the biologiced effect of such materials. The purpose of this study was to know the viability of fibroblast BHK-21cells to the surface of rapid heat cured acrylic resins. The sample of 5 mm in diameter and 1 mm thickness was cured in water bath for 20, 30, and 40 minutes at 100° C. BHK-21 cells were grown in medium eagle to be 2 × 105 cell/ml in 96 well micro titer plates as the added sample and incubated at 37° C for 24 hour. Five hours before the end of the incubation MTT solutionwas added from step one to each well containing cells. Viability cells were measured by spectrophotometer at 550 nm. The data were statistically analyzed by using one-way analysis of variance followed by LSD test. The result indicated that viability of fibroblast BHK-21 cells did not decrease to the surface of resin acrylic rapid heat cured.

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

    Science.gov (United States)

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

    2016-09-14

    We explored the changes in viability and lipid profile occurring in cancer cells, murine melanoma cells (B16F10 cells) and human cervical carcinoma cells (HeLa cells), when exposed to 24 h-treatments with an n-3 PUFA-rich oil obtained by supercritical extraction with CO2 from Mugil cephalus processed roe (bottarga). The composition of the major lipid classes of bottarga oil was determined by the (13)C NMR technique. Reversed-phase HPLC with DAD/ELSD detection was performed to analyze cells' total fatty acid profile and the levels of phospholipids, total/free cholesterol, triacylglycerols, and cholesteryl esters. Cell-based fluorescent measurements of intracellular membranes and lipid droplets were performed on bottarga oil-treated cells using the Nile red staining technique. The treatments of cancer cells with bottarga oil reduced the viability and affected the fatty acid profile, with a significant n-3 PUFA increase in treated cells. Mullet roe oil uptake modulated the cancer cell lipid composition, inducing a remarkable incorporation of health beneficial n-3 PUFA in the polar and neutral lipid fractions. Bottarga oil treatment influenced the synthesis of intracellular membranes and accumulation of cytoplasmic lipid droplets in cancer cells. PMID:27603212

  16. Effect of DNA methylation on protein-DNA interaction of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    何忠效; 白坚石; 张昱

    1999-01-01

    HL-60 cells have been induced with differentiation index 16 % by S-adenosyl-L-rnethionine (SAM) as inducer in the presence of optimum conceptration of 10 μmol/L. The methylation level of genorne DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma I, Sal I, XhoI and Hind Ⅲ which are sensitive to 5-methylcytosine were used to cleave the genorne DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.

  17. Pim2 is important for regulating DNA damage response in multiple myeloma cells

    Science.gov (United States)

    Ramachandran, J; Santo, L; Siu, K T; Panaroni, C; Raje, N

    2016-01-01

    Pan proviral integrations of Moloney virus (PIM) inhibition in multiple myeloma (MM) results in reduced cell viability in tested human-derived MM cell lines and reduces tumor burden in xenograft mouse models, making PIMs important therapeutic targets for the disease. PIM kinase inhibitors are currently being tested clinically in MM. We sought to elucidate the role of the various PIMs in MM. Our data demonstrate that Pim2 has a significant role in MM cell cytotoxicity. Our data provide evidence for a novel role for Pim2 in the regulation of the DNA damage response (DDR). Knockdown of Pim2 upregulates several downstream DDR markers, mimicking the effects of doxorubicin (Dox) treatment of MM cells, and suggesting a role for the kinase as a negative regulator of this pathway. Dox-induced DNA damage results in a decrease in Pim2 levels, placing the kinase directly downstream of the site of Dox-DNA binding. Overexpression of Pim2 confers a slight survival advantage against Dox through antiapoptotic activity, further underscoring its relevance in the DDR pathway. These data provide insights into a novel mechanism of PIM kinase activity and provide the framework for designing therapeutic approaches in MM. PMID:27564460

  18. Impairments of cells and genomic DNA by environmentally transformed engineered nanomaterials

    Science.gov (United States)

    Jones, Philip; Sugino, Sakiko; Yamamura, Shohei; Lacy, Fred; Biju, Vasudevanpillai

    2013-09-01

    Enormous increase in the production of nanomaterials and their growing applications in the device technology, biotechnology and biomedical areas suggest the need for developing models for predicting the environmental health and safety (EHS) risks posed by such nanomaterials. We hypothesize that CdSe quantum dots (QDs) and ZnO nanoparticles (NPs) encompassed in liposomes or not and transformed by simulated solar UV light can be model systems for studying the environmental toxicity of engineered nanomaterials. In this study, human lung epithelial adenocarcinoma cells (H1650) are exposed to photoirradiated CdSe QDs or ZnO nanopowder included or not in liposomes. The release of cadmium and zinc ions from the nanomaterials exposed to solar simulated UV radiation is detected and quantified by measuring the steady-state and time resolved fluorescence of the metal ion sensor tetracarboxyphenylporphyrin (TCPP) or the commercial Measure iT Pd/Cd sensor. Viability of cells treated with nanomaterials exposed to solar simulated UV radiation for different durations is measured by MTT assay. Enhanced etching of the nanoparticles exposed to solar simulated UV radiation results in the release of toxic levels of heavy metal ions, which considerably lower the viability of H1650 cells is due to the deactivation of DNA repair enzymes as evidenced by the pinching off of nuclear DNA in comet assays and DNA samples in electrophoresis. Results from this study highlight the need to obtain not only quantitative information about the environmental risks posed by engineered nanomaterials but also environment friendly nanomaterials for practical applications.Enormous increase in the production of nanomaterials and their growing applications in the device technology, biotechnology and biomedical areas suggest the need for developing models for predicting the environmental health and safety (EHS) risks posed by such nanomaterials. We hypothesize that CdSe quantum dots (QDs) and ZnO nanoparticles

  19. Prevention of DNA re-replication in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    Lan N. Truong; Xiaohua Wu

    2011-01-01

    DNA replication is a highly regulated process involving a number of licensing and replication factors that function in a carefully orchestrated manner to faithfully replicate DNA during every cell cycle. Loss of proper licensing control leads to deregulated DNA replication including DNA re-replication, which can cause genome instability and tumorigenesis. Eukaryotic organisms have established several conserved mechanisms to prevent DNA re-replication and to counteract its potentially harmful effects. These mechanisms include tightly controlled regulation of licensing factors and activation of cell cycle and DNA damage checkpoints.Deregulated licensing control and its associated compromised checkpoints have both been observed in tumor cells, indicating that proper functioning of these pathways is essential for maintaining genome stability. In this review, we discuss the regulatory mechanisms of licensing control, the deleterious consequences when both licensing and checkpoints are compromised, and present possible mechanisms to prevent re-replication in order to maintain genome stability.

  20. 2,2',4,4'-Tetrabromodiphenyl ether injures cell viability and mitochondrial function of mouse spermatocytes by decreasing mitochondrial proteins Atp5b and Uqcrc1.

    Science.gov (United States)

    Huang, Shaoping; Wang, Jing; Cui, Yiqiang

    2016-09-01

    Our object was to explore direct effects and mechanism of BDE47 on GC2 (immortalized mouse spermatocyte). GC2 were exposed to DMSO, 0.1, 1, 10, 100μM BDE47 for 48h. Cell viability was detected by trypan-blue exclusion; ultrastructure by electron-microscopy; cell cycle, mitochondrial membrane motential (MMP), reactive oxygen species (ROS) by flow-cytometry; ATP production by luminometer; Atp5b, Uqcrc1, Bcl-2 level by WB. To explore whether the decreased mitochondrial proteins play an important role in apoptosis, MMP and apoptosis were detected after Atp5b or Uqcrc1 knockdown in GC2. Results showed BDE47 reduced cell viability, caused condensation of nuclear and vacuolated mitochondria, decreased MMP and ATP, induced ROS, cell cycle arrest at S and G2/M phase, reduced Atp5b, Uqcrc1, Bcl-2 in GC2. Knockdown of Atp5b or Uqcrc1 decreased MMP, induced apoptosis in GC2. Results suggested that BDE47 reduced cell viability, injured mitochondria in spermatocytes probably by decreasing mitochondrial protein Atp5b and Uqcrc1. PMID:27525561

  1. Comparative study of the free radical and DNA break accumulation under gamma-irradiation of DNA solutions and cells

    International Nuclear Information System (INIS)

    The linear dependence between parameters of DNA radiation dmage is disclosed in the given paper in determining the concentration of free radicals and the number of DNA breaks in the same samples of irradiated frozen DNA solutions, cells and tissues

  2. DNA repair and radiation sensitivity in mammalian cells

    International Nuclear Information System (INIS)

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population

  3. CPT1α over-expression increases long-chain fatty acid oxidation and reduces cell viability with incremental palmitic acid concentration in 293T cells

    International Nuclear Information System (INIS)

    To test the cellular response to an increased fatty acid oxidation, we generated a vector for an inducible expression of the rate-limiting enzyme carnitine palmitoyl-transferase 1α (CPT1α). Human embryonic 293T kidney cells were transiently transfected and expression of the CPT1α transgene in the tet-on vector was activated with doxycycline. Fatty acid oxidation was measured by determining the conversion of supplemented, synthetic cis-10-heptadecenoic acid (C17:1n-7) to C15:ln-7. CPT1α over-expression increased mitochondrial long-chain fatty acid oxidation about 6-fold. Addition of palmitic acid (PA) decreased viability of CPT1α over-expressing cells in a concentration-dependent manner. Both, PA and CPT1α over-expression increased cell death. Interestingly, PA reduced total cell number only in cells over-expressing CPT1α, suggesting an effect on cell proliferation that requires PA translocation across the mitochondrial inner membrane. This inducible expression system should be well suited to study the roles of CPT1 and fatty acid oxidation in lipotoxicity and metabolism in vivo

  4. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds

    International Nuclear Information System (INIS)

    The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications. - Highlights: ► Critical point drying induces fibre bundling unlike lyophilisation. ► Cells positively expressed undifferentiated pluripotent stem cell markers. ► BCs were cytocompatible, supported cell adhesion, proliferation and differentiation ► Cells seeded on BC scaffolds were viable and metabolically active. ► Findings demonstrate that BC and EqMSCs are promising tissue engineered constructs

  5. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Favi, Pelagie M.; Benson, Roberto S. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Neilsen, Nancy R. [Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Hammonds, Ryan L. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Bates, Cassandra C. [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Stephens, Christopher P. [Department of Surgery, Graduate School of Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Center for Materials Processing, University of Tennessee, Knoxville, TN 37996 (United States); Dhar, Madhu S., E-mail: mdhar@utk.edu [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States)

    2013-05-01

    The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications. - Highlights: ► Critical point drying induces fibre bundling unlike lyophilisation. ► Cells positively expressed undifferentiated pluripotent stem cell markers. ► BCs were cytocompatible, supported cell adhesion, proliferation and differentiation ► Cells seeded on BC scaffolds were viable and metabolically active. ► Findings demonstrate that BC and EqMSCs are promising tissue engineered constructs.

  6. Quantitative analysis of cell-free DNA in ovarian cancer

    OpenAIRE

    Shao, Xuefeng; He, Yan; Ji, Min; Chen, Xiaofang; Qi, Jing; SHI, Wei; HAO, TIANBO; JU, SHAOQING

    2015-01-01

    The aim of the present study was to investigate the association between cell-free DNA (cf-DNA) levels and clinicopathological characteristics of patients with ovarian cancer using a branched DNA (bDNA) technique, and to determine the value of quantitative cf-DNA detection in assisting with the diagnosis of ovarian cancer. Serum specimens were collected from 36 patients with ovarian cancer on days 1, 3 and 7 following surgery, and additional serum samples were also collected from 22 benign ova...

  7. Dicer is associated with ribosomal DNA chromatin in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Lasse Sinkkonen

    Full Text Available BACKGROUND: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/- ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines. CONCLUSION/SIGNIFICANCE: We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays.

  8. Detection of hepatitis B virus DNA in mononuclear blood cells.

    OpenAIRE

    Pontisso, P; Poon, M C; Tiollais, P.; Brechot, C

    1984-01-01

    The Southern transfer hybridisation technique was used to test mononuclear blood cells for hepatitis B virus DNA. Viral DNA sequences were detected in mononuclear cells of 10 out of 16 patients with hepatitis B virus infection and in none of 21 normal controls. Blood contamination was excluded by the absence of hepatitis B virus DNA in the corresponding serum samples in all cases. Free monomeric hepatitis B virus DNA was found in three patients positive for hepatitis Be antigen (HBeAg) and on...

  9. Cell cycle control after DNA damage: arrest, recovery and adaptation

    International Nuclear Information System (INIS)

    DNA damage triggers surveillance mechanisms, the DNA checkpoints, that control the genome integrity. The DNA checkpoints induce several responses, either cellular or transcriptional, that favor DNA repair. In particular, activation of the DNA checkpoints inhibits cell cycle progression in all phases, depending on the stage when lesions occur. These arrests are generally transient and cells ultimately reenter the cell division cycle whether lesions have been repaired (this process is termed 'recovery') or have proved un-repairable (this option is called 'adaptation'). The mechanisms controlling cell cycle arrests, recovery and adaptation are largely conserved among eukaryotes, and much information is now available for the yeast Saccharomyces cerevisiae, that is used as a model organism in these studies. (author)

  10. 10-Acetylirciformonin B, A Sponge Furanoterpenoid, Induces DNA Damage and Apoptosis in Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Fu-Wen Kuo

    2012-10-01

    Full Text Available 10-Acetylirciformonin B, a furanoterpenoid derived from irciformonin B found in a marine sponge, has been reported to possess potent cytotoxic activity against several cancer cell lines. However, the mechanism of its apoptotic activity against human leukemia cells has never been reported. The purpose of this study was to investigate the cytotoxic effects of 10-acetylirciformonin B and its possible mechanism of action against leukemia HL 60 cells. We found that 10-acetylirciformonin B decreased cell viability through the inhibition of cell growth as well as the induction of DNA damage and apoptosis in a dose-dependent manner. The induction of DNA damage was mediated by the increase of p-CHK2 and γ-H2A.X, which was suggested from the increase of tail movement in the neutral Comet assay. Induction of apoptosis was mediated with the increase in caspases 8, 9 and 3 activation as well as PARP cleavage. In summary, our resultsindicate that 10-acetylirciformonin B treatment causes apoptosis in leukaemia cells; probably through a caspase-dependent regulatory pathway.

  11. Effect of All-Trans Retinoic Acid (ATRA on Viability, Proliferation, Activation and Lineage-Specific Transcription Factors of CD4+ T Cells

    Directory of Open Access Journals (Sweden)

    Katayoon Bidad

    2011-12-01

    Full Text Available All-trans retinoic acid (ATRA, as an active metabolite of vitamin A, has been shown to affect immune cells. This study was performed to evaluate the effect of ATRA on viability, proliferation, activation and lineage-specific transcription factors of CD4+ T cells. CD4+ T cells were  separated  from  heparinized  blood  of  healthy  donors  and  were  cultured  in conditions, some with, some without ATRA.Viability was assessed by PI  flowcytometry and proliferation was measured by MTT assay. CD69 expression was determined by flowcytometry as a measure of cell activation. Lineage-specific transcription  factors  (FOXP3,  RORγt  and  T-bet  were  examined  by intracellular staining and flowcytometry. High doses of ATRA (0.1-1 mM caused extensive cell death in both PBMCs and CD4+ T cells. Doses of ATRA equal to or lower than 10 µM did not  adversely affect cell viability and proliferation in comparison to  culture medium without ATRA.Doses of ATRA between 10 µM and 1nM significantly increased cell activation when compared  to  culture medium without  ATRA. ATRA could increase FOXP3+  and also FOXP3+RORγt+ T cells while it decreased RORγt+ and T-bet+ T cells. This study showed that doses of ATRA up to 10 µM are safe when using with CD4+  T cells in terms of cell viability, proliferation and activation.We  could  also  show  that  ATRA  diverts  the  human  immune  response  in  neutral conditions (without adding polarizing cytokines by increasing FOXP3+  cells and decreasing RORγt+  cells. ATRA could be regarded as a potential therapy in inflammatory conditions and autoimmunities.

  12. Influence of nanocrystalline structure and surface properties of TiO2 thin films on the viability of L929 cells

    Directory of Open Access Journals (Sweden)

    Osękowska Małgorzata

    2015-09-01

    Full Text Available In this work the physicochemical and biological properties of nanocrystalline TiO2 thin films were investigated. Thin films were prepared by magnetron sputtering method. Their properties were examined by X-ray diffraction, photoelectron spectroscopy, atomic force microscopy, optical transmission method and optical profiler. Moreover, surface wettability and scratch resistance were determined. It was found that as-deposited coatings were nanocrystalline and had TiO2-anatase structure, built from crystallites in size of 24 nm. The surface of the films was homogenous, composed of closely packed grains and hydrophilic. Due to nanocrystalline structure thin films exhibited good scratch resistance. The results were correlated to the biological activity (in vitro of thin films. Morphological changes of mouse fibroblasts (L929 cell line after contact with the surface of TiO2 films were evaluated with the use of a contrast-phase microscope, while their viability was tested by MTT colorimetric assay. The viability of cell line upon contact with the surface of nanocrystalline TiO2 film was comparable to the control sample. L929 cells had homogenous cytoplasm and were forming a confluent monofilm, while lysis and inhibition of cell growth was not observed. Moreover, the viability in contact with surface of examined films was high. This confirms non-cytotoxic effect of TiO2 film surface on mouse fibroblasts.

  13. RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage-induced cell senescence.

    Science.gov (United States)

    Cekan, Pavol; Hasegawa, Keisuke; Pan, Yu; Tubman, Emily; Odde, David; Chen, Jin-Qiu; Herrmann, Michelle A; Kumar, Sheetal; Kalab, Petr

    2016-04-15

    The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase-regulated nuclear-cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage-induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β-dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP-regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage. PMID:26864624

  14. Targeting DNA vaccines to myeloid cells using a small peptide.

    Science.gov (United States)

    Ye, Chunting; Choi, Jang Gi; Abraham, Sojan; Shankar, Premlata; Manjunath, N

    2015-01-01

    Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able to prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WNV-neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses.

  15. Glucosylceramides are critical for cell-type differentiation and organogenesis, but not for cell viability in Arabidopsis.

    Science.gov (United States)

    Msanne, Joseph; Chen, Ming; Luttgeharm, Kyle D; Bradley, Amanda M; Mays, Elizabeth S; Paper, Janet M; Boyle, Daniel L; Cahoon, Rebecca E; Schrick, Kathrin; Cahoon, Edgar B

    2015-10-01

    Glucosylceramides (GlcCer), glucose-conjugated sphingolipids, are major components of the endomembrane system and plasma membrane in most eukaryotic cells. Yet the quantitative significance and cellular functions of GlcCer are not well characterized in plants and other multi-organ eukaryotes. To address this, we examined Arabidopsis lines that were lacking or deficient in GlcCer by insertional disruption or by RNA interference (RNAi) suppression of the single gene for GlcCer synthase (GCS, At2g19880), the enzyme that catalyzes GlcCer synthesis. Null mutants for GCS (designated 'gcs-1') were viable as seedlings, albeit strongly reduced in size, and failed to develop beyond the seedling stage. Heterozygous plants harboring the insertion allele exhibited reduced transmission through the male gametophyte. Undifferentiated calli generated from gcs-1 seedlings and lacking GlcCer proliferated in a manner similar to calli from wild-type plants. However, gcs-1 calli, in contrast to wild-type calli, were unable to develop organs on differentiation media. Consistent with a role for GlcCer in organ-specific cell differentiation, calli from gcs-1 mutants formed roots and leaves on media supplemented with the glucosylated sphingosine glucopsychosine, which was readily converted to GlcCer independent of GCS. Underlying these phenotypes, gcs-1 cells had altered Golgi morphology and fewer cisternae per Golgi apparatus relative to wild-type cells, indicative of protein trafficking defects. Despite seedling lethality in the null mutant, GCS RNAi suppression lines with ≤2% of wild-type GlcCer levels were viable and fertile. Collectively, these results indicate that GlcCer are essential for cell-type differentiation and organogenesis, and plant cells produce amounts of GlcCer in excess of that required for normal development. PMID:26313010

  16. OPTIMIZATION OF ELECTROPORATION PARAMETERS FOR TRANSFECTION OF PLASMID DNA INTO MURINE BONE MARROW-DERIVED DENDRITIC CELL

    Institute of Scientific and Technical Information of China (English)

    KE Shan; CHEN Xue-hua; LI Hao; LI Jian-fang; GU Qin-long; ZHU Zheng-gang; LIU Bing-ya

    2006-01-01

    Objective To explore the optimal electroporation parameters for transfection of plasmid DNA into murine bone marrow-derived dendritic cells. Methods Murine bone marrow-derived dendritic cells (DCs) were electroporated with plasmid DNA in varied conditions, such as electrical voltage, pulse time,pre-electroporation cell condition and serum concentration in electrical buffer. Inverted fluorescence microscope and flow cytometer were used to determine the transfection efficiency. Some of the DCs genetically modified under different conditions were stained with trypan-blue and its viability was observed microscopically 48h after electroporation. Results Highest transfection efficiency (22.10%) could be reached when electrical voltage was 250V and pulse time was 20ms. Refreshing the culture medium pre-electroporation may help the cells recover more easily from gene transfer.Besides, electrical buffer containing serum may benefit the viability of DC after electroporation and temperature may has little influence on transfection efficiency. Conclusion Our observations demonstrated plasmid DNA could be efficiently transferred into murine bone marrow-derived DCs by electroporation. These data may helpful for cancer research related to murine DC transfection.

  17. Inhibition by 2-deoxy-D-ribose of DNA synthesis and growth in Raji cells

    International Nuclear Information System (INIS)

    When Raji cells were cultured for 3 days in serum-free medium, addition of 2-deoxy-D-ribose at the start of culture inhibited incorporation of [3H]thymidine and cell division. At deoxyribose concentrations between 1 and 5 mM, viability was 80% or greater after 3 days of culture even though 5 mM deoxyribose inhibited thymidine incorporation 95-99%. Inhibition by deoxyribose could be completely reversed if the culture medium was replaced with fresh medium up to 8 hr after the start of culture. The inhibition was specific for deoxyribose since other monosaccharides had no effect. Inhibition of DNA synthesis did not appear to be due to depletion of essential nutrients in the medium since the percentage inhibition of thymidine incorporation by cells cultured either in suboptimal serum-free media or in media supplemented with 0.025-5% human AB serum was similar. When DNA repair synthesis was measured as hydroxyurea-resistant thymidine incorporation, addition of deoxyribose to Raji cultures caused increased thymidine incorporation. These results, together with data from others,suggest that deoxyribose damages DNA

  18. Mitochondrial DNA sequence analysis of two mouse hepatocarcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Ji-Gang Dai; Xia Lei; Jia-Xin Min; Guo-Qiang Zhang; Hong Wei

    2005-01-01

    AIM: To study genetic difference of mitochondrial DNA (mtDNA)between two hepatocarcinoma cell lines (Hca-F and Hca-P)with diverse metastatic characteristics and the relationship between mtDNA changes in cancer cells and their oncogenic phenotype.METHODS: Mitochondrial DNA D-loop, tRNAMet+Glu+Ile and ND3gene fragments from the hepatocarcinoma cell lines with 1100, 1126 and 534 bp in length respectively were analysed by PCR amplification and restriction fragment length polymorphism techniques. The D-loop 3' end sequence of the hepatocarcinoma cell lines was determined by sequencing.RESULTS: No amplification fragment length polymorphism and restriction fragment length polymorphism were observed in tRNAMet+Glu+Ile,ND3 and D-loop of mitochondrial DNA of the hepatocarcinoma cells. Sequence differences between Hca-F and Hca-P were found in mtDNA D-loop.CONCLUSION: Deletion mutations of mitochondrial DNA restriction fragment may not play a significant role in carcinogenesis. Genetic difference of mtDNA D-loop between Hca-F and Hca-P, which may reflect the environmental and genetic influences during tumor progression, could be linked to their tumorigenic phenotypes.

  19. NucleoCounter-An efficient technique for the determination of cell number and viability in animal cell culture processes.

    Science.gov (United States)

    Shah, Dimpalkumar; Naciri, Mariam; Clee, Paul; Al-Rubeai, Mohamed

    2006-05-01

    The NucleoCounter is a novel, portable cell counting device based on the principle of fluorescence microscopy. The present work establishes its use with animal cells and checks its reliability, consistency and accuracy in comparison with other cytometric techniques. The main advantages of this technique are its ability to handle a large number of samples with a high degree of precision and its simplicity and specificity in detecting viable cells quantitatively in a heterogeneous culture. The work addresses and overcomes the problems of subjectivity, and some of the inherent sampling errors associated with using the traditional haemocytometer and Trypan Blue exclusion method. NucleoCounter offers reduced intra- and inter-observer variation as well as consistency in repetitive analysis that establishes it as an efficient and highly potential device for at-line monitoring of animal cell processes. Furthermore, since the only manual steps required are sample aspiration and mixing with two reagents, it is feasible that the whole method could be automated and brought on-line for process monitoring and control. PMID:19002893

  20. Circulating Cell Free DNA in the Diagnosis of Trophoblastic Tumors

    Science.gov (United States)

    Openshaw, Mark R.; Harvey, Richard A.; Sebire, Neil J.; Kaur, Baljeet; Sarwar, Naveed; Seckl, Michael J.; Fisher, Rosemary A.

    2015-01-01

    Gestational trophoblastic neoplasia (GTN) represents a group of diseases characterized by production of human chorionic gonadotropin (hCG). Since non-gestational tumors may occasionally secrete hCG, histopathological diagnosis is important for appropriate clinical management. However, a histopathological diagnosis is not always available. We therefore investigated the feasibility of extracting cell free DNA (cfDNA) from the plasma of women with GTN for use as a “liquid biopsy” in patients without histopathological diagnosis. cfDNA was prepared from the plasma of 20 women with a diagnosis of GTN and five with hCG-secreting tumors of unknown origin. Genotyping of cfDNA from the patient, genomic DNA from her and her partner and DNA from the tumor tissue identified circulating tumor DNA (ctDNA) (from 9% to 53% of total cfDNA) in 12 of 20 patients with GTN. In one case without a tissue diagnosis, ctDNA enabled a diagnosis of GTN originating in a non-molar conception and in another a diagnosis of non-gestational tumor, based on the high degree of allelic instability and loss of heterozygosity in the ctDNA. In summary ctDNA can be detected in the plasma of women with GTN and can facilitate the diagnosis of both gestational and non-gestational trophoblastic tumors in cases without histopathological diagnosis. PMID:26981554

  1. Circulating Cell Free DNA in the Diagnosis of Trophoblastic Tumors

    Directory of Open Access Journals (Sweden)

    Mark R. Openshaw

    2016-02-01

    Full Text Available Gestational trophoblastic neoplasia (GTN represents a group of diseases characterized by production of human chorionic gonadotropin (hCG. Since non-gestational tumors may occasionally secrete hCG, histopathological diagnosis is important for appropriate clinical management. However, a histopathological diagnosis is not always available. We therefore investigated the feasibility of extracting cell free DNA (cfDNA from the plasma of women with GTN for use as a “liquid biopsy” in patients without histopathological diagnosis. cfDNA was prepared from the plasma of 20 women with a diagnosis of GTN and five with hCG-secreting tumors of unknown origin. Genotyping of cfDNA from the patient, genomic DNA from her and her partner and DNA from the tumor tissue identified circulating tumor DNA (ctDNA (from 9% to 53% of total cfDNA in 12 of 20 patients with GTN. In one case without a tissue diagnosis, ctDNA enabled a diagnosis of GTN originating in a non-molar conception and in another a diagnosis of non-gestational tumor, based on the high degree of allelic instability and loss of heterozygosity in the ctDNA. In summary ctDNA can be detected in the plasma of women with GTN and can facilitate the diagnosis of both gestational and non-gestational trophoblastic tumors in cases without histopathological diagnosis.

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

    OpenAIRE

    Ji, Min-Kyung; Park, Sang-Won; Lee, Kwangmin; Kang, In-Chol; Yun, Kwi-Dug; Kim, Hyun-Seung; Lim, Hyun-Pil

    2015-01-01

    PURPOSE The aim of this study was to evaluate antibacterial activity and osteoblast-like cell viability according to the ratio of titanium nitride and zirconium nitride coating on commercially pure titanium using an arc ion plating system. MATERIALS AND METHODS Polished titanium surfaces were used as controls. Surface topography was observed by scanning electron microscopy, and surface roughness was measured using a two-dimensional contact stylus profilometer. Antibacterial activity was evalu...

  3. Mitochondrial DNA sequence variation in single cells from leukemia patients

    OpenAIRE

    Yao, Yong-Gang; Ogasawara, Yoji; Kajigaya, Sachiko; Molldrem, Jeffrey J.; Falcão, Roberto P; Pintão, Maria-Carolina; McCoy, J. Philip; Rizzatti, Edgar Gil; Young, Neal S

    2007-01-01

    A high frequency of mtDNA somatic mutation has been observed in many tumors as well as in aging tissues. In this study, we analyzed the mtDNA control region sequence variation in 3534 single normal cells and individual blasts from 18 patients with leukemia and 10 healthy donors, to address the mutation process in leukemic cells. We found significant differences in mtDNA sequence, as represented by the number of haplotypes and the mean number of cells with each nonaggregate haplotype in a popu...

  4. Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    and mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces cell cycle synchronization in different human osteosarcoma cell lines. The UV pulse also has a destabilizing......Imbalanced dNTP pools are highly mutagenic due to a deleterious effect on DNA polymerase fidelity. Mitochondrial DNA defects, including mutations and deletions, are commonly found in a wide variety of different cancer types. In order to further study the interconnection between dNTP pools......-synthase. As a result of this the rho0 cells have much lower ATP levels than rho+ cells. In order to mimic the ATP situation in rho0 cells the rho+ cells are incubated with the ATP synthase inhibitor, oligomycin. Similar to the rho0 cells the oligomycin incubated rho+ cells display destabilized dNTP pools upon UV...

  5. DNA profiles generated from minute amounts of single cells

    OpenAIRE

    Wenäll, Lovisa

    2011-01-01

    The genetic code in our cells is built up by deoxyribonucleic acid (DNA) with a sequence that is individual and unique to each person. A cell’s origin can be decided by comparing an established DNA profile with a known profile. The most publicly known application is in the forensic field and its use for identification and for establishing a connection between perpetrators and victims or crime scenes. DNA profiling is also commonly used for kinship investigations. The information embedded in t...

  6. 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.

  7. Effects of two fast-setting calcium-silicate cements on cell viability and angiogenic factor release in human pulp-derived cells.

    Science.gov (United States)

    Chung, Chooryung J; Kim, Euiseong; Song, Minju; Park, Jeong-Won; Shin, Su-Jung

    2016-05-01

    Mineral trioxide aggregate (MTA) is considered a pulp-capping agent of choice, but has the drawback of a long setting time. This study aimed to assess two different types of calcium-silicate cements as pulp-capping agents, by investigating their in vitro cytotoxicity and angiogenic effects in human pulp cells. ProRoot MTA, Endocem Zr, and Retro MTA were prepared as set or freshly mixed pellets. Human pulp-derived cells were grown in direct contact with these three cements, Dycal, or no cement, for 7 days. Initial cell attachment, viability, calcium release, and the levels of vascular endothelial growth factor (VEGF), angiogenin, and basic fibroblast growth factor (FGF-2) were evaluated statistically using a linear mixed model (P calcium concentration compared with the control group (P  0.05). We demonstrate that Retro MTA, which has a short setting time, has similar biocompatibility and angiogenic effects on human pulp cells, and can therefore potentially be as effective in pulp capping as ProRoot MTA. Endocem Zr showed intermittent cytotoxicity and elicited lower levels of VEGF and angiogenin expression. PMID:25596932

  8. Solid lipid nanoparticles for hydrophilic biotech drugs: optimization and cell viability studies (Caco-2 & HEPG-2 cell lines).

    Science.gov (United States)

    Severino, Patrícia; Andreani, Tatiana; Jäger, Alessandro; Chaud, Marco V; Santana, Maria Helena A; Silva, Amélia M; Souto, Eliana B

    2014-06-23

    Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

  9. Organically Modified Silica Nanoparticles Interaction with Macrophage Cells: Assessment of Cell Viability on the Basis of Physicochemical Properties.

    Science.gov (United States)

    Kumar, Dhiraj; Mutreja, Isha; Keshvan, Prashant C; Bhat, Madhusudan; Dinda, Amit K; Mitra, Susmita

    2015-11-01

    Silica nanoparticles have drawn a lot of attention for nanomedicine application, and this is attributed to their biocompatibility and ease of surface functionalization. However, successful utilization of these inorganic systems for biomedical application depends on their physicochemical properties. This study, therefore, discusses in vitro toxicity of organically modified silica nanoparticles on the basis of size, shape, and surface properties of silica nanoparticles. Spherical- and oval-shaped nanoparticles having hydroxyl and amine groups were synthesized in Tween 80 micelles using different organosilanes. Nanoparticles of similar size and morphology were considered for comparative assessment. "As-prepared" nanoparticles were characterized in terms of size, shape, and surface properties using ZetaSizer, transmission electron microscopy, and Fourier transform infrared to establish the above parameters. In vitro analysis in terms of nanoparticle-based toxicity was performed on J-774 (macrophage) cell line using propidium iodide-4',6-diamidino-2-phenylindol and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Fluorescent dye-entrapped nanoparticles were used to visualize the uptake of the nanoparticles by macrophage cells. Results from cell studies suggested low levels of toxicity for different nanoparticle formulations studied, therefore are suitable for nanocarrier application for poorly soluble molecules. On the contrary, the nanoparticles of similar size and shape, having amine groups and low net negative charge, do not exhibit any in vitro cytotoxicity.

  10. Controls to validate plasma samples for cell free DNA quantification

    DEFF Research Database (Denmark)

    Pallisgaard, Niels; Spindler, Karen-Lise Garm; Andersen, Rikke Fredslund;

    2015-01-01

    Recent research has focused on the utility of cell free DNA (cfDNA) in serum and plasma for clinical application, especially in oncology. The literature holds promise of cfDNA as a valuable tumour marker to be used for treatment selection, monitoring and follow-up. The results, however, are diver......Recent research has focused on the utility of cell free DNA (cfDNA) in serum and plasma for clinical application, especially in oncology. The literature holds promise of cfDNA as a valuable tumour marker to be used for treatment selection, monitoring and follow-up. The results, however......, are diverging due to methodological differences with lack of standardisation and definition of sensitivity. The new biological information has not yet come into routine use. The present study presents external standardisation by spiking with non-human DNA fragments to control for loss of DNA during sample...... preparation and measurement. It also suggests a method to control for admixture of DNA from normal lymphocytes by utilizing the unique immunoglobulin gene rearrangement in the B-cells. The results show that this approach improves the quality of the analysis and lowers the risk of falsely increased values...

  11. The DNA methylome of human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Li, Yingrui; Zhu, Jingde; Tian, Geng;

    2010-01-01

    DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per...

  12. Analysis of epigenetic modifications of DNA in human cells

    DEFF Research Database (Denmark)

    Kristensen, Lasse Sommer; Treppendahl, Marianne Bach; Grønbæk, Kirsten

    2013-01-01

    Epigenetics, the study of somatically heritable changes in gene expression not related to changes in the DNA sequence, is a rapidly expanding research field that plays important roles in healthy as well as in diseased cells. DNA methylation and hydroxymethylation are epigenetic modifications found...

  13. DNA repair in human xeroderma pigmentosum and chinese hamster cells

    NARCIS (Netherlands)

    Zelle, B.

    1980-01-01

    An important feature of living cells is their capacity to maintain the integrity of their hereditary material, the DNA. DNA can be damaged by a variety of physical and chemical agents, among which ultraviolet radiation (UV), ion1z1ng radiation and chemical carcinogens as 4-nitroquinoline-1-oxide (4N

  14. DNA repair in human xeroderma pigmentosum and Chinese hamster cells

    NARCIS (Netherlands)

    B. Zelle (Bauke)

    1980-01-01

    textabstractAn important feature of living cells is their capacity to maintain the integrity of their hereditary material, the DNA. DNA can be damaged by a variety of physical and chemical agents, among which ultraviolet radiation (UV), ion1z1ng radiation and chemical carcinogens as 4-nitroquinoline

  15. Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    and mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces an S phase delay in different human osteosarcoma cell lines. The UV pulse also has a destabilizing effect......Imbalanced dNTP pools are highly mutagenic due to a deleterious effect on DNA polymerase fidelity. Mitochondrial DNA defects, including mutations and deletions, are commonly found in a wide variety of different cancer types. In order to further study the interconnection between dNTP pools...... of this the rho0 cells have much lower ATP levels than rho+ cells. In order to mimic the ATP situation in rho0 cells the rho+ cells are incubated with the ATP synthase inhibitor, oligomycin. Similar to the rho0 cells the oligomycin incubated rho+ cells display increased dNTP pools upon UV irradiation. Our data...

  16. Cell viability, pigments and photosynthetic performance of Arctic phytoplankton in contrasting ice-covered and open-water conditions during the spring-summer transition

    KAUST Repository

    Alou-Font, E

    2015-12-02

    © Inter-Research 2016. We examined phytoplankton biomass and community composition (mostly based on pigments) as well as cell viability with the cell digestion assay in surface waters of the Canadian Beaufort Sea during the spring-summer transition. Our aim was to understand phytoplankton responses to the large environmental changes (irradiance, temperature and nutrients) occurring during this period. Two categories of stations were visited in May and June 2008: ice-covered (IC), exposed to low irradiances, and open-water (OW), exposed to higher irradiances. We observed a large variation in the percentage of living cells (%LC) relative to the total community. No relationship was found between %LC and nitrate concentration (the nutrient potentially limiting in this environment). The in situ irradiance influenced the status of the cells at OW stations. Mean surface mixed layer irradiances >600 μmol photons m-2 s-1 were associated with low cell viability and a decline in photosynthetic performance (Fv/Fm). For IC stations, %LC declined at temperatures above 0°C, whereas for OW stations, it increased, suggesting that ice melting resulted in the release into surface waters of unhealthy cells from the bottom ice in one case, and that seasonal warming favored the communities present in open waters. A chlorophyll degradation pigment tentatively identified as pyropheophorbide a-\\'like\\' showed a significant negative relationship between its concentration (relative to chlorophyll a) and the %LC and Fv/Fm. Our results suggest that the melting conditions influence the distribution of this pigment and that it may be useful as a marker for low cell viability of ice algae being released into surface waters.

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

    Science.gov (United States)

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

    2016-02-01

    This study examined the effect of the immobilization of the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide on titanium dioxide (TiO2) nanotube via chemical grafting on osteoblast-like cell (MG-63) viability and differentiation. The specimens were divided into two groups; TiO2 nanotubes and GRGDS-immobilized TiO2 nanotubes. The surface characteristics of GRGDS-immobilized TiO2 nanotubes were observed by using X-ray photoelectron spectroscopy (XPS) and a field emission scanning electron microscope (FE-SEM). The morphology of cells on specimens was observed by FE-SEM after 2 hr and 24 hr. The level of cell viability was investigated via a tetrazolium (XTT) assay after 2 and 4 days. Alkaline phosphatase (ALP) activity was evaluated to measure the cell differentiation after 4 and 7 days. The presence of nitrogen up-regulation or C==O carbons con- firmed that TiO2 nanotubes were immobilized with GRGDS peptides. Cell adhesion was enhanced on the GRGDS-immobilized TiO2 nanotubes compared to TiO2 nanotubes. Furthermore, significantly increased cell spreading and proliferation were observed with the cells grown on GRGDS-immobilized TiO2 nanotubes (P nanotubes and TiO2 nanotubes. These results suggest that the GRGDS-immobilized TiO2 nanotubes might be effective in improving the osseointegration of dental implants.

  18. DNA Charge Transport: from Chemical Principles to the Cell.

    Science.gov (United States)

    Arnold, Anna R; Grodick, Michael A; Barton, Jacqueline K

    2016-01-21

    The DNA double helix has captured the imagination of many, bringing it to the forefront of biological research. DNA has unique features that extend our interest into areas of chemistry, physics, material science, and engineering. Our laboratory has focused on studies of DNA charge transport (CT), wherein charges can efficiently travel long molecular distances through the DNA helix while maintaining an exquisite sensitivity to base pair π-stacking. Because DNA CT chemistry reports on the integrity of the DNA duplex, this property may be exploited to develop electrochemical devices to detect DNA lesions and DNA-binding proteins. Furthermore, studies now indicate that DNA CT may also be used in the cell by, for example, DNA repair proteins, as a cellular diagnostic, in order to scan the genome to localize efficiently to damage sites. In this review, we describe this evolution of DNA CT chemistry from the discovery of fundamental chemical principles to applications in diagnostic strategies and possible roles in biology. PMID:26933744

  19. DNA breaks early in replication in B cell cancers

    Science.gov (United States)

    Research by scientists at the NCI has identified a new class of DNA sites in cells that break early in the replication process. They found that these break sites correlate with damage often seen in B cell cancers, such as diffuse large B cell lymphoma.

  20. DNA-mediated gene transfer into ataxia-telangiectasia cells

    International Nuclear Information System (INIS)

    The complete description of the genetic lesion(s) underlying the AT mutation might, therefore, highlight not only a DNA-repair pathwa, but also an important aspect of the physiology of lymphocytes. DNA-mediated gene transfer into eukaryotic cells has proved a powerful tool for the molecular cloning of certain mammalian genes. The possibility to clone a given gene using this technology depends, basically, on the availability of a selectable marker associated with the expression of the transfected gene in the recipient cell. Recently, a human DNA repair gene has been cloned in CHO mutant cells by taking advantage of the increased resistance to ultraviolet radiation of the transformants. As a preliminary step toward the molecular cloning of the AT gene(s), the authors have attempted to confer radioresistance to AT cells by transfection with normal human DNA

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

    Science.gov (United States)

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

    2013-11-01

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

  2. Cell-free DNA screening and sex chromosome aneuploidies.

    Science.gov (United States)

    Mennuti, Michael T; Chandrasekaran, Suchitra; Khalek, Nahla; Dugoff, Lorraine

    2015-10-01

    Cell-free DNA (cfDNA) testing is increasingly being used to screen pregnant women for fetal aneuploidies. This technology may also identify fetal sex and can be used to screen for sex chromosome aneuploidies (SCAs). Physicians offering this screening will need to be prepared to offer comprehensive prenatal counseling about these disorders to an increasing number of patients. The purpose of this article is to consider the source of information to use for counseling, factors in parental decision-making, and the performance characteristics of cfDNA testing in screening for SCAs. Discordance between ultrasound examination and cfDNA results regarding fetal sex is also discussed.

  3. Decreased serum cell-free DNA levels in rheumatoid arthritis

    OpenAIRE

    Dunaeva, Marina; Buddingh’, Bastiaan C.; René E M Toes; Luime, Jolanda J.; Lubberts, Erik; Pruijn, Ger J. M.

    2015-01-01

    Purpose Recent studies have demonstrated that serum/plasma DNA and RNA molecules in addition to proteins can serve as biomarkers. Elevated levels of these nucleic acids have been found not only in acute, but also in chronic conditions, including autoimmune diseases. The aim of this study was to assess cell-free DNA (cfDNA) levels in sera of rheumatoid arthritis (RA) patients compared to controls. Methods cfDNA was extracted from sera of patients with early and established RA, relapsing-remitt...

  4. Cloning of monomeric human papillomavirus type 16 DNA integrated within cell DNA from a cervical carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Matsukura, T.; Kanda, T.; Furuno, A.; Yoshikawa, H.; Kawana, T.; Yoshiike, K.

    1986-06-01

    The authors have molecularly cloned and characterized monomeric human papillomavirus type 16 DNA with flanking cell DNA sequences from a cervical carcinoma. Determination of nucleotide sequence around the junctions of human papillomavirus and cell DNAs revealed that at the site of integration within cell DNA the cloned viral DNA had a deletion between nucleotides 1284 and 4471 (numbering system from K. Seedorf, G. Kraemmer, M. Duerst, S. Suhai, and W.G. Roewkamp), which includes the greater part of E1 gene and the entire E2 gene. In the remaining part of the E1 gene, three guanines were found at the location where two guanines at nucleotides 1137 and 1138 have been recorded. This additional guanine shifted the reading frame and erased an interruption in the E1 gene. The data strongly suggest that, like other papillomaviruses, human papillomavirus type 16 has an uninterrupted E1 gene.

  5. Nonhomologous DNA End Joining in Cell-Free Extracts

    Directory of Open Access Journals (Sweden)

    Sheetal Sharma

    2010-01-01

    Full Text Available Among various DNA damages, double-strand breaks (DSBs are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems.

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

    Science.gov (United States)

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

    2016-07-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

  8. UV stimulation of DNA-mediated transformation of human cells

    International Nuclear Information System (INIS)

    Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells, which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome

  9. Low-dose formaldehyde delays DNA damage recognition and DNA excision repair in human cells.

    Directory of Open Access Journals (Sweden)

    Andreas Luch

    Full Text Available OBJECTIVE: Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions. METHODOLOGY/PRINCIPAL FINDINGS: The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding and XPC (xeroderma pigmentosum group C was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (<100 μM formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. CONCLUSIONS/SIGNIFICANCE: A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks.

  10. The DNA glycosylases OGG1 and NEIL3 influence differentiation potential, proliferation, and senescence-associated signs in neural stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Reis, Amilcar [Linnaeus Center in Developmental Biology for Regenerative Medicine (DBRM), Department of Neuroscience, Karolinska Institutet, SE 17177 Stockholm (Sweden); Hermanson, Ola, E-mail: ola.hermanson@ki.se [Linnaeus Center in Developmental Biology for Regenerative Medicine (DBRM), Department of Neuroscience, Karolinska Institutet, SE 17177 Stockholm (Sweden)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer DNA glycosylases OGG1 and NEIL3 are required for neural stem cell state. Black-Right-Pointing-Pointer No effect on cell viability by OGG1 or NEIL3 knockdown in neural stem cells. Black-Right-Pointing-Pointer OGG1 or NEIL3 RNA knockdown result in decreased proliferation and differentiation. Black-Right-Pointing-Pointer Increased HP1{gamma} immunoreactivity after NEIL3 knockdown suggests premature senescence. -- Abstract: Embryonic neural stem cells (NSCs) exhibit self-renewal and multipotency as intrinsic characteristics that are key parameters for proper brain development. When cells are challenged by oxidative stress agents the resulting DNA lesions are repaired by DNA glycosylases through the base excision repair (BER) pathway as a means to maintain the fidelity of the genome, and thus, proper cellular characteristics. The functional roles for DNA glycosylases in NSCs have however remained largely unexplored. Here we demonstrate that RNA knockdown of the DNA glycosylases OGG1 and NEIL3 decreased NSC differentiation ability and resulted in decreased expression of both neuronal and astrocytic genes after mitogen withdrawal, as well as the stem cell marker Musashi-1. Furthermore, while cell survival remained unaffected, NEIL3 deficient cells displayed decreased cell proliferation rates along with an increase in HP1{gamma} immunoreactivity, a sign of premature senescence. Our results suggest that DNA glycosylases play multiple roles in governing essential neural stem cell characteristics.

  11. Photoenzyme probes of photodamage to cells and cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, B. M.

    1979-01-01

    Development of photoenzyme probes for detection of ultraviolet damage to cells and DNA is reviewed with special emphasis on a process using polyethylene glycol to induce cell fusion. Polyethylene glycol is easy to obtain and handle, is gentle to the cells and does not induce latent or productive virus infection; therefore, it may be a general method for insertion of exogenous enzymes into mammalian cells. (PCS)

  12. Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division.

    Science.gov (United States)

    Oliva, María A

    2016-01-01

    Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex. PMID:27668216

  13. Viability of human adenovirus from hospital fomites.

    Science.gov (United States)

    Ganime, Ana Carolina; Carvalho-Costa, Filipe A; Santos, Marisa; Costa Filho, Rubens; Leite, José Paulo G; Miagostovich, Marize P

    2014-12-01

    The monitoring of environmental microbial contamination in healthcare facilities may be a valuable tool to determine pathogens transmission in those settings; however, such procedure is limited to bacterial indicators. Viruses are found commonly in those environments and are rarely used for these procedures. The aim of this study was to assess distribution and viability of a human DNA virus on fomites in an Adult Intensive Care Unit of a private hospital in Rio de Janeiro, Brazil. Human adenoviruses (HAdV) were investigated in 141 fomites by scraping the surface area and screening by quantitative PCR (qPCR) using TaqMan® System (Carlsbad, CA). Ten positive samples were selected for virus isolation in A549 and/or HEp2c cell lines. A total of 63 samples (44.7%) were positive and presented viral load ranging from 2.48 × 10(1) to 2.1 × 10(3) genomic copies per millilitre (gc/ml). The viability was demonstrated by integrated cell culture/nested-PCR in 5 out of 10 samples. Nucleotide sequencing confirmed all samples as HAdV and characterized one of them as specie B, serotype 3 (HAdV-3). The results indicate the risk of nosocomial transmission via contaminated fomites and point out the use of HAdV as biomarkers of environmental contamination.

  14. Sulforaphane induces DNA single strand breaks in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sestili, Piero, E-mail: piero.sestili@uniurb.it [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Paolillo, Marco [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Lenzi, Monia [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy); Colombo, Evelin; Vallorani, Luciana; Casadei, Lucia; Martinelli, Chiara [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Fimognari, Carmela [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy)

    2010-07-07

    Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1-3 h treatments with 10-30 {mu}M SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC-/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which - since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents - might help to exploit the therapeutic value

  15. Chromatin factors affecting DNA repair in mammalian cell nuclei

    International Nuclear Information System (INIS)

    We are investigating chromatin factors that participate in the incision step of DNA repair in eukaryotic cells. Localization of repair activity within nuclei, the stability and extractability of activity, the specificity for recognizing damage in chromatin or purified DNA as substrates are of interest in this investigation of human cells, CHO cells, and their radiation sensitive mutants. We have developed procedures that provide nuclei in which their DNA behaves as a collection of circular molecules. The integrity of the DNA in human nuclei can be maintained during incubation in appropriate buffers for as long as 60 minutes. When cells or nuclei are exposed to uv light prior to incubation, incisions presumably associated with DNA repair can be demonstrated. Incision activity is stable to prior extraction of nuclei with 0.6 M NaCl, which removes many nonhistone proteins. Our studies are consistent with an hypothesis that factors responsible for initiating DNA repair are localized in the nuclear matrix. 18 references, 3 figures

  16. Quantitative analysis of cell-free DNA in ovarian cancer

    Science.gov (United States)

    SHAO, XUEFENG; He, YAN; JI, MIN; CHEN, XIAOFANG; QI, JING; SHI, WEI; HAO, TIANBO; JU, SHAOQING

    2015-01-01

    The aim of the present study was to investigate the association between cell-free DNA (cf-DNA) levels and clinicopathological characteristics of patients with ovarian cancer using a branched DNA (bDNA) technique, and to determine the value of quantitative cf-DNA detection in assisting with the diagnosis of ovarian cancer. Serum specimens were collected from 36 patients with ovarian cancer on days 1, 3 and 7 following surgery, and additional serum samples were also collected from 22 benign ovarian tumor cases, and 19 healthy, non-cancerous ovaries. bDNA techniques were used to detect serum cf-DNA concentrations. All data were analyzed using SPSS version 18.0. The cf-DNA levels were significantly increased in the ovarian cancer group compared with those of the benign ovarian tumor group and healthy ovarian group (P<0.01). Furthermore, cf-DNA levels were significantly increased in stage III and IV ovarian cancer compared with those of stages I and II (P<0.01). In addition, cf-DNA levels were significantly increased on the first day post-surgery (P<0.01), and subsequently demonstrated a gradual decrease. In the ovarian cancer group, the area under the receiver operating characteristic curve of cf-DNA and the sensitivity were 0.917 and 88.9%, respectively, which was higher than those of cancer antigen 125 (0.724, 75%) and human epididymis protein 4 (0.743, 80.6%). There was a correlation between the levels of serum cf-DNA and the occurrence and development of ovarian cancer in the patients evaluated. bDNA techniques possessed higher sensitivity and specificity than other methods for the detection of serum cf-DNA in patients exhibiting ovarian cancer, and bDNA techniques are more useful for detecting cf-DNA than other factors. Thus, the present study demonstrated the potential value for the use of bDNA as an adjuvant diagnostic method for ovarian cancer. PMID:26788153

  17. Live cell imaging reveals at novel view of DNA

    International Nuclear Information System (INIS)

    Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) that are the most severe form of DNA damages. Recently, live cell imaging techniques coupled with laser micro-irradiation were used to analyze the spatio-temporal behavior of the NHEJ core factors upon DSB induction in living cells. Based on the live cell imaging studies, we proposed a novel two-phase model for DSB sensing and protein assembly in the NHEJ pathway. This new model provides a novel view of the dynamic protein behavior on DSBs and broad implications for the molecular mechanism of NHEJ. (author)

  18. Silver nanoparticle induced cytotoxicity, oxidative stress, and DNA damage in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Awasthi, Kumud Kant [University of Rajasthan, Department of Zoology (India); Awasthi, Anjali; Kumar, Narender; Roy, Partha [Indian Institute of Technology Roorkee, Department of Biotechnology (India); Awasthi, Kamlendra, E-mail: kamlendra.awasthi@gmail.com [Malaviya National Institute of Technology, Department of Physics (India); John, P. J., E-mail: placheriljohn@yahoo.com [University of Rajasthan, Department of Zoology (India)

    2013-09-15

    Silver nanoparticles (Ag NPs) are being used increasingly in wound dressings, catheters, and in various household products due to their antimicrobial activity. The present study reports the toxicity evaluation of synthesized and well characterized Ag NPs using Chinese hamster ovary (CHO) cells. The UV-Vis spectroscopy reveals the formation of silver nanoparticles by exhibiting the typical surface plasmon absorption maxima at 408-410 nm. Transmission electron microscopy (TEM) reveals that the average diameter of silver nanoparticles is about 5.0 {+-} 1.0 nm and that they have spherical shape. Cell visibility and cell viability percentage show dose-dependent cellular toxicity of Ag NPs. The half maximal inhibitory concentration (IC{sub 50}) for CHO cells is 68.0 {+-} 2.65 {mu}g/ml after 24 h Ag NPs exposure. Toxicity evaluations, including cellular morphology, mitochondrial function (MTT assay), reactive oxygen species (ROS), and DNA fragmentation assay (Ladder pattern) were assessed in unexposed CHO cells (control) and the cells exposed to Ag NPs concentrations of 15, 30, and 60 {mu}g/ml for 24 h. The findings may assist in the designing of Ag NPs for various applications and provide insights into their toxicity.

  19. Silver nanoparticle induced cytotoxicity, oxidative stress, and DNA damage in CHO cells

    International Nuclear Information System (INIS)

    Silver nanoparticles (Ag NPs) are being used increasingly in wound dressings, catheters, and in various household products due to their antimicrobial activity. The present study reports the toxicity evaluation of synthesized and well characterized Ag NPs using Chinese hamster ovary (CHO) cells. The UV–Vis spectroscopy reveals the formation of silver nanoparticles by exhibiting the typical surface plasmon absorption maxima at 408–410 nm. Transmission electron microscopy (TEM) reveals that the average diameter of silver nanoparticles is about 5.0 ± 1.0 nm and that they have spherical shape. Cell visibility and cell viability percentage show dose-dependent cellular toxicity of Ag NPs. The half maximal inhibitory concentration (IC50) for CHO cells is 68.0 ± 2.65 μg/ml after 24 h Ag NPs exposure. Toxicity evaluations, including cellular morphology, mitochondrial function (MTT assay), reactive oxygen species (ROS), and DNA fragmentation assay (Ladder pattern) were assessed in unexposed CHO cells (control) and the cells exposed to Ag NPs concentrations of 15, 30, and 60 μg/ml for 24 h. The findings may assist in the designing of Ag NPs for various applications and provide insights into their toxicity

  20. Scintillometric determination of DNA repair in human cell lines

    International Nuclear Information System (INIS)

    The ability of a variety of chemical and physical agents to stimulate DNA repair synthesis in human cell cultures was tested by a simplified scintillometric procedure, with the use of hydroxyurea (HU) to suppress DNA replicative synthesis. After incubation with [3H]thymidine, the radioactivity incorporated into DNA was determined in controls (C) and treated (T) cultures and in the corresponding HU series (Csub(HU), Tsub(HU)). The ratios Tsub(HU)/Csub(HU) and Tsub(HU)/T:Csub(HU)/C, indicating absolute and relative increases of DNA radioactivity, were calculated. When both ratios were significantly higher than 1, they were taken as indices of DNA repair stimulation. (orig./AJ)

  1. Drosophila Ctf4 is essential for efficient DNA replication and normal cell cycle progression

    Directory of Open Access Journals (Sweden)

    Christensen Tim W

    2011-04-01

    Full Text Available Abstract Background Proper coordination of the functions at the DNA replication fork is vital to the normal functioning of a cell. Specifically the precise coordination of helicase and polymerase activity is crucial for efficient passage though S phase. The Ctf4 protein has been shown to be a central member of the replication fork and links the replicative MCM helicase and DNA polymerase α primase. In addition, it has been implicated as a member of a complex that promotes replication fork stability, the Fork Protection Complex (FPC, and as being important for sister chromatid cohesion. As such, understanding the role of Ctf4 within the context of a multicellular organism will be integral to our understanding of its potential role in developmental and disease processes. Results We find that Drosophila Ctf4 is a conserved protein that interacts with members of the GINS complex, Mcm2, and Polymerase α primase. Using in vivo RNAi knockdown of CTF4 in Drosophila we show that Ctf4 is required for viability, S phase progression, sister chromatid cohesion, endoreplication, and coping with replication stress. Conclusions Ctf4 remains a central player in DNA replication. Our findings are consistent with what has been previously reported for CTF4 function in yeast, Xenopus extracts, and human tissue culture. We show that Ctf4 function is conserved and that Drosophila can be effectively used as a model to further probe the precise function of Ctf4 as a member of the replication fork and possible roles in development.

  2. Toxicity DNA damage and inhibition of DNA repair synthesis in human melanoma cells by concentrated sunlight

    International Nuclear Information System (INIS)

    A water lens was used to focus solar radiation, giving an 8-fold concentration of the total spectrum and a cytocidal flux similar to that of laboratory UV sources. Survival curves for human melanoma cells were similar for sunlight and 254 nm UV. An xeroderma pigmentosum lymphoblastoid line was equally sensitive to both agents and human cell lines sensitive to ionizing radiation (lymphoblastoid lines), crosslinking agents or monofunctional alkylating agents (melanoma lines) had the same 254 nm UV and solar survival responses as appropriate control lines. Two melanoma sublines derived separately by 16 cycles of treatment with sunlight or 254 nm UV were crossresistant to both agents. In one melanoma cell line, DNA strand breaks and DNA protein crosslinking were induced in melanoma cells by sunlight but pyrimidine dimers and DNA interstrand crosslinking could not be detected. The solar fluence response of DNA repair synthesis was much less than that from equitoxic 254 nm UV, reaching a maximum near the D0 value and then declining; but semiconservative DNA synthesis remained high. These effects were not due to changes in thymidine pool sizes. Solar exposure did not have a major effect on 254 nm UV-induced repair synthesis. (author)

  3. Effect of Tinospora cordifolia on the reduction of ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells.

    Science.gov (United States)

    Masuma, Runa; Okuno, Tsutomu; Kabir Choudhuri, Mohammad Shahabuddin; Saito, Takeshi; Kurasaki, Masaaki

    2014-01-01

    The safety of Tinospora cordifolia and its potential to protect against ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells were investigated. To evaluate the safety of T. cordifolia, cell viability and agarose gel electrophoresis were carried out using PC12 cells treated with 0 to 100 μg mL(-1) of methanol extract of T. cordifolia. T. cordifolia extracts did not show cytotoxicity ranging 0 to 100 μg mL(-1). In addition, T. cordifolia extracts significantly increased cell viability at 1 ng, 10 ng and 1 μg mL(-1) concentrations in serum-deprived medium compared to control. To confirm the protective role against UV-induced damage, PC12 cells alone or in the presence of 10 ng, 100 ng, or 1 μg mL(-1) of T. cordifolia extract were exposed to 250, 270 and 290 nm of UV radiation, which corresponded to doses of 120, 150 and 300 mJ cm(-2), respectively. Treatment with T. cordifolia extracts significantly increased the cell survival rate irradiated at 290 nm. In addition, T. cordifolia extracts significantly reduced cyclobutane pyrimidine dimer formation induced by UV irradiation at all wavelengths. In conclusion, T. cordifolia is not toxic and safe for cells. Our findings can support its application as phototherapy in the medical sector. PMID:24762179

  4. Lipid nanocapsules functionalized with polyethyleneimine for plasmid DNA and drug co-delivery and cell imaging

    Science.gov (United States)

    Skandrani, Nadia; Barras, Alexandre; Legrand, Dominique; Gharbi, Tijani; Boulahdour, Hatem; Boukherroub, Rabah

    2014-06-01

    The paper reports on the preparation of lipid nanocapsules (LNCs) functionalized with poly(ethyleneimine) (PEI) moieties and their successful use as drug and gene delivery systems. The cationic LNCs were produced by a phase inversion process with a nominal size of 25 nm and subsequently modified with PEI chains using a transacylation reaction. The functionalization process allowed good control over the nanoscale particle size (26.2 +/- 3.9 nm) with monodisperse size characteristics (PI < 0.2) and positive surface charge up to +18.7 mV. The PEI-modified LNCs (LNC25-T) displayed good buffering capacity. Moreover, the cationic LNC25-T were able to condense DNA and form complexes via electrostatic interactions in a typical weight ratio-dependent relationship. It was found that the mean diameter of LNC25-T/pDNA complexes increased to ~40-50 nm with the LNC25-T/pDNA ratio from 1 to 500. Gel electrophoresis and cell viability experiments showed that the LNC25-T/pDNA complexes had high stability with no cytotoxicity due to the anchored PEI polymers on the surface of LNCs. Finally, the transfection efficiency of the LNC25-T/pDNA complexes was studied and evaluated on HEK cell lines in comparison with free PEI/pDNA polyplexes. The combination of cationic LNCs with pDNA exhibited more than a 2.8-fold increase in transfection efficiency compared to the standard free PEI/pDNA polyplexes at the same PEI concentrations. Moreover, we have demonstrated that LNC25-T/pDNA loaded with a hydrophobic drug, paclitaxel, showed high drug efficacy. The high transfection efficiency combined with the potential of simultaneous co-delivery of hydrophobic drugs, relatively small size of LNC25-T/pDNA complexes, and fluorescence imaging can be crucial for gene therapy, as small particle sizes may be more favorable for in vivo studies.The paper reports on the preparation of lipid nanocapsules (LNCs) functionalized with poly(ethyleneimine) (PEI) moieties and their successful use as drug and gene

  5. A novel Atoh1 "self-terminating" mouse model reveals the necessity of proper Atoh1 level and duration for hair cell differentiation and viability.

    Directory of Open Access Journals (Sweden)

    Ning Pan

    Full Text Available Atonal homolog1 (Atoh1 is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO mouse line using Tg(Atoh1-cre, in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to "self-terminate" its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.

  6. Study of DNA uptake locations in single E. coli cells

    Science.gov (United States)

    Xu, C. Shan; Meadow Anderson, L.; Yang, Haw

    2006-03-01

    Artificial gene transfer of bacteria, such as E. coli, has become the main stream technique in genetic engineering and molecular cell biology studies. In spite of the great improvements in transformation efficiency, some fundamental questions remained to be answered. For instance, what are the DNA uptake channels and how do they form and function under external stimuli? Furthermore, where are these channels located on the cell membrane? Here we report a study aimed at DNA uptake locations in the two widely used gene transformation techniques: electroporation and heat shock. A direct visualization of the settling location of single DNA molecules inside individual E. coli cells was obtained by fluorescence imaging and spectroscopy. Electroporation and heat shock exhibit two distinct characteristics of DNA uptake locations. A preferential distribution toward cell poles during electroporation is consistent with earlier experiments and previously proposed models. However, the result from heat shock is unanticipated in which the majority of DNA enters the cell near the center. Such observation suggests that uptake channels form preferentially where newly-synthesized membrane is located under cation and low temperature treatment

  7. Mitochondrial DNA mutations in oxyphilic and chief cell parathyroid adenomas

    Directory of Open Access Journals (Sweden)

    Roth Sanford I

    2007-10-01

    Full Text Available Abstract Background The potential pathogenetic significance of mitochondrial DNA (mtDNA mutations in tumorigenesis is controversial. We hypothesized that benign tumorigenesis of a slowly replicating tissue like the human parathyroid might constitute an especially fertile ground on which a selective advantage conferred by mtDNA mutation could be manifested and might contribute to the oxyphilic phenotype observed in a subset of parathyroid tumors. Methods We sought acquired mitochondrial DNA mutations by sequencing the entire 16.6 kb mitochondrial genome of each of thirty sporadic parathyroid adenomas (18 chief cell and 12 oxyphil cell, eight independent, polyclonal, parathyroid primary chief cell hyperplasias plus corresponding normal control samples, five normal parathyroid glands, and one normal thyroid gland. Results Twenty-seven somatic mutations were identified in 15 of 30 (9 of 12 oxyphil adenomas, 6 of 18 chief cell parathyroid adenomas studied. No somatic mutations were observed in the hyperplastic parathyroid glands. Conclusion Features of the somatic mutations suggest that they may confer a selective advantage and contribute to the molecular pathogenesis of parathyroid adenomas. Importantly, the statistically significant differences in mutation prevalence in oxyphil vs. chief cell adenomas also suggest that mtDNA mutations may contribute to the oxyphil phenotype.

  8. Continuous cytokine exposure of colonic epithelial cells induces DNA damage

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    Chronic inflammatory diseases of the intestinal tract are associated with an increased risk of colorectal cancer. As an example ulcerative colitis (UC) is associated with a production of reactive oxygen species (ROS), including nitrogen monoxide (NO), which is produced in high amounts by inducibl...... nitrogen oxide synthase (iNOS). NO as well as other ROS are potential DNA damaging agents. The aim was to determine the effect of long-term cytokine exposure on NO formation and DNA damage in epithelial cells....

  9. Circulating Cell Free DNA in the Diagnosis of Trophoblastic Tumors

    OpenAIRE

    Openshaw, Mark R.; Harvey, Richard A.; Sebire, Neil J; Baljeet Kaur; Naveed Sarwar; Michael J Seckl; Fisher, Rosemary A.

    2015-01-01

    Gestational trophoblastic neoplasia (GTN) represents a group of diseases characterized by production of human chorionic gonadotropin (hCG). Since non-gestational tumors may occasionally secrete hCG, histopathological diagnosis is important for appropriate clinical management. However, a histopathological diagnosis is not always available. We therefore investigated the feasibility of extracting cell free DNA (cfDNA) from the plasma of women with GTN for use as a “liquid biopsy” in patients wit...

  10. [Profiles of DNA methylation in normal and cancer cells].

    Science.gov (United States)

    Weber, Michaël

    2008-01-01

    In eukaryotes, the epigenetic mark DNA methylation is found exclusively at cytosine residues in the CpG islands of genes, transposons and intergenic DNA. Among functional roles, DNA methylation is essential for mammalian embryonic development, and is classically thought to function by stably silencing promoter activity. However, until recently, understanding of the distribution of cytosine methylation in the whole genome - and hence, identification of its targets - was very limited. High-throughput methodologies, including methylated DNA immunoprecipitation, have recently revealed genome-wide mapping of DNA methylation, and provided new and unexpected data. Clearly DNA methylation is selectively associated with some key promoters- and is not a prerequisite for promoter inactivation, since strong CpG island promoters are mostly unmethylated, even when inactive. Most germline-specific genes are methylated and permanently silenced in somatic cells, suggesting a role of this mark in maintaining somatic cellular identity. These large scale studies will also help understanding the deregulation of DNA methylation associated with cancer, among which unmethylation of germinal cells genes, and recent observtion of large hypomethylated regions in tumoral specimens. The next challenge will be to understand if these methylation changes occur randomly, or more likely are specified by oncogenes or linked to environmental pressure. PMID:18789220

  11. EGFR-targeted plasmonic magnetic nanoparticles suppress lung tumor growth by abrogating G2/M cell-cycle arrest and inducing DNA damage

    Directory of Open Access Journals (Sweden)

    Kuroda S

    2014-08-01

    Full Text Available Shinji Kuroda,1 Justina Tam,2 Jack A Roth,1 Konstantin Sokolov,2 Rajagopal Ramesh3–5 1Department of Thoracic and Cardiovascular Surgery, 2Department of Imaging Physics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 3Department of Pathology, 4Graduate Program in Biomedical Sciences, 5Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Background: We have previously demonstrated the epidermal growth factor receptor (EGFR-targeted hybrid plasmonic magnetic nanoparticles (225-NP produce a therapeutic effect in human lung cancer cell lines in vitro. In the present study, we investigated the molecular mechanism of 225-NP-mediated antitumor activity both in vitro and in vivo using the EGFR-mutant HCC827 cell line. Methods: The growth inhibitory effect of 225-NP on lung tumor cells was determined by cell viability and cell-cycle analysis. Protein expression related to autophagy, apoptosis, and DNA-damage were determined by Western blotting and immunofluorescence. An in vivo efficacy study was conducted using a human lung tumor xenograft mouse model. Results: The 225-NP treatment markedly reduced tumor cell viability at 72 hours compared with the cell viability in control treatment groups. Cell-cycle analysis showed the percentage of cells in the G2/M phase was reduced when treated with 225-NP, with a concomitant increase in the number of cells in Sub-G1 phase, indicative of cell death. Western blotting showed LC3B and PARP cleavage, indicating 225-NP-treatment activated both autophagy- and apoptosis-mediated cell death. The 225-NP strongly induced γH2AX and phosphorylated histone H3, markers indicative of DNA damage and mitosis, respectively. Additionally, significant γH2AX foci formation was observed in 225-NP-treated cells compared with control treatment groups, suggesting 225-NP induced cell death by triggering DNA damage. The 225-NP-mediated DNA damage involved abrogation of the

  12. Differential sensitivity to aphidicolin of replicative DNA synthesis and ultraviolet-induced unscheduled DNA synthesis in vivo in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Seki,Shuji

    1984-06-01

    Full Text Available In vivo in mammalian cells, ultraviolet-induced unscheduled DNA synthesis was less sensitive to aphidicolin than was replicative DNA synthesis. Replicative DNA synthesis in HeLa, HEp-2, WI-38 VA-13 and CV-1 cells was inhibited more than 97% by aphidicolin at 10 micrograms/ml, whereas aphidicolin inhibition of DNA synthesis in ultraviolet-irradiated cells varied between 30% and 90% depending on cell types and assay conditions. Aphidicolin inhibition of unscheduled DNA synthesis (UDS in HeLa cells increased gradually with increasing aphidicolin concentration and reached approximately 90% at 100 micrograms/ml aphidicolin. A significant fraction of UDS in ultraviolet-irradiated HEp-2 cells was resistant to aphidicolin even at 300 micrograms/ml. Considered along with related information reported previously, the present results suggest that both aphidicolin-sensitive and insensitive DNA polymerases, DNA polymerase alpha and a non-alpha DNA polymerase (possibly DNA polymerase beta, are involved in in situ UDS in these ultraviolet-irradiated cells. Comparison of staphylococcal nuclease sensitivity between DNAs repaired in the presence and in the absence of aphidicolin in HEp-2 cells suggested that the involvement of DNA polymerase alpha in UDS favored DNA synthesis in the intranucleosomal region.

  13. [Transfer of T-DNA from agrobacteria into plant cells through cell walls and membranes].

    Science.gov (United States)

    Chumakov, M I

    2001-01-01

    Discusses probable routes of agrobacterial penetration through the plant integumental tissues, cell wall, and plant cell plasmodesma. Analyzes the contribution of extracellular structures of agrobacteria in penetration through barriers of a plant cell, primary contact (adhesion), and during DNA transfer from bacterial (E. coli, A. tumefaciens) to recipient (bacterial or plant) cells. Discusses the relationship between donor cell adhesion to recipient cell surface and the infectious and conjugation processes. Considers the probable role of piles in conjugative transfer of agrobacterial DNA through membranes of donor and recipient (bacterial and plant) cells. Analyzes the contribution of the plant cell cytoskeleton to T-DNA transfer. Suggests a model of transport of T-DNA-VirD2 complex and VirE2 proteins through independent channels consisting of vir-coded proteins. PMID:11236737

  14. Troglitazone, but not rosiglitazone, damages mitochondrial DNA and induces mitochondrial dysfunction and cell death in human hepatocytes

    International Nuclear Information System (INIS)

    Thiazolidinediones (TZDs), such as troglitazone (TRO) and rosiglitazone (ROSI), improve insulin resistance by acting as ligands for the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ). TRO was withdrawn from the market because of reports of serious hepatotoxicity. A growing body of evidence suggests that TRO caused mitochondrial dysfunction and induction of apoptosis in human hepatocytes but its mechanisms of action remain unclear. We hypothesized that damage to mitochondrial DNA (mtDNA) is an initiating event involved in TRO-induced mitochondrial dysfunction and hepatotoxicity. Primary human hepatocytes were exposed to TRO and ROSI. The results obtained revealed that TRO, but not ROSI at equimolar concentrations, caused a substantial increase in mtDNA damage and decreased ATP production and cellular viability. The reactive oxygen species (ROS) scavenger, N-acetyl cystein (NAC), significantly diminished the TRO-induced cytotoxicity, suggesting involvement of ROS in TRO-induced hepatocyte cytotoxicity. The PPARγ antagonist (GW9662) did not block the TRO-induced decrease in cell viability, indicating that the TRO-induced hepatotoxicity is PPARγ-independent. Furthermore, TRO induced hepatocyte apoptosis, caspase-3 cleavage and cytochrome c release. Targeting of a DNA repair protein to mitochondria by protein transduction using a fusion protein containing the DNA repair enzyme Endonuclease III (EndoIII) from Escherichia coli, a mitochondrial translocation sequence (MTS) and the protein transduction domain (PTD) from HIV-1 TAT protein protected hepatocytes against TRO-induced toxicity. Overall, our results indicate that significant mtDNA damage caused by TRO is a prime initiator of the hepatoxicity caused by this drug.

  15. The DNA methylation profile of activated human natural killer cells.

    Science.gov (United States)

    Wiencke, John K; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A; Siegel, Derick; Houseman, E Andres; Kelsey, Karl T

    2016-05-01

    Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states. PMID:26967308

  16. DNA mismatch repair efficiency and fidelity are elevated during DNA synthesis in human cells

    International Nuclear Information System (INIS)

    DNA mismatch repair (MMR) within human cells is hypothesized to occur primarily at the replication fork. However, experimental models measuring MMR activity at specific phases of the cell cycle and during genomic DNA synthesis are lacking. We have investigated MMR activity within the nuclear environment of HeLa cells after enriching for G1, S and G2/M phase of the cell cycle by centrifugal elutriation. This approach preserves physiologically normal MMR activity in cell populations subdivided into different phases of the cell cycle. Here we have shown that nuclear protein concentration of hMutSα and hMutLα increases as cells progress into S phase during routine cell culture. MMR activity, as measured by both in vitro and in vivo approaches, increases during S phase to the highest extent within normally growing cells. Both fidelity and activity of MMR are highest on actively replicating templates within intact cells during S phase. The MMR pathway however, is also active at lower levels at other phases of the cell cycle, and on nonreplicating templates

  17. Cellular aging of mitochondrial DNA-depleted cells

    International Nuclear Information System (INIS)

    We have reported that mitochondrial DNA-depleted ρ0 cells are resistant to cell death. Because aged cells have frequent mitochondrial DNA mutations, the resistance of ρ0 cells against cell death might be related to the apoptosis resistance of aged cells and frequent development of cancers in aged individuals. We studied if ρ0 cells have features simulating aged cells. SK-Hep1 hepatoma ρ0 cells showed typical morphology associated with aging such as increased size and elongated appearance. They had increased senescence-associated β-Gal activity, lipofuscin pigment, and plasminogen activator inhibitor-1 expression. Consistent with their decreased proliferation, the expression of mitotic cyclins was decreased and that of cdk inhibitors was increased. Rb hypophosphorylation and decreased telomerase activity were also noted. Features simulating aged cells were also observed in MDA-MB-435 ρ0 cells. These results support the mitochondrial theory of aging, and suggest that ρ0 cells could serve as an in vitro model for aged cells

  18. Tumor-Related Methylated Cell-Free DNA and Circulating Tumor Cells in Melanoma

    OpenAIRE

    Salvianti, Francesca; Orlando, Claudio; Massi, Daniela; DE GIORGI, VINCENZO; Grazzini, Marta; Pazzagli, Mario; Pinzani, Pamela

    2016-01-01

    Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma. The aim of the pres...

  19. Toxic effect of silica nanoparticles on endothelial cells through DNA damage response via Chk1-dependent G2/M checkpoint.

    Directory of Open Access Journals (Sweden)

    Junchao Duan

    Full Text Available Silica nanoparticles have become promising carriers for drug delivery or gene therapy. Endothelial cells could be directly exposed to silica nanoparticles by intravenous administration. However, the underlying toxic effect mechanisms of silica nanoparticles on endothelial cells are still poorly understood. In order to clarify the cytotoxicity of endothelial cells induced by silica nanoparticles and its mechanisms, cellular morphology, cell viability and lactate dehydrogenase (LDH release were observed in human umbilical vein endothelial cells (HUVECs as assessing cytotoxicity, resulted in a dose- and time- dependent manner. Silica nanoparticles-induced reactive oxygen species (ROS generation caused oxidative damage followed by the production of malondialdehyde (MDA as well as the inhibition of superoxide dismutase (SOD and glutathione peroxidase (GSH-Px. Both necrosis and apoptosis were increased significantly after 24 h exposure. The mitochondrial membrane potential (MMP decreased obviously in a dose-dependent manner. The degree of DNA damage including the percentage of tail DNA, tail length and Olive tail moment (OTM were markedly aggravated. Silica nanoparticles also induced G2/M arrest through the upregulation of Chk1 and the downregulation of Cdc25C, cyclin B1/Cdc2. In summary, our data indicated that the toxic effect mechanisms of silica nanoparticles on endothelial cells was through DNA damage response (DDR via Chk1-dependent G2/M checkpoint signaling pathway, suggesting that exposure to silica nanoparticles could be a potential hazards for the development of cardiovascular diseases.

  20. DNA precursor compartmentation in mammalian cells: metabolic and antimetabolic studies of nuclear and mitochondrial DNA synthesis

    International Nuclear Information System (INIS)

    HeLa cells were used for the quantitation of cellular and mitochondrial deoxyribonucleoside triphosphate (dNTP) and ribonucleoside triphosphate (rNTP) pools and of changes in pools in response to treatment with the antimetabolites methotrexate (mtx) and 5-fluorodeoxyuridine (FUdR). Use of an enzymatic assay of dNTPs and of improved nucleotide extraction methods allowed quantitation of mitochondrial dNTP pools. All four mitochondrial dNTP pools expand following treatment with mtx or FUdR whereas cellular dTTP and dGTP pools are depleted. Mitochrondrial rNTP pools were also found to expand in response to these antimetabolites. Mouse L-cells were used to determine the relative contributions of an exogenously supplied precursor to nuclear and mitochrondrial DNA replication. Cells were labeled to near steady state specific activities with 32P-orthophosphate and subsequently labeled with [3H]uridine, a general pyrimidine precursor, in the continuing presence of 32P. Deoxyribonucleoside monophosphates derived from these DNAs were separated by HPLC and the 3H/32P ratio in each pyrimidine determined. The dCMP residues in mitochondrial DNA (mtDNA) were found to be derived exclusively from the exogenous supplied uridine. The dTMP residues from nuclear and mtDNA and the dCMP residues from nuclear DNA were seen to be synthesized partly from exogenous sources and partly from other sources, presumably de novo pyrimidine synthesis

  1. 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 appl

  2. Epigenetic DNA Demethylation Causes Inner Ear Stem Cell Differentiation into Hair Cell-Like Cells

    Science.gov (United States)

    Zhou, Yang; Hu, Zhengqing

    2016-01-01

    The DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-aza) causes genomic demethylation to regulate gene expression. However, it remains unclear whether 5-aza affects gene expression and cell fate determination of stem cells. In this study, 5-aza was applied to mouse utricle sensory epithelia-derived progenitor cells (MUCs) to investigate whether 5-aza stimulated MUCs to become sensory hair cells. After treatment, MUCs increased expression of hair cell genes and proteins. The DNA methylation level (indicated by percentage of 5-methylcytosine) showed a 28.57% decrease after treatment, which causes significantly repressed DNMT1 protein expression and DNMT activity. Additionally, FM1-43 permeation assays indicated that the permeability of 5-aza-treated MUCs was similar to that of sensory hair cells, which may result from mechanotransduction channels. This study not only demonstrates a possible epigenetic approach to induce tissue specific stem/progenitor cells to become sensory hair cell-like cells, but also provides a cell model to epigenetically modulate stem cell fate determination. PMID:27536218

  3. DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

    International Nuclear Information System (INIS)

    Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process. A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry. Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone. Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair

  4. The Cell Cycle Timing of Human Papillomavirus DNA Replication.

    Science.gov (United States)

    Reinson, Tormi; Henno, Liisi; Toots, Mart; Ustav, Mart; Ustav, Mart

    2015-01-01

    Viruses manipulate the cell cycle of the host cell to optimize conditions for more efficient viral genome replication. One strategy utilized by DNA viruses is to replicate their genomes non-concurrently with the host genome; in this case, the viral genome is amplified outside S phase. This phenomenon has also been described for human papillomavirus (HPV) vegetative genome replication, which occurs in G2-arrested cells; however, the precise timing of viral DNA replication during initial and stable replication phases has not been studied. We developed a new method to quantitate newly synthesized DNA levels and used this method in combination with cell cycle synchronization to show that viral DNA replication is initiated during S phase and is extended to G2 during initial amplification but follows the replication pattern of cellular DNA during S phase in the stable maintenance phase. E1 and E2 protein overexpression changes the replication time from S only to both the S and G2 phases in cells that stably maintain viral episomes. These data demonstrate that the active synthesis and replication of the HPV genome are extended into the G2 phase to amplify its copy number and the duration of HPV genome replication is controlled by the level of the viral replication proteins E1 and E2. Using the G2 phase for genome amplification may be an important adaptation that allows exploitation of changing cellular conditions during cell cycle progression. We also describe a new method to quantify newly synthesized viral DNA levels and discuss its benefits for HPV research. PMID:26132923

  5. The Cell Cycle Timing of Human Papillomavirus DNA Replication.

    Directory of Open Access Journals (Sweden)

    Tormi Reinson

    Full Text Available Viruses manipulate the cell cycle of the host cell to optimize conditions for more efficient viral genome replication. One strategy utilized by DNA viruses is to replicate their genomes non-concurrently with the host genome; in this case, the viral genome is amplified outside S phase. This phenomenon has also been described for human papillomavirus (HPV vegetative genome replication, which occurs in G2-arrested cells; however, the precise timing of viral DNA replication during initial and stable replication phases has not been studied. We developed a new method to quantitate newly synthesized DNA levels and used this method in combination with cell cycle synchronization to show that viral DNA replication is initiated during S phase and is extended to G2 during initial amplification but follows the replication pattern of cellular DNA during S phase in the stable maintenance phase. E1 and E2 protein overexpression changes the replication time from S only to both the S and G2 phases in cells that stably maintain viral episomes. These data demonstrate that the active synthesis and replication of the HPV genome are extended into the G2 phase to amplify its copy number and the duration of HPV genome replication is controlled by the level of the viral replication proteins E1 and E2. Using the G2 phase for genome amplification may be an important adaptation that allows exploitation of changing cellular conditions during cell cycle progression. We also describe a new method to quantify newly synthesized viral DNA levels and discuss its benefits for HPV research.

  6. Does 'Immortal DNA strand' exist in 'immortal' stem cells?

    Institute of Scientific and Technical Information of China (English)

    Linheng Li

    2007-01-01

    @@ Stem cells function to generate differentiated cells,and,at the same time,are maintained as‘immortal'cells through self-renewal.Accumulated evidence indicates that adult stem cells may be the most common precursor for cancers in adult mammalian tissue[1],and this concept is gaining increasing support[2,3].Meanwhile,this also raises the question of how stem cells,which have a much longer lifespan compared to other progenitor and mature ceils,can avoid accumulating mutations from DNA replica-tion errors.

  7. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells.

    Science.gov (United States)

    Yedjou, Clement G; Tchounwou, Hervey M; Tchounwou, Paul B

    2016-01-01

    In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO₃)₂] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO₃)₂ for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p rupture by Pb(NO₃)₂ compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05) in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO₃)₂ exposure significantly (p < 0.05) increased the proportion of caspase-3 positive cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO₃)₂ exposure caused cell cycle arrest at the G₀/G₁ checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO₃)₂ inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G₀/G₁ checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO₃)₂ exposure and its associated adverse health effects. PMID:26703663

  8. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

    Directory of Open Access Journals (Sweden)

    José J. Gaforio

    2011-10-01

    Full Text Available Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A or breast cancer cells (MDA-MB-231 and MCF7. We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  9. IL-1beta-Induced iNOS Expression, NO Release and Loss in Metabolic Cell Viability Are Resistant to Inhibitors of Ceramide Synthase and Sphingomyelinase in INS 832/13 Cells

    Directory of Open Access Journals (Sweden)

    Rajakrishnan Veluthakal

    2006-11-01

    Full Text Available Context Emerging evidence indicates regulatory roles for ceramide in the metabolic dysfunction of the islet beta cell. Recently, potential similarities between IL-1beta and ceramide on their effects on islet beta cell have been reported, including reduction in mitochondrial membrane potential and loss in metabolic cell viability.Objective Herein, we investigated whether IL-1beta-induced nitric oxide synthetase (iNOS expression, nitric oxide (NO release and loss in metabolic cell viability require ceramide biosynthesis either via the activation of sphingomyelinase or ceramide synthase.Setting Insulin-secreting INS 832/13 cells.Results We found that two structurally-distinct inhibitors of sphingomyelinase activation (e.g., 3-O-methylsphingomyelin or desipramine or ceramide biosynthesis inhib-itor (e.g., fumonisin failed to exert clear effects on IL-1beta-induced iNOS expression, NO release and loss in cell viability.Conclusions Taken together, our findings indicate that neither the sphingomyelinase nor the ceramide synthase activation is required for IL-1beta-induced metabolic abnormalities in insulin-secreting INS 832/13 cells.

  10. Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield.

    Directory of Open Access Journals (Sweden)

    Angela N Barrett

    Full Text Available OBJECTIVE: Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used. METHODS: Microfluidic digital PCR was performed to precisely quantify male (fetal DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA. Real-time qPCR was used to assay for the presence of male SRY signal in samples. RESULTS: Total cell-free DNA quantity increased significantly with time in samples stored in K(3EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4 °C did not prevent these changes. CONCLUSION: When samples can be processed within eight hours of blood draw, K(3EDTA tubes can be used. Prolonged transfer times in K(3EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests.

  11. Far-infrared radiation protects viability in a cell model of Spinocerebellar Ataxia by preventing polyQ protein accumulation and improving mitochondrial function

    Science.gov (United States)

    Chang, Jui-Chih; Wu, Shey-Lin; Hoel, Fredrik; Cheng, Yu-Shan; Liu, Ko-Hung; Hsieh, Mingli; Hoel, August; Tronstad, Karl Johan; Yan, Kuo-Chia; Hsieh, Ching-Liang; Lin, Wei-Yong; Kuo, Shou-Jen; Su, Shih-Li; Liu, Chin-San

    2016-01-01

    Far infrared radiation (FIR) is currently investigated as a potential therapeutic strategy in various diseases though the mechanism is unknown. Presently, we tested if FIR mediates beneficial effects in a cell model of the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3). SCA3 is caused by a mutation leading to an abnormal polyglutamine expansion (PolyQ) in ataxin-3 protein. The consequent aggregation of mutant ataxin-3 results in disruption of vital cell functions. In this study, neuroblastoma cells (SK-N-SH) was transduced to express either non-pathogenic ataxin-3-26Q or pathogenic ataxin-3-78Q proteins. The cells expressing ataxin-3-78Q demonstrated decreased viability, and increased sensitivity to metabolic stress in the presence rotenone, an inhibitor of mitochondrial respiration. FIR exposure was found to protect against these effects. Moreover, FIR improved mitochondrial respiratory function, which was significantly compromised in ataxin-3-78Q and ataxin-3-26Q expressing cells. This was accompanied by decreased levels of mitochondrial fragmentation in FIR treated cells, as observed by fluorescence microscopy and protein expression analysis. Finally, the expression profile LC3-II, Beclin-1 and p62 suggested that FIR prevent the autophagy inhibiting effects observed in ataxin-3-78Q expressing cells. In summary, our results suggest that FIR have rescuing effects in cells expressing mutated pathogenic ataxin-3, through recovery of mitochondrial function and autophagy. PMID:27469193

  12. Far-infrared radiation protects viability in a cell model of Spinocerebellar Ataxia by preventing polyQ protein accumulation and improving mitochondrial function.

    Science.gov (United States)

    Chang, Jui-Chih; Wu, Shey-Lin; Hoel, Fredrik; Cheng, Yu-Shan; Liu, Ko-Hung; Hsieh, Mingli; Hoel, August; Tronstad, Karl Johan; Yan, Kuo-Chia; Hsieh, Ching-Liang; Lin, Wei-Yong; Kuo, Shou-Jen; Su, Shih-Li; Liu, Chin-San

    2016-01-01

    Far infrared radiation (FIR) is currently investigated as a potential therapeutic strategy in various diseases though the mechanism is unknown. Presently, we tested if FIR mediates beneficial effects in a cell model of the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3). SCA3 is caused by a mutation leading to an abnormal polyglutamine expansion (PolyQ) in ataxin-3 protein. The consequent aggregation of mutant ataxin-3 results in disruption of vital cell functions. In this study, neuroblastoma cells (SK-N-SH) was transduced to express either non-pathogenic ataxin-3-26Q or pathogenic ataxin-3-78Q proteins. The cells expressing ataxin-3-78Q demonstrated decreased viability, and increased sensitivity to metabolic stress in the presence rotenone, an inhibitor of mitochondrial respiration. FIR exposure was found to protect against these effects. Moreover, FIR improved mitochondrial respiratory function, which was significantly compromised in ataxin-3-78Q and ataxin-3-26Q expressing cells. This was accompanied by decreased levels of mitochondrial fragmentation in FIR treated cells, as observed by fluorescence microscopy and protein expression analysis. Finally, the expression profile LC3-II, Beclin-1 and p62 suggested that FIR prevent the autophagy inhibiting effects observed in ataxin-3-78Q expressing cells. In summary, our results suggest that FIR have rescuing effects in cells expressing mutated pathogenic ataxin-3, through recovery of mitochondrial function and autophagy. PMID:27469193

  13. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells.

    Science.gov (United States)

    Yedjou, Clement G; Tchounwou, Hervey M; Tchounwou, Paul B

    2016-01-01

    In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO₃)₂] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO₃)₂ for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p cell death in Pb(NO₃)₂-treated cells, indicative of membrane rupture by Pb(NO₃)₂ compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO₃)₂ exposure caused cell cycle arrest at the G₀/G₁ checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO₃)₂ inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G₀/G₁ checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO₃)₂ exposure and its associated adverse health effects.

  14. Antimicrobial and cell viability measurement of bovine serum albumin capped silver nanoparticles (Ag/BSA) loaded collagen immobilized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film.

    Science.gov (United States)

    Bakare, Rotimi; Hawthrone, Samantha; Vails, Carmen; Gugssa, Ayele; Karim, Alamgir; Stubbs, John; Raghavan, Dharmaraj

    2016-03-01

    Bacterial infection of orthopedic devices has been a major concern in joint replacement procedures. Therefore, this study is aimed at formulating collagen immobilized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film loaded with bovine serum albumin capped silver nanoparticles (Ag/BSA NPs) to inhibit bacterial growth while retaining/promoting osteoblast cells viability. The nanoparticles loaded collagen immobilized PHBV film was characterized for its composition by X-ray Photoelectron Spectroscopy and Anodic Stripping Voltammetry. The extent of loading of Ag/BSA NPs on collagen immobilized PHBV film was found to depend on the chemistry of the functionalized PHBV film and the concentration of Ag/BSA NPs solution used for loading nanoparticles. Our results showed that more Ag/BSA NPs were loaded on higher molecular weight collagen immobilized PHEMA-g-PHBV film. Maximum loading of Ag/BSA NPs on collagen immobilized PHBV film was observed when 16ppm solution was used for adsorption studies. Colony forming unit and optical density measurements showed broad antimicrobial activity towards Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa at significantly lower concentration i.e., 0.19 and 0.31μg/disc, compared to gentamicin and sulfamethoxazole trimethoprim while MTT assay showed that released nanoparticles from Ag/BSA NPs loaded collagen immobilized PHBV film has no impact on MCTC3-E1 cells viability.

  15. Cryopreservation of Bone Marrow Mononuclear Cells Alters Their Viability and Subpopulation Composition but Not Their Treatment Effects in a Rodent Stroke Model

    Directory of Open Access Journals (Sweden)

    Bing Yang

    2016-01-01

    Full Text Available The systemic administration of autologous bone marrow (BM derived mononuclear cells (MNCs is under investigation as a novel therapeutic modality for the treatment of ischemic stroke. Autologous applications raise the possibility that MNCs could potentially be stored as a banked source. There have been no studies that investigate the effects of cryopreservation of BM-MNCs on their functional abilities in stroke models. In the present study, C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAo for 60 minutes and then divided into two treatment groups: fresh MNCs versus cryopreserved MNCs. BM-MNCs were collected at 22 hours after MCAo and were stored in liquid nitrogen for 12 months in cryopreserved MNCs group. BM-MNCs cellular viability, composition, and phenotype of the various subpopulations of mice BM-MNCs were evaluated by flow cytometry, and the behavioral recovery of stroke animals was tested with freshly harvested MNCs versus cryopreserved MNCs by corner test and ladder rung test. We found that long-term cryopreservation negatively impacts the cellular viability of bone marrow MNCs. Cryopreservation also alters the cellular composition of various subpopulations within the MNCs. However, despite the changes observed in cryopreserved cells, both fresh and frozen MNCs have similar beneficial effect on behavioral and histological outcomes.

  16. The DNA damage-induced cell death response: a roadmap to kill cancer cells.

    Science.gov (United States)

    Matt, Sonja; Hofmann, Thomas G

    2016-08-01

    Upon massive DNA damage cells fail to undergo productive DNA repair and trigger the cell death response. Resistance to cell death is linked to cellular transformation and carcinogenesis as well as radio- and chemoresistance, making the underlying signaling pathways a promising target for therapeutic intervention. Diverse DNA damage-induced cell death pathways are operative in mammalian cells and finally culminate in the induction of programmed cell death via activation of apoptosis or necroptosis. These signaling routes affect nuclear, mitochondria- and plasma membrane-associated key molecules to activate the apoptotic or necroptotic response. In this review, we highlight the main signaling pathways, molecular players and mechanisms guiding the DNA damage-induced cell death response. PMID:26791483

  17. Two New Faces of Amifostine: Protector from DNA Damage in Normal Cells and Inhibitor of DNA Repair in Cancer Cells.

    Science.gov (United States)

    Hofer, Michal; Falk, Martin; Komůrková, Denisa; Falková, Iva; Bačíková, Alena; Klejdus, Bořivoj; Pagáčová, Eva; Štefančíková, Lenka; Weiterová, Lenka; Angelis, Karel J; Kozubek, Stanislav; Dušek, Ladislav; Galbavý, Štefan

    2016-04-14

    Amifostine protects normal cells from DNA damage induction by ionizing radiation or chemotherapeutics, whereas cancer cells typically remain uninfluenced. While confirming this phenomenon, we have revealed by comet assay and currently the most sensitive method of DNA double strand break (DSB) quantification (based on γH2AX/53BP1 high-resolution immunofluorescence microscopy) that amifostine treatment supports DSB repair in γ-irradiated normal NHDF fibroblasts but alters it in MCF7 carcinoma cells. These effects follow from the significantly lower activity of alkaline phosphatase measured in MCF7 cells and their supernatants as compared with NHDF fibroblasts. Liquid chromatography-mass spectrometry confirmed that the amifostine conversion to WR-1065 was significantly more intensive in normal NHDF cells than in tumor MCF cells. In conclusion, due to common differences between normal and cancer cells in their abilities to convert amifostine to its active metabolite WR-1065, amifostine may not only protect in multiple ways normal cells from radiation-induced DNA damage but also make cancer cells suffer from DSB repair alteration. PMID:26978566

  18. cDNA expression cloning in mammalian cells.

    Science.gov (United States)

    Hoffman, B J

    2001-05-01

    This unit contains protocols for expression cloning in mammalian cells. Either calcium phosphate- or liposome-mediated transfection of mammalian cells, or virus infection and liposome-mediated transfection are used to screen pools derived from a cDNA library. cDNA pools are prepared for cloning from library-transformed E. coli grown in liquid culture medium or on antibiotic-containing selection plates. Results of screening assays for expression can be detected using autoradiography of dishes of cultured cells to identify clones, direct visualization of radiolabeled cells on emulsion-coated and developed chamber slides, detection and quantification of gene activity by a functional (transport) assay with scintillation counting, or detection using a filter-based assay for binding of radioligand to membranes or whole cells. The most critical step of any cDNA cloning project is the establishment of the screening protocol. Therefore, the bioassay for the gene product must be established prior to executing any of these protocols, including construction of the cDNA library. PMID:18428491

  19. Impaired DNA replication within progenitor cell pools promotes leukemogenesis.

    Directory of Open Access Journals (Sweden)

    Ganna Bilousova

    2005-12-01

    Full Text Available Impaired cell cycle progression can be paradoxically associated with increased rates of malignancies. Using retroviral transduction of bone marrow progenitors followed by transplantation into mice, we demonstrate that inhibition of hematopoietic progenitor cell proliferation impairs competition, promoting the expansion of progenitors that acquire oncogenic mutations which restore cell cycle progression. Conditions that impair DNA replication dramatically enhance the proliferative advantage provided by the expression of Bcr-Abl or mutant p53, which provide no apparent competitive advantage under conditions of healthy replication. Furthermore, for the Bcr-Abl oncogene the competitive advantage in contexts of impaired DNA replication dramatically increases leukemogenesis. Impaired replication within hematopoietic progenitor cell pools can select for oncogenic events and thereby promote leukemia, demonstrating the importance of replicative competence in the prevention of tumorigenesis. The demonstration that replication-impaired, poorly competitive progenitor cell pools can promote tumorigenesis provides a new rationale for links between tumorigenesis and common human conditions of impaired DNA replication such as dietary folate deficiency, chemotherapeutics targeting dNTP synthesis, and polymorphisms in genes important for DNA metabolism.

  20. G4-Tetra DNA Duplex Induce Lung Cancer Cell Apoptosis in A549 Cells

    Science.gov (United States)

    Xu, Xiaobo; Zhao, YiZhuo; Lu, Hu; Fu, Cuiping; Li, Xiao; Jiang, Liyan; Li, Shanqun

    2016-10-01

    The specific DNA is typically impermeable to the plasma membrane due to its natural characters, but DNA tetra structures (DTNs) can be readily uptake by cells in the absence of transfection agents, providing a new strategy to deliver DNA drugs. In this research, the delivery efficiency of tetrahedral DNA nanostructures was measured on adenocarcinomic human alveolar basal epithelial (A549) cells via delivering AS1411 (G4). The DNA tetra-AS1411 complex was rapidly and abundantly uptake by A549 cells, and the induced apoptosis was enhanced. Furthermore, biodistribution in mouse proved the rapid clearance from non-targeted organs in vivo. This study improved the understanding of potential function in DNA-based drug delivery and proved that DTNs-AS1411 could be potentially useful for the treatment of lung cancer.

  1. ATM-deficient human fibroblast cells are resistant to low levels of DNA double-strand break induced apoptosis and subsequently undergo drug-induced premature senescence

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jun; Jo, Yong Hwa; Cho, Chang Hoon; Choe, Wonchae; Kang, Insug; Baik, Hyung Hwan [Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of); Yoon, Kyung-Sik, E-mail: sky9999@khu.ac.kr [Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer A-T cells were not hypersensitive to low levels of DNA DSBs. Black-Right-Pointing-Pointer A-T cells have enhanced Akt but defect in activation of p53 and apoptotic proteins. Black-Right-Pointing-Pointer A-T cells underwent premature senescence after DNA damage accumulated. Black-Right-Pointing-Pointer Chemotherapeutic effect in cancer therapy may be associated with premature senescence. -- Abstract: DNA DSBs are induced by IR or radiomimetic drugs such as doxorubicin. It has been indicated that cells from ataxia-telangiectasia patients are highly sensitive to radiation due to defects in DNA repair, but whether they have impairment in apoptosis has not been fully elucidated. A-T cells showed increased sensitivity to high levels of DNA damage, however, they were more resistant to low doses. Normal cells treated with combination of KU55933, a specific ATM kinase inhibitor, and doxorubicin showed increased resistance as they do in a similar manner to A-T cells. A-T cells have higher viability but more DNA breaks, in addition, the activations of p53 and apoptotic proteins (Bax and caspase-3) were deficient, but Akt expression was enhanced. A-T cells subsequently underwent premature senescence after treatment with a low dose of doxorubicin, which was confirmed by G2 accumulation, senescent morphology, and SA-{beta}-gal positive until 15 days repair incubation. Finally, A-T cells are radio-resistant at low doses due to its defectiveness in detecting DNA damage and apoptosis, but the accumulation of DNA damage leads cells to premature senescence.

  2. Strategies for Implementing Cell-Free DNA Testing.

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    Cuckle, Howard

    2016-06-01

    Maternal plasma cell-free (cf) DNA testing has higher discriminatory power for aneuploidy than any conventional multi-marker screening test. Several strategies have been suggested for introducing it into clinical practice. Secondary cfDNA, restricted only to women with positive conventional screening test, is generally cost saving and minimizes the need for invasive prenatal diagnosis but leads to a small loss in detection. Primary cfDNA, replacing conventional screening or retaining the nuchal translucency scan, is not currently cost-effective for third-party payers. Contingent cfDNA, testing about 20% of women with the highest risks based on a conventional test, is the preferred approach. PMID:27235907

  3. Transplantation of progenitor cells after reperfused acute myocardial infarction: evaluation of perfusion and myocardial viability with FDG-PET and thallium SPECT

    Energy Technology Data Exchange (ETDEWEB)

    Doebert, Natascha; Berner, Uwe; Menzel, Christian; Hamscho, Nadja; Gruenwald, Frank [Department of Nuclear Medicine, University of Frankfurt (Germany); Britten, Martina; Assmus, Birgit; Lehmann, Ralf; Schaechinger, Volker; Zeiher, Andreas M. [Department of Cardiology, University of Frankfurt (Germany); Dimmeler, Stefanie [Department of Molecular Cardiology, University of Frankfurt (Germany)

    2004-08-01

    Clinical outcome after myocardial infarction depends on the extent of irreversibly damaged myocardium. Implantation of bone marrow-/circulating blood-derived progenitor cells has been shown to improve contractile cardiac function after myocardial infarction in both experimental and initial clinical studies. In the present study, first observations of the effect of local intracoronary progenitor cell infusion on the regeneration of infarcted cardiac tissue after acute myocardial infarction was evaluated by means of {sup 18}F-fluorodeoxyglucose positron emission tomography (PET) and {sup 201}Tl single-photon emission computed tomography (SPECT). Twenty-six patients underwent intracoronary infusion of bone marrow-derived (BMCs) (15 patients) or circulating blood-derived endothelial progenitor cells (EPCs) (11 patients) 4{+-}2 days after acute myocardial infarction. Based on a left ventricular segmentation model (17 segments), mean signal intensities as a parameter of viability and perfusion in the infarct zone and non-infarct areas were calculated quantitatively by PET and SPECT at baseline and at 4 months of follow-up. Transplantation of progenitor cells was associated with a significant increase in the mean signal intensity (MSI) in the infarct zone from 54.5% (25th and 75th percentiles: 47.7%, 60.0%) to 58.0% (52.7%, 66.7%) on PET (P=0.013) and from 58.0% (49.5%, 63.0%) to 61.5% (52.5%, 70.2%) on SPECT (P=0.005). Global left ventricular ejection fraction (LVEF) increased from 53.5% (42.6%, 60.0%) to 58.0% (53.0%, 65.8%) (P<0.001). In the five patients without an increase in MSI on PET, LVEF changed from 60.0% (50.0%, 64.0%) to 72.0% (64.0%, 75.5%) at follow-up. PET and SPECT did not show any significant changes in MSI in the non-infarct areas [from 73% (68.5%, 76.2%) to 73% (69.7%, 78.0%) for PET and from 72.0% (66.5%, 77.6%) to 73.0% (67.5%, 78.2%) for SPECT]. There were no significant differences in myocardial viability and perfusion between BMC and EPC infusion

  4. Variation of DNA Methylome of Zebrafish Cells under Cold Pressure.

    Science.gov (United States)

    Han, Bingshe; Li, Wenhao; Chen, Zuozhou; Xu, Qiongqiong; Luo, Juntao; Shi, Yingdi; Li, Xiaoxia; Yan, Xiaonan; Zhang, Junfang

    2016-01-01

    DNA methylation is an essential epigenetic mechanism involved in multiple biological processes. However, the relationship between DNA methylation and cold acclimation remains poorly understood. In this study, Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) was performed to reveal a genome-wide methylation profile of zebrafish (Danio rerio) embryonic fibroblast cells (ZF4) and its variation under cold pressure. MeDIP-seq assay was conducted with ZF4 cells cultured at appropriate temperature of 28°C and at low temperature of 18°C for 5 (short-term) and 30 (long-term) days, respectively. Our data showed that DNA methylation level of whole genome increased after a short-term cold exposure and decreased after a long-term cold exposure. It is interesting that metabolism of folate pathway is significantly hypomethylated after short-term cold exposure, which is consistent with the increased DNA methylation level. 21% of methylation peaks were significantly altered after cold treatment. About 8% of altered DNA methylation peaks are located in promoter regions, while the majority of them are located in non-coding regions. Methylation of genes involved in multiple cold responsive biological processes were significantly affected, such as anti-oxidant system, apoptosis, development, chromatin modifying and immune system suggesting that those processes are responsive to cold stress through regulation of DNA methylation. Our data indicate the involvement of DNA methylation in cellular response to cold pressure, and put a new insight into the genome-wide epigenetic regulation under cold pressure. PMID:27494266

  5. Blood cell mitochondrial DNA content and premature ovarian aging.

    Directory of Open Access Journals (Sweden)

    Marco Bonomi

    Full Text Available Primary ovarian insufficiency (POI is a critical fertility defect characterized by an anticipated and silent impairment of the follicular reserve, but its pathogenesis is largely unexplained. The frequent maternal inheritance of POI together with a remarkable dependence of ovarian folliculogenesis upon mitochondrial biogenesis and bioenergetics suggested the possible involvement of a generalized mitochondrial defect. Here, we verified the existence of a significant correlation between blood and ovarian mitochondrial DNA (mtDNA content in a group of women undergoing ovarian hyperstimulation (OH, and then aimed to verify whether mtDNA content was significantly altered in the blood cells of POI women. We recruited 101 women with an impaired ovarian reserve: 59 women with premature ovarian failure (POF and 42 poor responders (PR to OH. A Taqman copy number assay revealed a significant mtDNA depletion (P<0.001 in both POF and PR women in comparison with 43 women of similar age and intact ovarian reserve, or 53 very old women with a previous physiological menopause. No pathogenic variations in the mitochondrial DNA polymerase γ (POLG gene were detected in 57 POF or PR women with low blood mtDNA content. In conclusion, blood cell mtDNA depletion is a frequent finding among women with premature ovarian aging, suggesting that a still undetermined but generalized mitochondrial defect may frequently predispose to POI which could then be considered a form of anticipated aging in which the ovarian defect may represent the first manifestation. The determination of mtDNA content in blood may become an useful tool for the POI risk prediction.

  6. Variation of DNA Methylome of Zebrafish Cells under Cold Pressure

    Science.gov (United States)

    Xu, Qiongqiong; Luo, Juntao; Shi, Yingdi; Li, Xiaoxia; Yan, Xiaonan; Zhang, Junfang

    2016-01-01

    DNA methylation is an essential epigenetic mechanism involved in multiple biological processes. However, the relationship between DNA methylation and cold acclimation remains poorly understood. In this study, Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) was performed to reveal a genome-wide methylation profile of zebrafish (Danio rerio) embryonic fibroblast cells (ZF4) and its variation under cold pressure. MeDIP-seq assay was conducted with ZF4 cells cultured at appropriate temperature of 28°C and at low temperature of 18°C for 5 (short-term) and 30 (long-term) days, respectively. Our data showed that DNA methylation level of whole genome increased after a short-term cold exposure and decreased after a long-term cold exposure. It is interesting that metabolism of folate pathway is significantly hypomethylated after short-term cold exposure, which is consistent with the increased DNA methylation level. 21% of methylation peaks were significantly altered after cold treatment. About 8% of altered DNA methylation peaks are located in promoter regions, while the majority of them are located in non-coding regions. Methylation of genes involved in multiple cold responsive biological processes were significantly affected, such as anti-oxidant system, apoptosis, development, chromatin modifying and immune system suggesting that those processes are responsive to cold stress through regulation of DNA methylation. Our data indicate the involvement of DNA methylation in cellular response to cold pressure, and put a new insight into the genome-wide epigenetic regulation under cold pressure. PMID:27494266

  7. Failure of 5-thio-d-glucose to alter the viability or radiation response of EMT6 tumor cells in vivo

    International Nuclear Information System (INIS)

    The glucose analogue 5-thio-d-glucose (5TG) was toxic to hypoxic EMT6 Rw mouse mammary tumor cells in exponential growth in cell culture, at a concentration of 5mM and with incubation times of 2-6 hrs. These same treatments were not toxic to aerated EMT6 cells. When hypoxic or aerobic cultures were preincubated with 5mM 5TG for 2 hors before irradiation, only insignificant changes in the radiation dose-response curves were observed. Mice bearing solid intradermal EMT6 tumors were treated with single or multiple injections of 5TG predicted to produce 5TG levels similar to those used during in vitro experiments. These 5TG treatments produced no significant changes in tumor cell viability, either in unirradiated tumors or in tumors irradiated with 15Gy of x-rays to deplete the aerobic tumor cell population. Fasting the mice before treatment to lower blood glucose levels increased the toxicity of 5TG, but did not allow more efficacious treatment of the tumors. These data provide no evidence that 5TG can be used effectively in the treatment of solid tumors, either as a cytotoxic agent which selectively kills hypoxic cells or as a hypoxic cell radiosensitizer

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

    Science.gov (United States)

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

    2016-01-01

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

  9. Nimotuzumab enhances radiation sensitivity of NSCLC H292 cells in vitro by blocking epidermal growth factor receptor nuclear translocation and inhibiting radiation-induced DNA damage repair

    Directory of Open Access Journals (Sweden)

    Teng K

    2015-04-01

    Full Text Available Kai Teng,1,2,* Yong Zhang,1,* Xiaoyan Hu,1 Yihui Ding,1 Rui Gong,1 Li Liu1,* 1Department of Thoracic Oncology, Cancer Center of Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China; 2Department of Radiation Oncology, Hainan Cancer Hospital, Haikou, Hainan, People’s Republic of China *These authors contributed equally to this work Background: The epidermal growth factor receptor (EGFR signaling pathway plays a significant role in radiation resistance. There is evidence that EGFR nuclear translocation is associated with DNA double-strand breaks (DSB repair. Nimotuzumab has shown the effect of radiosensitization in various cancer cells, but little is known about the relationship between nimotuzumab and EGFR nuclear translocation in non-small cell lung cancer (NSCLC cell lines. In this study, we selected two NSCLC cell lines, namely, H292 (with high EGFR expression and H1975 (with low EGFR expression and explored the mechanisms underlying radiation sensitivity.Methods: MTT assay, clonogenic survival assay, and flow cytometry were performed separately to test cell viability, radiation sensitivity, cell cycle distribution, and apoptosis. Protein γ-H2AX, DNA-PK/p-DNA-PK, and EGFR/p-EGFR expression were further compared both in the cytoplasm and the nucleus with the western blot.Results: Nimotuzumab reduced the viability of H292 cells and sensitized H292 cells to ionizing radiation. The radiation sensitivity enhancement ratio (SER was 1.304 and 1.092 for H292 and H1975 cells, respectively. H292 cells after nimotuzumab administration were arrested at the G0/G1 phase in response to radiation. Apoptosis was without statistical significance in both cell lines. γ-H2AX formation in the combination group (nimotuzumab and radiation increased both in the cytoplasm and the nucleus along with the decreased expression of nuclear EGFR/p-EGFR and p-DNA-PK in H292 cells (P<0.05 that

  10. Bio-green synthesis of Ag-GO, Au-GO and Ag-Au-GO nanocomposites using Azadirachta indica: its application in SERS and cell viability

    Science.gov (United States)

    Hareesh, K.; Williams, J. F.; Dhole, N. A.; Kodam, K. M.; Bhoraskar, V. N.; Dhole, S. D.

    2016-07-01

    Silver-graphene oxide, Gold-graphene oxide, Silver-Gold-graphene oxide nanocomposites were synthesized from Neem leaves (Azadirachta indica) extract using a bio-green one-pot method. The synthesized bio-green nanocomposites were characterized by UV-visible spectroscopy, x-ray diffractogram, transmission electron microscopy, x-ray photoelectron spectroscopy and Raman spectroscopy. The results indicated the decoration of ˜10 nm of silver, ˜20 nm of gold and ˜15 nm of silver-gold nanoparticles on a graphene oxide sheet. The synthesized nanocomposites showed enhancement in surface enhanced Raman spectroscopy with alizarin and also enhancement in cell viability of Chang liver cell lines which may be due to a synergetic effect of nanoparticles and graphene oxide.

  11. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma

    Science.gov (United States)

    Burnham, Philip; Kim, Min Seong; Agbor-Enoh, Sean; Luikart, Helen; Valantine, Hannah A.; Khush, Kiran K.; De Vlaminck, Iwijn

    2016-01-01

    Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10−5, Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10−5) and microbial cfDNA (71.3x, p 10−5). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods. PMID:27297799

  12. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma.

    Science.gov (United States)

    Burnham, Philip; Kim, Min Seong; Agbor-Enoh, Sean; Luikart, Helen; Valantine, Hannah A; Khush, Kiran K; De Vlaminck, Iwijn

    2016-01-01

    Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10(-5), Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10(-5)) and microbial cfDNA (71.3x, p 10(-5)). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods. PMID:27297799

  13. Multiparametric analysis of cell-free DNA in melanoma patients.

    Directory of Open Access Journals (Sweden)

    Francesca Salvianti

    Full Text Available Cell-free DNA in blood (cfDNA represents a promising biomarker for cancer diagnosis. Total cfDNA concentration showed a scarce discriminatory power between patients and controls. A higher specificity in cancer diagnosis can be achieved by detecting tumor specific alterations in cfDNA, such as DNA integrity, genetic and epigenetic modifications.The aim of the present study was to identify a sequential multi-marker panel in cfDNA able to increase the predictive capability in the diagnosis of cutaneous melanoma in comparison with each single marker alone. To this purpose, we tested total cfDNA concentration, cfDNA integrity, BRAF(V600E mutation and RASSF1A promoter methylation associated to cfDNA in a series of 76 melanoma patients and 63 healthy controls. The chosen biomarkers were assayed in cfDNA samples by qPCR. Comparison of biomarkers distribution in cases and controls was performed by a logistic regression model in both univariate and multivariate analysis. The predictive capability of each logistic model was investigated by means of the area under the ROC curve (AUC. To aid the reader to interpret the value of the AUC, values between 0.6 and 0.7, between 0.71 and 0.8 and greater than 0.8 were considered as indicating a weak predictive, satisfactory and good predictive capacity, respectively. The AUC value for each biomarker (univariate logistic model was weak/satisfactory ranging between 0.64 (BRAF(