Plasmacytoid dendritic cells play a major role in apoptotic leukocyte-induced immune modulation.

PubMed

Bonnefoy, Francis; Perruche, Sylvain; Couturier, Mélanie; Sedrati, Abdeslem; Sun, Yunwei; Tiberghien, Pierre; Gaugler, Béatrice; Saas, Philippe

2011-05-15

Several APCs participate in apoptotic cell-induced immune modulation. Whether plasmacytoid dendritic cells (PDCs) are involved in this process has not yet been characterized. Using a mouse model of allogeneic bone marrow engraftment, we demonstrated that donor bone marrow PDCs are required for both donor apoptotic cell-induced engraftment and regulatory T cell (Treg) increase. We confirmed in naive mice receiving i.v. syngeneic apoptotic cell infusion that PDCs from the spleen induce ex vivo Treg commitment. We showed that PDCs did not interact directly with apoptotic cells. In contrast, in vivo macrophage depletion experiments using clodronate-loaded liposome infusion and coculture experiments with supernatant from macrophages incubated with apoptotic cells showed that PDCs required macrophage-derived soluble factors--including TGF-β--to exert their immunomodulatory functions. Overall, PDCs may be considered as the major APC involved in Treg stimulation/generation in the setting of an immunosuppressive environment obtained by apoptotic cell infusion. These findings show that like other APCs, PDC functions are influenced, at least indirectly, by exposure to blood-borne apoptotic cells. This might correspond with an additional mechanism preventing unwanted immune responses against self-antigens clustered at the cell surface of apoptotic cells occurring during normal cell turnover.

Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering

PubMed Central

Yang, Yunzhi Peter

2015-01-01

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies. PMID:26407291

Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering.

PubMed

Ker, Dai Fei Elmer; Sharma, Rashmi; Wang, Evelyna Tsi Hsin; Yang, Yunzhi Peter

2015-01-01

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies.

Effects of hypergravity on immunologic function

NASA Technical Reports Server (NTRS)

Sonnenfeld, G.; Koebel, D. A.; Davis, S.

1995-01-01

The purpose of this study was to compare the effects of hypergravity exposure (2g) with those of exposure to space flight in the Cosmos 2044 flight. To do so, rats were centrifuged continuously for 14 days. Two different experiments were carried out on tissue obtained from the centrifuged rats. In the first experiment, rat bone marrow cells were examined for their response to recombinant murine colony stimulating factor-granulocyte/monocyte (GM-CSF). In the second experiment, rat spleen and bone marrow cells were stained in with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and analyzed on a flow cytometer. The results of the studies indicated that bone marrow cells from centrifuged rats showed no significant change in response to GM-CSF as compared to bone marrow cells from control rats. Spleen cells from flown rats showed some statistically significant changes in leukocytes subset distribution, but no differences that appeared to be of biological significance. These results indicate that hypergravity did not greatly affect the same immunological parameters affected by space flight in the Cosmos 2044 mission.

Effects of hypergravity on immunologic function

NASA Technical Reports Server (NTRS)

Sonnenfeld, G.; Koebel, D. A.; Davis, S.

1994-01-01

The purpose of this study was to compare the effects of hypergravity exposure (2g) with those of exposure to space flight in the Cosmos 2044 flight. To do so, rats were centrifuged continuously for 14 days. Two different experiments were carried out on tissue obtained from the centrifuged rats. In the first experiment, rat bone marrow cells were examined for their response to recombinant murine colony stimulating factor-granulocyte/monocyte (GM-CSF). In the second experiment, rate spleen and bone marrow cells were stained in with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and analyzed on a flow cytometer. The results of the studies indicated that bone marrow cells from centrifuged rats showed no significant change in response to GM-CSF as compared to bone marrow cells from control rats. Spleen cells from flown rats showed some statistically significant changes in leukocytes subset distribution, but no differences that appeared to be of biological significance. These results indicate that hypergravity did not greatly affect the same immunological parameters affected by space flight in the Cosmos 2044 mission.

Propionibacterium acnes inhibits FOXM1 and induces cell cycle alterations in human primary prostate cells.

PubMed

Sayanjali, Behnam; Christensen, Gitte J M; Al-Zeer, Munir A; Mollenkopf, Hans-Joachim; Meyer, Thomas F; Brüggemann, Holger

2016-11-01

Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant on human skin. A knock-out mutant lacking the gene encoding the berninamycin-like peptide precursor was unable to downregulate FOXM1 and to halt the cell cycle. Our study reveals a novel host cell-interacting activity of P. acnes. Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.

Activation and proliferation of lymphocytes and other mammalian cells in microgravity

NASA Technical Reports Server (NTRS)

Cogoli, A.; Cogoli-Greuter, M.

1997-01-01

The experimental findings reviewed in this chapter support the following conclusions: Proliferation. Human T-lymphocytes, associated with monocytes as accessory cells, show dramatic changes in the centrifuge, in the clinostat and in space. In free-floating cells the mitogenic response is depressed by 90% in microgravity, whereas in cells attached to a substratum activation is enhanced by 100% compared to 1-G ground and inflight controls. The duration of phase G1 of the mitotic cycle of HeLa cells is reduced in hypergravity, resulting in an increased proliferation rate. Other systems like Friend cells and WI38 human embryonic lung cells do not show significant changes. Genetic expression and signal transduction. T-lymphocytes and monocytes show important changes in the expression of cytokines like interleukin-1, interleukin-2, interferon-gamma and tumor necrosis factor. The data from space experiments in Spacelab, Space Shuttle mid-deck, and Biokosmos have helped to clarify certain aspects of the mechanism of T-cell activation. Epidermoid A431 cells show changes in the genetic expression of the proto-oncogenes c-fos and c-jun in the clinostat and in sounding rockets. Membrane function, in particular the binding of ligates as first messengers of a signal, is not changed in most of the cell systems in microgravity. Morphology and Mortility. Free cells, lymphocytes in particular, are able to move and form aggregates in microgravity, indicating that cell-cell contacts and cell communications do take place in microgravity. Dramatic morphological and ultrastructural changes are not detected in cells cultured in microgravity. Important experiments with single mammalian cells, including immune cells, were carried out recently in three Spacelab flights, (SL-J, D-2, and IML-2 in 1992, 1993, and 1994, respectively). The results of the D-2 mission have been published in ref. 75; those of the IML-2 mission in ref. 76. Finally, many cell biology experiments in space have suffered in the past from a lack of adequate controls (like 1-G centrifuges) and of proper experimental conditions (like well-controlled temperature). In this respect the availability of Biorack, outfitted with proper incubators with 1-G control centrifuge as well as a glovebox with a microscope, is a great advantage. It is also desirable that cell biology experiments in space are accompanied or even preceded by a program of ground-based investigations in the fast rotating clinostat and in the centrifuge, and that preparatory experiments be done in parabolic flights and sounding rockets, whenever possible. Proper publication of the results of space experiments is another important need. A great number of data have been published in proceedings and reports that are not available to the broad scientific community. To guarantee the credibility and the international recognition of space biology it is important that the results be published in international, peer reviewed journals.

Activity-Induced Remodeling of Olfactory Bulb Microcircuits Revealed by Monosynaptic Tracing

PubMed Central

Arenkiel, Benjamin R.; Hasegawa, Hiroshi; Yi, Jason J.; Larsen, Rylan S.; Wallace, Michael L.; Philpot, Benjamin D.; Wang, Fan; Ehlers, Michael D.

2011-01-01

The continued addition of new neurons to mature olfactory circuits represents a remarkable mode of cellular and structural brain plasticity. However, the anatomical configuration of newly established circuits, the types and numbers of neurons that form new synaptic connections, and the effect of sensory experience on synaptic connectivity in the olfactory bulb remain poorly understood. Using in vivo electroporation and monosynaptic tracing, we show that postnatal-born granule cells form synaptic connections with centrifugal inputs and mitral/tufted cells in the mouse olfactory bulb. In addition, newly born granule cells receive extensive input from local inhibitory short axon cells, a poorly understood cell population. The connectivity of short axon cells shows clustered organization, and their synaptic input onto newborn granule cells dramatically and selectively expands with odor stimulation. Our findings suggest that sensory experience promotes the synaptic integration of new neurons into cell type-specific olfactory circuits. PMID:22216277

Experiment K-6-23. Effect of spaceflight on levels and function of immune cells

NASA Technical Reports Server (NTRS)

Mandel, A. D.; Sonnenfeld, G.; Berry, W.; Taylor, G.; Wellhausen, S. R.; Konstantinova, I.; Lesnyak, A.; Fuchs, B.

1990-01-01

Two different immunology experiments were performed on samples received from rats flown on Cosmos 1887. In the first experiment, rat bone marrow cells were examined in Moscow for their response to colony stimulating factor-M. In the second experiment, rat spleen and bone marrow cells were stained in Moscow with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and shipped to the United States where they were subjected to analysis on a flow cytometer. The results of the studies indicate that bone marrow cells from flown rats showed a decreased response to colony stimulating factor than did bone marrow cells from control rats. There was a higher percentage of spleen cells from flown rats staining positively for pan-T-cell, suppressor-T-cell and innate interleukin-2 receptor antigens than from control animals. In addition, a higher percentage of cells that appeared to be part of the myelogenous population of bone marrow cells from flown rats stained positively for surface immunoglobulin than did equivalent cells from control rats.

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

PubMed Central

Carson, Cheryl; Raman, Pichai; Tullai, Jennifer; Xu, Lei; Henault, Martin; Thomas, Emily; Yeola, Sarita; Lao, Jianmin; McPate, Mark; Verkuyl, J. Martin; Marsh, George; Sarber, Jason; Amaral, Adam; Bailey, Scott; Lubicka, Danuta; Pham, Helen; Miranda, Nicolette; Ding, Jian; Tang, Hai-Ming; Ju, Haisong; Tranter, Pamela; Ji, Nan; Krastel, Philipp; Jain, Rishi K.; Schumacher, Andrew M.; Loureiro, Joseph J.; George, Elizabeth; Berellini, Giuliano; Ross, Nathan T.; Bushell, Simon M.; Erdemli, Gül; Solomon, Jonathan M.

2015-01-01

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels. PMID:26098886

Effects of temperature and cellular interactions on the mechanics and morphology of human cancer cells investigated by atomic force microscopy.

PubMed

Li, Mi; Liu, LianQing; Xi, Ning; Wang, YueChao; Xiao, XiuBin; Zhang, WeiJing

2015-09-01

Cell mechanics plays an important role in cellular physiological activities. Recent studies have shown that cellular mechanical properties are novel biomarkers for indicating the cell states. In this article, temperature-controllable atomic force microscopy (AFM) was applied to quantitatively investigate the effects of temperature and cellular interactions on the mechanics and morphology of human cancer cells. First, AFM indenting experiments were performed on six types of human cells to investigate the changes of cellular Young's modulus at different temperatures and the results showed that the mechanical responses to the changes of temperature were variable for different types of cancer cells. Second, AFM imaging experiments were performed to observe the morphological changes in living cells at different temperatures and the results showed the significant changes of cell morphology caused by the alterations of temperature. Finally, by co-culturing human cancer cells with human immune cells, the mechanical and morphological changes in cancer cells were investigated. The results showed that the co-culture of cancer cells and immune cells could cause the distinct mechanical changes in cancer cells, but no significant morphological differences were observed. The experimental results improved our understanding of the effects of temperature and cellular interactions on the mechanics and morphology of cancer cells.

Purification and Cultivation of Human Pituitary Growth Hormones Secreting Cells

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Todd, P.; Grindeland, R.; Lanham, W.; Morrison, D.

1985-01-01

The rat and human pituitary gland contains a mixture of hormone producing cell types. The separation of cells which make growth hormone (GH) is attempted for the purpose of understanding how the hormone molecule is made within the pituitary cell; what form(s) it takes within the cell; and what form(s) GH assumes as it leaves the cell. Since GH has a number of biological targets (e.g., muscle, liver, bone), the assessment of the activities of the intracellular/extracellular GH by new and sensitive bioassays. GH cells contained in the mixture was separated by free flow electrophoresis. These experiments show that GH cells have different electrophoretic mobilities. This is relevant to NASA since a lack of GH could be a prime causative factor in muscle atrophy. Further, GH has recently been implicated in the etiology of motion sickness in space. Continous flow electrophoresis experiment on STS-8 showed that GH cells could be partially separated in microgravity. However, definitive cell culture studies could not be done due to insufficient cell recoveries.

Impact of pseudo-continuous fermentation on the ethanol tolerance of Scheffersomyces stipitis.

PubMed

Liang, Meng; Kim, Min Hea; He, Qinghua Peter; Wang, Jin

2013-09-01

In this work we conducted the pseudo-continuous fermentation, i.e., continuous fermentation with cell retention, using Scheffersomyces stipitis, and studied its effect on ethanol tolerance of the strain. During the fermentation experiments, S. stipitis was adapted to a mild concentration of ethanol (20-26 g/L) for two weeks. Two substrates (glucose and xylose) were used in different fermentation experiments. After fermentation, various experiments were performed to evaluate the ethanol tolerance of adapted cells and unadapted cells. Compared to the unadapted cells, the viability of adapted cells increased by 8 folds with glucose as the carbon source and 6 folds with xylose as the carbon source following exposure to 60 g/L ethanol for 2 h. Improved ethanol tolerance of the adapted cells was also revealed in the effects of ethanol on plasma membrane permeability, extracellular alkalization and acidification. The mathematical modeling of cell leakage, extracellular alkalization and acidification revealed that cells cultured on glucose show better ethanol tolerance than cells cultured on xylose but the differences become smaller for adapted cells. The results show that pseudo-continuous fermentation can effectively improve cell's ethanol tolerance due to the environmental pressure during the fermentation process. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The UoSAT-5 solar cell experiment: First year in orbit

NASA Technical Reports Server (NTRS)

Goodbody, C.

1993-01-01

The results for the first year in orbit of the DRA solar cell experiment flying on the Surrey University UoSAT-5 satellite are described. Several problems were identified with the measured data, which are discussed along with the techniques used to remove or minimize the effect of the problems. After 1 year in orbit the majority of the cells flying on the experiment have undergone little or no degradation. The exception to this are all the ITO/InP cells, supplied by two different manufacturers, they are showing more degradation than the GaAs cells. This result is unexpected and currently unexplainable. It will be necessary to retrieve data from the experiment for several years to obtain the best results due to the relatively benign radiation environment.

Retinal changes in rats flown on Cosmos 936 - A cosmic ray experiment

NASA Technical Reports Server (NTRS)

Philpott, D. E.; Corbett, R.; Turnbill, C.; Black, S.; Dayhoff, D.; Mcgourty, J.; Lee, R.; Harrison, G.; Savik, L.

1980-01-01

Ten rats, five centrifuged during flight to simulate gravity and five stationary in flight and experiencing hypogravity, orbited the Earth. No differences were noted between flight-stationary and flight-centrifuged animals, but changes were seen between these two groups and ground controls. Morphological alterations were observed comparable to those in the experiment flown on Cosmos 782 and to the retinal cells exposed to high-energy particles at Berkeley. Affected cells in the outer nuclear layer showed swelling, clearing of cytoplasm, and disruption of the membranes. Tissue channels were again found, similar to those seen on 782. After space flight, preliminary data indicated an increase in cell size in montages of the nuclear layer of both groups of flight animals. This experiment shows that weightlessness and environmental conditions other than cosmic radiation do not contribute to the observed damage of retinal cells.

Colocalization of Mating-Induced Fos and D2-Like Dopamine Receptors in the Medial Preoptic Area: Influence of Sexual Experience.

PubMed

Nutsch, Victoria L; Will, Ryan G; Robison, Christopher L; Martz, Julia R; Tobiansky, Daniel J; Dominguez, Juan M

2016-01-01

Dopamine in the medial preoptic area (mPOA) stimulates sexual activity in males. This is evidenced by microdialysis and microinjection experiments revealing that dopamine receptor antagonists in the mPOA inhibit sexual activity, whereas agonists facilitate behavior. Microdialysis experiments similarly show a facilitative role for dopamine, as levels of dopamine in the mPOA increase with mating. While the majority of evidence suggests an important role for dopamine receptors in the mPOA in the regulation of male sexual behaviors, whether sexual activity or sexual experience influence dopamine receptor function in the mPOA has not been previously shown. Here we used immunohistochemical assays to determine whether varying levels of sexual activity or experience influence the number of cells containing Fos or D2 receptor immunoreactivity. Results show that sexual experience facilitated subsequent behavior, namely experience decreased latencies. Moreover, the number of cells with immunoreactivity for Fos or D2 correlated with levels of sexual experience and sexual activity. Sexual activity increased Fos immunoreactivity. Sexually experienced animals also had significantly more D2-positive cells. Sexually inexperienced animals copulating for the first time had a larger percentage of D2-positive cells containing Fos, when compared to sexually experienced animals. Finally, regardless of experience, animals that had sex prior to sacrifice had significantly more D2-positive cells that contained Fos, vs. animals that did not copulate. These findings are noteworthy because sexually experienced animals display increased sexual efficiency. The differences in activation of D2 and changes in receptor density may play a role in this efficiency and other behavioral changes across sexual experience.

RanGTPase regulates the interaction between the inner nuclear membrane proteins, Samp1 and Emerin.

PubMed

Vijayaraghavan, Balaje; Figueroa, Ricardo A; Bergqvist, Cecilia; Gupta, Amit J; Sousa, Paulo; Hallberg, Einar

2018-06-01

Samp1, spindle associated membrane protein 1, is a type II integral membrane protein localized in the inner nuclear membrane. Recent studies have shown that the inner nuclear membrane protein, Emerin and the small monomeric GTPase, Ran are direct binding partners of Samp1. Here we addressed the question whether Ran could regulate the interaction between Samp1 and Emerin in the inner nuclear membrane. To investigate the interaction between Samp1 and Emerin in live cells, we performed FRAP experiments in cells overexpressing YFP-Emerin. We compared the mobility of YFP-Emerin in Samp1 knock out cells and cells overexpressing Samp1. The results showed that the mobility of YFP-Emerin was higher in Samp1 knock out cells and lower in cells overexpressing Samp1, suggesting that Samp1 significantly attenuates the mobility of Emerin in the nuclear envelope. FRAP experiments using tsBN2 cells showed that the mobility of Emerin depends on RanGTP. Consistently, in vitro binding experiments showed that the affinity between Samp1 and Emerin is decreased in the presence of Ran, suggesting that Ran attenuates the interaction between Samp1 and Emerin. This is the first demonstration that Ran can regulate the interaction between two proteins in the nuclear envelope. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Morphological and functional studies on the epidermal cells of amphioxus ( Branchiostoma belcheri tsingtauense) at different developmental stages

NASA Astrophysics Data System (ADS)

Mao, Bing-Yu; Sun, Xiao-Yang; Zhang, Hong-Wei; Zhang, Shi-Cui; Wu, Xian-Han

1997-09-01

Epidermal cells of amphioxus at different developmental stages were investigated by electron microscopy and colloidal carbon tracing experiments. Amphioxus epidermal cells showed different ultrastructural characteristics at larval and adult stages. The epidermal cells at all larval stages studied (24 96 h) had numerous vesicles containing electron dense materials in their apical cytoplasm. In tracing experiments, carbon particles were found in apical vesicles and interoellular spaces. Under scanning electron microscope, many crater-like protrusions were observed on the surface of the cells. These results indicated that amphioxus larval epidermal cells may be capable of endocytosis. The epidermal cells of 3-month and adult amphioxus were obviously secretory ones characterized by well-developed peripheral filaments, a prominent Golgi apparatus and abundant apical secretory vesicles. This study also showed that adult amphioxus body surface mucus contained lectin that could agglutinate human red blood cells. The authors propose that the epidermal cells of amphioxus larva and adult may contribute to the immune defense of the amimal by different means.

Lipotoxicity in HepG2 cells triggered by free fatty acids

PubMed Central

Yao, Hong-Rui; Liu, Jun; Plumeri, Daniel; Cao, Yong-Bing; He, Ting; Lin, Ling; Li, Yu; Jiang, Yuan-Ying; Li, Ji; Shang, Jing

2011-01-01

The goal of this study was to investigate the lipid accumulation and lipotoxicity of free fatty acids (FFAs) induced in HepG2 cells. HepG2 cells were co-incubated with various concentrations of FFAs for 24h and the intracellular lipid contents were observed by Oil Red O and Nile Red staining methods. The lipotoxicity of HepG2 cells were then detected by Hoechest 33342/PI, Annexin V-FITC/PI double-staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-di phenyltetrazolium bromide (MTT) experiment tests. The experiments showed a lipid accumulation and lipotoxicity by increasing FFA concentration gradients. Through cell morphological observation and quantitative analysis, FFAs have shown to increase in a dose-dependent manner compared with the control group. The data collected from hoechst 33342/PI, annexin V-FITC/PI double staining and also MTT experiments showed that cell apoptosis and necrosis significantly increased with increasing FFA concentrations. Apoptosis was not obvious in the 1 mM FFAs-treated group compared to the other two groups. In a certain concentration range, FFAs induced intracellular lipid accumulation and lipotoxicity of HepG2 cells in a dose-dependent manner. PMID:21654881

Merkel cells are long-lived cells whose production is stimulated by skin injury✰

PubMed Central

Wright, Margaret C.; Logan, Gregory J.; Bolock, Alexa M.; Kubicki, Adam C.; Hemphill, Julie A.; Sanders, Timothy A.; Maricich, Stephen M.

2017-01-01

Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. PMID:27998808

Merkel cells are long-lived cells whose production is stimulated by skin injury.

PubMed

Wright, Margaret C; Logan, Gregory J; Bolock, Alexa M; Kubicki, Adam C; Hemphill, Julie A; Sanders, Timothy A; Maricich, Stephen M

2017-02-01

Mechanosensitive Merkel cells are thought to have finite lifespans, but controversy surrounds the frequency of their replacement and which precursor cells maintain the population. We found by embryonic EdU administration that Merkel cells undergo terminal cell division in late embryogenesis and survive long into adulthood. We also found that new Merkel cells are produced infrequently during normal skin homeostasis and that their numbers do not change during natural or induced hair cycles. In contrast, live imaging and EdU experiments showed that mild mechanical injury produced by skin shaving dramatically increases Merkel cell production. We confirmed with genetic cell ablation and fate-mapping experiments that new touch dome Merkel cells in adult mice arise from touch dome keratinocytes. Together, these independent lines of evidence show that Merkel cells in adult mice are long-lived, are replaced rarely during normal adult skin homeostasis, and that their production can be induced by repeated shaving. These results have profound implications for understanding sensory neurobiology and human diseases such as Merkel cell carcinoma. Copyright © 2016 Elsevier Inc. All rights reserved.

Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station

NASA Astrophysics Data System (ADS)

Yano, Sachiko; Kasahara, Haruo; Masuda, Daisuke; Tanigaki, Fumiaki; Shimazu, Toru; Suzuki, Hiromi; Karahara, Ichirou; Soga, Kouichi; Hoson, Takayuki; Tayama, Ichiro; Tsuchiya, Yoshikazu; Kamisaka, Seiichiro

2013-03-01

In 2004, Japan Aerospace Exploration Agency developed the engineered model of the Plant Experiment Unit and the Cell Biology Experiment Facility. The Plant Experiment Unit was designed to be installed in the Cell Biology Experiment Facility and to support the seed-to-seed life cycle experiment of Arabidopsis plants in space in the project named Space Seed. Ground-based experiments to test the Plant Experiment Unit showed that the unit needed further improvement of a system to control the water content of a seedbed using an infrared moisture analyzer and that it was difficult to keep the relative humidity inside the Plant Experiment Unit between 70 and 80% because the Cell Biology Experiment Facility had neither a ventilation system nor a dehumidifying system. Therefore, excess moisture inside the Cell Biology Experiment Facility was removed with desiccant bags containing calcium chloride. Eight flight models of the Plant Experiment Unit in which dry Arabidopsis seeds were fixed to the seedbed with gum arabic were launched to the International Space Station in the space shuttle STS-128 (17A) on August 28, 2009. Plant Experiment Unit were installed in the Cell Biology Experiment Facility with desiccant boxes, and then the Space Seed experiment was started in the Japanese Experiment Module, named Kibo, which was part of the International Space Station, on September 10, 2009 by watering the seedbed and terminated 2 months later on November 11, 2009. On April 19, 2010, the Arabidopsis plants harvested in Kibo were retrieved and brought back to Earth by the space shuttle mission STS-131 (19A). The present paper describes the Space Seed experiment with particular reference to the development of the Plant Experiment Unit and its actual performance in Kibo onboard the International Space Station. Downlinked images from Kibo showed that the seeds had started germinating 3 days after the initial watering. The plants continued growing, producing rosette leaves, inflorescence stems, flowers, and fruits in the Plant Experiment Unit. In addition, the senescence of rosette leaves was found to be delayed in microgravity.

The natural history of fetal cells in postpartum murine maternal lung and bone marrow: a two-stage phenomenon.

PubMed

Pritchard, Stephanie; Peter, Inga; Johnson, Kirby L; Bianchi, Diana W

2012-01-01

During pregnancy, fetal cells cross into the maternal organs where they reside postpartum. Evidence from multiple laboratories suggests that these microchimeric fetal cells contribute to maternal tissue repair after injury. In mouse models, most injury experiments are performed during pregnancy; however, in a clinical setting most injuries or diseases occur postpartum. Therefore, experiments using animal models should be designed to address questions in the time period following delivery. In order to provide a baseline for such experiments, we analyzed the natural history of fetal cells in the postpartum maternal organs. Female C57BL/6J mice were mated to males homozygous for the enhanced green fluorescent protein gene. Fetal cells in the maternal lungs and bone marrow were identified by their green fluorescence using in a high-speed flow cytometer and their counts were compared between the lung and bone marrow. Spearman correlation analysis was used to identify relationships between the duration of time postpartum and the cell counts and ratio of live and dead cells. Our results show that fetal cells persist in these organs until at least three months postpartum in healthy female mice. We show a two-stage decline, with an initial two and a half-week rapid clearance followed by a trend of gradual decrease. Additionally, an increase in the ratio of live to dead cells within the lung over time suggests that these cells may replicate in vivo. The results presented here will inform the design of future experiments and may have implications for women's health.

Multi-scale modelling of the dynamics of cell colonies: insights into cell-adhesion forces and cancer invasion from in silico simulations.

PubMed

Schlüter, Daniela K; Ramis-Conde, Ignacio; Chaplain, Mark A J

2015-02-06

Studying the biophysical interactions between cells is crucial to understanding how normal tissue develops, how it is structured and also when malfunctions occur. Traditional experiments try to infer events at the tissue level after observing the behaviour of and interactions between individual cells. This approach assumes that cells behave in the same biophysical manner in isolated experiments as they do within colonies and tissues. In this paper, we develop a multi-scale multi-compartment mathematical model that accounts for the principal biophysical interactions and adhesion pathways not only at a cell-cell level but also at the level of cell colonies (in contrast to the traditional approach). Our results suggest that adhesion/separation forces between cells may be lower in cell colonies than traditional isolated single-cell experiments infer. As a consequence, isolated single-cell experiments may be insufficient to deduce important biological processes such as single-cell invasion after detachment from a solid tumour. The simulations further show that kinetic rates and cell biophysical characteristics such as pressure-related cell-cycle arrest have a major influence on cell colony patterns and can allow for the development of protrusive cellular structures as seen in invasive cancer cell lines independent of expression levels of pro-invasion molecules.

Multi-scale modelling of the dynamics of cell colonies: insights into cell-adhesion forces and cancer invasion from in silico simulations

PubMed Central

Schlüter, Daniela K.; Ramis-Conde, Ignacio; Chaplain, Mark A. J.

2015-01-01

Studying the biophysical interactions between cells is crucial to understanding how normal tissue develops, how it is structured and also when malfunctions occur. Traditional experiments try to infer events at the tissue level after observing the behaviour of and interactions between individual cells. This approach assumes that cells behave in the same biophysical manner in isolated experiments as they do within colonies and tissues. In this paper, we develop a multi-scale multi-compartment mathematical model that accounts for the principal biophysical interactions and adhesion pathways not only at a cell–cell level but also at the level of cell colonies (in contrast to the traditional approach). Our results suggest that adhesion/separation forces between cells may be lower in cell colonies than traditional isolated single-cell experiments infer. As a consequence, isolated single-cell experiments may be insufficient to deduce important biological processes such as single-cell invasion after detachment from a solid tumour. The simulations further show that kinetic rates and cell biophysical characteristics such as pressure-related cell-cycle arrest have a major influence on cell colony patterns and can allow for the development of protrusive cellular structures as seen in invasive cancer cell lines independent of expression levels of pro-invasion molecules. PMID:25519994

Growth regulation of the mammalian ocular lens by vitreous humor.

PubMed

Banerjee, A; Parafina, J; Bagchi, M

1992-05-01

Experiments were performed in our laboratory to study the effects of a mammalian 8 kD vitreous humor (VH) factor on the DNA synthesis and mitosis of the epithelial cells of organ cultured rabbit lens. The 8 kD polypeptide factor was purified from mature rabbit vitreous humor by liquid chromatography. Proliferative activities of the epithelial cells of organ cultured lenses were stimulated by 3% rabbit serum. The data from our experiments depicted that the 8 kD VH factor effectively inhibits DNA synthesis and mitosis by the epithelial cells of the organ cultured lens. Our experiments also showed that this 8 kD VH factor can maintain its growth inhibitory activity even when heated for 3 min at 95 degrees C. The growth inhibitory effect of the 8 kD VH factor was dose dependent. Using iodinated vitreal proteins it was demonstrated that the VH proteins are able to enter or bind to lens epithelial cells. The growth inhibitory effect of the 8 kD VH factor was also tested on tissue cultured lens epithelial cells. These experiments showed that the 8 kD VH factor has no growth inhibitory effect on the tissue cultured lens epithelial cells. This experiment has been repeated many times using different concentrations of the factor. These observations suggest that the 8 kD VH factor may have receptors in the lens capsular material (extracellular matrix) and the factor-receptor binding is essential for the growth inhibitory effect.

Bioprocessing in Microgravity: Applications of Continuous Flow Electrophoresis to Rat Anterior Pituitary Particles

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Salada, T.; Cenci, R.; Krishnan, K.; Seaman, G. V. F.; Snyder, R.; Matsumiya, H.; Nagaoka, S.

1996-01-01

In this report we describe the results of a continuous flow electrophoresis (CFE) experiment done on STS-65 in which we tested the idea that intracellular growth hormone (GH) particles contained in a cell lysate prepared from cultured rat anterior pituitary cells in microgravity might have different electrophoretic mobilities from those in a synchronous ground control cell lysate. Collectively, the results suggested that CFE processing in microgravity was better than on earth; more samples could be processed at a time (6 x) and more variant forms of GH molecules could be resolved as well. We had also hoped to carry out a pituitary cell CFE experiment, but failure of the hardware required that the actual cell electrophoresis trials be done on earth shortly after Shuttle landing. Data from these experiments showed that space-flown cells possessed a higher electrophoretic mobility than ground control cells, thereby offering evidence for the idea that exposure of cultured cells to microgravity can change their net surface charge-density especially when the cells are fed. Collectively, the results from this pituitary cell experiment document the advantage of using coupled cell culture and CFE techniques in the microgravity environment.

Electrodeposition in microgravity: Ground-based experiments

NASA Technical Reports Server (NTRS)

Riley, C.; Coble, H. D.

1982-01-01

Electrodeposition was studied at one-hundreth g and compared with bench studies at 1 g. The low gravity was achieved during KC-135 aircraft parobolic flights. Flow in a simple cobalt cell (1 M CoSO4) operating under typical commercial conditions (10 to 20 mA/sq cm and 1 V) was monitored with a Schlieren optical system. Natural convection was absent at one-hundreth g. Quantitative comparisons on a cobalt cell with shielded electrodes using interferometry were carried out. Fringe shift differences indicate greater semi-infinite linear diffusion at 1 g than at one-hundreth g for cobalt. Since a shielded electrode operates under diffusion controlled conditions, no differences between 1 g and one-hundreth g would be expected. Similar comparisons on a shielded electrode copper cell were inconclusive. Bench codeposition experiments using polystyrene neutral buoyancy particles coupled with a shielded electrode cobalt cell were begun. Tracking of 12 micron particles showed no measurable difference between thermal/Brownian motion when the cell was operational or nonoperational. Initial experiments on codeposition quality showed a strong dependence upon cathode surface preparation in a shielded electrode configuration.

Simian virus 40-related antigens in three human meningiomas with defined chromosome loss.

PubMed Central

Weiss, A F; Portmann, R; Fischer, H; Simon, J; Zang, K D

1975-01-01

Two out of seven meningiomas tested in early cell cultures by indirect immunofluorescence staining showed simian virus 40 (SV40)-related tumor (T) antigen. In one tumor 90% of the cells were positive. An additional SV40-related antigen (U) was found in 10% of cells of a third tumor. These findings indicate that the meningioma cells showing a positive reaction are transformed by a papova virus that has at least partly the same antigenic properties as SV40 virus. SV40-related viral capsid (V) antigen was absent in all the meningiomas tested. No virus infectious for African green monkey kidney (AGMK) cells could be isolated. The tumors positive for T and U antigens showed the chromosome aberration typical for human meningiomas, i.e., the loss of one chromosome, G-22. The T-antigen-positive tumors showed further hypodiploidization. Experiments to rescue virus from the T-antigen-positive tumors showed further hypodiploidization. Experiments to rescue virus from the T-antigen-positive meningioma cells were performed: fusion of cells pretreated with 8-azaguanine with cells premissive for SV40 led to a low percentage (0.01-0.05%) of V-antigen-positive nuclei in heterokaryon cultures. On the basis of these results, the possibility of a correlation between the meningioma, a relatively common intracranial tumor in man, and an SV40-related papova virus must be considered. It remains to be shown whether this virus is a causative agent for human meningiomas. Images PMID:164660

Experiment "Regeneration" Performed Aboard the Russian Spacecraft Foton-M2 in 2005

NASA Technical Reports Server (NTRS)

Grigoryan, Elonora; Almeida, Eduardo; Domaratskaya, Elena; Poplinskaya, Valentina; Aleinikova, Karina; Tairbekov, Murad; Mitashov, Victor

2006-01-01

The experiments on the newts performed earlier aboard Russian biosate llites showed that the rate of lens and tail regeneration in space wa s greater than on the ground. In parallel it was found that the numbe r of cells in S-phase was greater in space-flown animals than in the ground controls. However, it was unclear whether cell proliferation stimulation was induced by micro-g per se. Molecular mechanisms under lying the change also remained obscure. These issues were addressed b y the joint Russian-American experiment "Regeneration" flown on Foton -M2 in 2005. The method for in-flight delivering DNA precursor BrdU was developed. The experiment showed that during the flight the numbe r of S-phase cells in the regenerating eyes and tails increased. Thes e data together with those obtained earlier suggest that cell prolife ration increases in response to the effects of both micro-g and 1-g a fter return to Earth. The expression of bFGF in regenerating tissues of "flown" newts and ground controls was examined using immuno-histo chemistry. Obtained results suggest that this growth factor is a part icipant of the promotional effect of space flight upon cell prolifera tion in lens and tail regenerates.

The cardioprotective and antiarrhythmic effects of Nardostachys chinensis in animal and cell experiments.

PubMed

Li, Min; Xu, Xue; Yang, Xinyu; Kwong, Joey S W; Shang, Hongcai

2017-08-10

Cardiovascular disease (CVD) is the leading cause of premature death throughout the world. An estimated 17.5 million people died from CVD in 2012, representing 31% of all global deaths. Nardostachys chinensis (NC), a typical traditional Chinese medicine (TCM), plays a crucial role in the management of patients with CVD, especially for those with cardiac arrhythmia. The purpose of this study was to evaluate the cardioprotective and antiarrhythmic effects of NC in animal and cell experiments. To review the cardioprotective and antiarrhythmic effects of NC, studies of NC on cardiovascular diseases in animal and cell experiments were identified from five databases through April 2016. Two investigators independently conducted the literature search, study selection, and data extraction. A total of 16 studies were identified, including five animal experiments and eleven cell experiments. Four studies showed significant effects of NC on myocardial protection by inhibiting myocardial apoptosis, inflammation and oxidative stress. Twelve studies indicated significant beneficial effects of NC in cardiac arrhythmia primarily through the modulation of ion channels (I k , I k1 , I Na , I Ca-L , I to ). The above findings showed the possible efficacy of NC via its cardioprotective and antiarrhythmic effects, but the results should be interpreted with caution due to the limitations and the deficiencies in the studies.

Studies on penetration of antibiotic in bacterial cells in space conditions (7-IML-1)

NASA Technical Reports Server (NTRS)

Tixador, R.

1992-01-01

The Cytos 2 experiment was performed aboard Salyut 7 in order to test the antibiotic sensitivity of bacteria cultivated in vitro in space. An increase of the Minimal Inhibitory Concentration (MIC) in the inflight cultures (i.e., an increase of the antibiotic resistance) was observed. Complementary studies of the ultrastructure showed a thickening of the cell envelope. In order to confirm the results of the Cytos 2 experiment, we performed the ANTIBIO experiment during the D1 mission to try to differentiate, by means of the 1 g centrifuge in the Biorack, between the biological effects of cosmic rays and those caused by microgravity conditions. The originality of this experiment was in the fact that it was designed to test the antibiotic sensitivity of bacteria cultivated in vitro during the orbital phase of the flight. The results show an increase in resistance to Colistin in in-flight bacteria. The MIC is practically double in the in-flight cultures. A cell count of living bacteria in the cultures containing the different Colistin concentrations showed a significant difference between the cultures developed during space flight and the ground based cultures. The comparison between the 1 g and 0 g in-flight cultures show similar behavior for the two sets. Nevertheless, a small difference between the two sets of ground based control cultures was noted. The cultures developed on the ground centrifuge (1.4 g) present a slight decrease in comparison with the cultures developed in the static rack (1 g). In order to approach the mechanisms of the increase of antibiotic resistance on bacteria cultivated in vitro in space, we have proposed the study on penetration of antibiotics in bacterial cells in space conditions. This experiment was selected for the International Microgravity Laboratory 1 (IML-1) mission.

The differentiation directions of the bone marrow stromal cells under modeling microgravity

NASA Astrophysics Data System (ADS)

Nesterenko, Olga; Rodionova, Natalia; Katkova, Olena

Within experiments on rats simulating microgravity by base load remove from back limbs (duration of the experiment 1,5 months) on marrow stromal cells cultures (ex vivo, in vitro) comprising osteogenic cells-predecessors, extracted from femurs, studied their peculiarities of the colony formation ablity, the cell structure, some cytological and ultra-structural characteristics and differentiation direction. It was found that that under microgravity conditions there is a decline of the stromal cells colony formation intensity, decrease of the colonies size and cells mitotic activity that indicates decrease of their growth potential. Both in control and in experiment the colonies were presented by population of low-differentiated cells, differentiated cells and mature cells. The comparative cytological and morphometric analysis have shown that the studied stromal cells in colonies have the smaller sizes, more elongated shape, and higher nucleocytoplasmic ratio. Cells composition in the experiment colonies is reliably different by the ratio of the low-differentiating to being differentiated cells; a ratio of low-differentiated to already differentiated cells; ratio of differentiated cells to total number of all cells. In comparison with control group, amount of the cells passed trough a differentiation stage and mature cells in colonies is decreased by 3 to 4 times. Among the differentiated stromal cells in colonies increasing amount of adipocytes was revealed. The analysis of electron microscope microphotographs showed that in osteogenic cells differentiated under microgravity conditions, there is a reduction of the specific volume of a granular endoplasmic reticulum, Golgi's complex and quantity of nuclei reduction that indicates depression of the specific biosyntheses process intensity in cells. The increase of lysosomes and myelinic structures quantity is linked to organelles partial reduction. Consolidation of mitochondrias is an evidence of the cells’ energy metabolism disorder. In differentiated cells, disorganization and a cytoskeleton destruction was observed. Results showed that under microgravity conditions proliferative and differentiation (including osteogenic) potentialities of low-differentiated marrow stromal cells decreased, induction of their adipocytic differentiation was observes as well. Obtained results make a new contribution into gravitation sensitivity mechanisms understanding for stromal cells of the bone marrow which contain osteogenic cells- predecessors, features of the osteoporosis development.

A minimal physical model for crawling cells

NASA Astrophysics Data System (ADS)

Tiribocchi, Adriano; Tjhung, Elsen; Marenduzzo, Davide; Cates, Michael E.

Cell motility in higher organisms (eukaryotes) is fundamental to biological functions such as wound healing or immune response, and is also implicated in diseases such as cancer. For cells crawling on solid surfaces, considerable insights into motility have been gained from experiments replicating such motion in vitro. Such experiments show that crawling uses a combination of actin treadmilling (polymerization), which pushes the front of a cell forward, and myosin-induced stress (contractility), which retracts the rear. We present a simplified physical model of a crawling cell, consisting of a droplet of active polar fluid with contractility throughout, but treadmilling connected to a thin layer near the supporting wall. The model shows a variety of shapes and/or motility regimes, some closely resembling cases seen experimentally. Our work supports the view that cellular motility exploits autonomous physical mechanisms whose operation does not need continuous regulatory effort.

A minimal physical model captures the shapes of crawling cells

NASA Astrophysics Data System (ADS)

Tjhung, E.; Tiribocchi, A.; Marenduzzo, D.; Cates, M. E.

2015-01-01

Cell motility in higher organisms (eukaryotes) is crucial to biological functions ranging from wound healing to immune response, and also implicated in diseases such as cancer. For cells crawling on hard surfaces, significant insights into motility have been gained from experiments replicating such motion in vitro. Such experiments show that crawling uses a combination of actin treadmilling (polymerization), which pushes the front of a cell forward, and myosin-induced stress (contractility), which retracts the rear. Here we present a simplified physical model of a crawling cell, consisting of a droplet of active polar fluid with contractility throughout, but treadmilling connected to a thin layer near the supporting wall. The model shows a variety of shapes and/or motility regimes, some closely resembling cases seen experimentally. Our work strongly supports the view that cellular motility exploits autonomous physical mechanisms whose operation does not need continuous regulatory effort.

Effects of Space Flight, Clinorotation, and Centrifugation on the Growth and Metabolism of Escherichia Coli

NASA Technical Reports Server (NTRS)

Brown, Robert B.

1999-01-01

Previous experiments have shown that space flight stimulates bacterial growth and metabolism. An explanation for these results is proposed, which may eventually lead to improved terrestrial pharmaceutical production efficiency. It is hypothesized that inertial acceleration affects bacterial growth and metabolism by altering the transport phenomena in the cells external fluid environment. It is believed that this occurs indirectly through changes in the sedimentation rate acting on the bacteria and buoyancy-driven convection acting on their excreted by-products. Experiments over a broad range of accelerations consistently supported this theory. Experiments at I g indicated that higher concentrations of excreted by products surrounding bacterial cells result in a shorter lag phase. Nineteen additional experiments simulated 0 g and 0.5 g using a clinostat, and achieved 50 g, 180 g, and 400 g using a centrifuge. These experiments showed that final cell density is inversely related to the level of acceleration. The experiments also consistently showed that acceleration affects the length of the lag phase in a non-monotonic, yet predictable, manner. Additional data indicated that E. coli metabolize glucose less efficiently at hypergravity, and more efficiently at hypogravity. A space-flight experiment was also performed. Samples on orbit had a statistically significant higher final cell density and more efficient metabolism than did ground controls. These results. which were similar to simulations of 0 g using a clinostat, support the theory that gravity only affects bacterial growth and metabolism indirectly, through changes in the bacteria's fluid environment.

The Effect of Micro-Gravity on in vitro Calcification

NASA Technical Reports Server (NTRS)

Boskey; Stiner; Binderman; Mendelsohn; Doty, S. B.

1997-01-01

The experiment focuses on mineral deposition or calcification of cartilage. The experiments were used to compare the mineral formed in the microgravity of space with that formed on earth. Results of these experiments were anticipated to provide direct insight into how calcification in cartridge and bone may be controlled in space. In the C-2 experiment (STS 66), we found that mineralization started later in the cartridges (both on the ground and in hypo-gravity) than in plastic, and that mineralization appeared to be retarded in hypo-gravity. The flight experiments also showed that the cells differentiated normally, but more slowly than the ground controls, and that the matrix produced was not different from that made on the ground. The purpose of the C-5 experiment was to confirm these findings. The C-5 experiment was flown on STS-72. Because of a computer problem, cells received no gases and no nutrition. The C-7 was flown on STS-77. Ground controls were repeated a week later, however, because there was a problem with the temperature control during the flight, the concurrent ground controls were performed at a different temperature. Despite these problems, the results of the C-2 experiment were confirmed. The cells in the flight cultures did not mature, formed few cartilage nodules, and showed no evidence of mineral deposition up to a culture age of 28 days. Ground controls showed the presence of mineral (based on chemical, spectroscopic, and histochemical analyses) by 21 days. The mineral in these cultures was analogous to that found in calcifying cartilage of young chicks.

[Effects of stable ANO1 overexpression on biological behaviors of human laryngeal squamous cell carcinoma Hep-2 cells in vitro].

PubMed

Li, Yadong; Zhang, Jinsong; Yang, Kai; Zhang, Fujun; Chen, Rui; Chen, Dan

2014-02-01

To detect the effects of ANO1 overexpression on the biological behaviors of human laryngeal squamous cell carcinoma Hep-2 cells. A Hep-2 cell line stably overexpressing ANO1 were examined with flow cytometry, soft agar assay, wound healing assay, siRNA experiments, and chloride channel block with DIDS to observe the effect of ANO1 overexpression on the growth, migration and invasion of the cells. Flow cytometry revealed a comparable cell percentage in G0/G1 phase between ANO1-overexpressing cells and the control cells (P>0.05). The two cells showed no significant difference in soft agar assay (P>0.05), but in wound healing experiments, ANO1-overexpressing cells showed significantly accelerated migration (P<0.05), whereas siRNA-mediated silencing of ANO1 significantly inhibited the cell migration (P<0.05). Treatment with DIDS resulted in an effective block of the ANO1 chloride channel activity and obviously decreased the migration speed of Hep-2 cells. ANO1 overexpression does not significantly affect the proliferation of cancer cells, but can enhance the migration ability of head and neck squamous cell carcinoma, suggesting the value of ANO1 as a new gene therapy target for head and neck squamous cell carcinoma.

The transplantation of neural stem cells and predictive factors in hematopoietic recovery in irradiated mice.

PubMed

Filip, S; Mokrý, J; Karbanová, J; Vávrová, J; Vokurková, J; Bláha, M; English, D

2005-04-01

A number of surprising observations have shown that stem cells, in suitable conditions, have the ability to produce a whole spectrum of cell types, regardless, whether these tissues are derived from the same germ layer or not. This phenomenon is called stem cell plasticity, which means that tissue-specific stem cells are mutually interchangeable. In our experiments, as a model, we used neural stem cells (NSCs) harvested from fetal (E14-15) neocortex and beta-galactosidase positive. In the first experiment we found that on days 12 and 30 after sub-lethal irradiation (LD 8.5 Gy) and (beta-galactosidase(+)) NSCs transplantation all mice survived, just as the group with bone marrow transplantation. Moreover, the bone marrow of mice transplanted NSCs contained the number of CFU-GM colonies with beta-galactosidase(+) cells which was as much as 50% higher. These differences were statistically significant, p<0.001. In the second experiment, we studied kinetics of (beta-galactosidase(+)) NSCs after their transplantation to sub-lethally irradiated mice. Histochemistry of tissues was performed on days 12 and 30 post-transplantation, and beta-galactosidase(+) cells were detected with the help of histochemical examination of removed tissues (lung, liver, spleen, thymus, and skeletal muscle). In tissues removed on day 12 post-transplantation, we found a significantly higher number of beta-galactosidase(+) cells in the spleen and thymus on day 30. While we presumed the presence beta-galactosidase(+) cells in the spleen, as spleen and reticuloendothelial system represent an important retaining system for different cell types, the presence of beta-galactosidase(+) cells in the thymus was rather surprising but very interesting. This indicates a certain mutual and close interconnection of transplanted stem cells and immune system in an adult organism. In the third experiment, we verified the mutual interchange of Sca-1 surface antigen in the bone marrow cells and NSCs before transplantation. Analysis of this antigen showed 24.8% Sca-1 positive cells among the bone marrow cells, while NSCs were Sca-1 negative. Our experiments show that NSCs share hemopoietic identity and may significantly influence the recovery of damaged hematopoiesis but do not have typical superficial markers as HSCs. This result is important for the determination of predictive factors for hemopoiesis recovery, for stem cell plasticity and for their use in the cell therapy.

Automated analysis of siRNA screens of cells infected by hepatitis C and dengue viruses based on immunofluorescence microscopy images

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

2008-03-01

We present an image analysis approach as part of a high-throughput microscopy siRNA-based screening system using cell arrays for the identification of cellular genes involved in hepatitis C and dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in the neighborhood of segmented cell nuclei, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment and single images. In particular, we propose a novel approach for the localization of regions of transfected cells within cell array images, which combines model-based circle fitting and grid fitting. By this scheme we integrate information from single cell array images and knowledge from the complete cell arrays. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behaviour of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

Handheld Diffusion Test Cells

NASA Technical Reports Server (NTRS)

2001-01-01

This photo shows an individual cell from the Handheld Diffusion Test Cell (HH-DTC) apparatus flown on the Space Shuttle. Similar cells will be used in the Observable Protein Crystal Growth Apparatus (OPCGA) to be operated aboard the International Space Station (ISS). The principal investigator is Dr. Alex McPherson of the University of California, Irvine. Each individual cell comprises two sample chambers with a rotating center section that isolates the two from each other until the start of the experiment and after it is completed. The cells are made from optical-quality quartz glass to allow photography and interferometric observations. Each cell has a small light-emitting diode and lens to back-light the solution. In protein crystal growth experiments, a precipitating agent such as a salt solution is used to absorb and hold water but repel the protein molecules. This increases the concentration of protein until the molecules nucleate to form crystals. This cell is one of 96 that make up the experiment module portion of the OPCGA.

Development of Fixed-Point Cells at the SMU

NASA Astrophysics Data System (ADS)

Ďuriš, S.; Ranostaj, J.; Palenčár, R.

2008-06-01

One of the research programs realized at the thermometry laboratory of the Slovak Institute of Metrology (SMU) in recent years has focused on the development of fixed-point cells. In the frame of this research, several primary cells for realization of the International Temperature Scale of 1990 (ITS-90) and several secondary cells for industrial thermometer calibrations were built and studied. This article discusses primary cells for the gallium and mercury fixed points and miniature cells for the zinc point that were developed at the SMU. Information about the cell designs is provided, the materials that were used are specified, and the procedures for their manufacture are described. Briefly, the realization of the fixed points of mercury, gallium, and zinc by using these cells is also described. Many experiments were carried out to study the characteristics of these cells. One of the gallium cells was compared with the circulating transfer cell during the key comparison CCT-K3, and it and the mercury cell were used for the EUROMET Project No. 552. The results of the experiments together with the results of the comparisons show the high quality of these cells. Secondary zinc-point cells were compared against SMU primary zinc-point cells. The comparison shows agreement within 0.12 mK.

Subcellular targeting and interactions among the Potato virus X TGB proteins.

PubMed

Samuels, Timmy D; Ju, Ho-Jong; Ye, Chang-Ming; Motes, Christy M; Blancaflor, Elison B; Verchot-Lubicz, Jeanmarie

2007-10-25

Potato virus X (PVX) encodes three proteins named TGBp1, TGBp2, and TGBp3 which are required for virus cell-to-cell movement. To determine whether PVX TGB proteins interact during virus cell-cell movement, GFP was fused to each TGB coding sequence within the viral genome. Confocal microscopy was used to study subcellular accumulation of each protein in virus-infected plants and protoplasts. GFP:TGBp2 and TGBp3:GFP were both seen in the ER, ER-associated granular vesicles, and perinuclear X-bodies suggesting that these proteins interact in the same subdomains of the endomembrane network. When plasmids expressing CFP:TGBp2 and TGBp3:GFP were co-delivered to tobacco leaf epidermal cells, the fluorescent signals overlapped in ER-associated granular vesicles indicating that these proteins colocalize in this subcellular compartment. GFP:TGBp1 was seen in the nucleus, cytoplasm, rod-like inclusion bodies, and in punctate sites embedded in the cell wall. The puncta were reminiscent of previous reports showing viral proteins in plasmodesmata. Experiments using CFP:TGBp1 and YFP:TGBp2 or TGBp3:GFP showed CFP:TGBp1 remained in the cytoplasm surrounding the endomembrane network. There was no evidence that the granular vesicles contained TGBp1. Yeast two hybrid experiments showed TGBp1 self associates but failed to detect interactions between TGBp1 and TGBp2 or TGBp3. These experiments indicate that the PVX TGB proteins have complex subcellular accumulation patterns and likely cooperate across subcellular compartments to promote virus infection.

[Essential oil from Artemisia lavandulaefolia induces apoptosis and necrosis of HeLa cells].

PubMed

Zhang, Lu-min; Lv, Xue-wei; Shao, Lin-xiang; Ma, Yan-fang; Cheng, Wen-zhao; Gao, Hai-tao

2013-12-01

To investigate the effects of Artemisia lavandulaefolia essential oil on apoptosis and necrosis of HeLa cells. Cell viability was assayed using MTT method. The morphological and structure alterations in HeLa cells were observed by microscopy. Furthermore, cell apoptosis was measured by DNA Ladder and flow cytometry. DNA damage was measured by comet assay, and the protein expression was examined by Western blot analysis. MTT assay displayed essential oil from Artemisia lavandulaefolia could inhibit the proliferation of HeLa cells in a dose-dependent manner. After treated with essential oil of Artemisia lavadulaefolia for 24 h, HeLa cells in 100 and 200 microg/mL experiment groups exhibited the typical morphology changes of undergoing apoptosis, such as cell shrinkage and nucleus chromatin condensed. However, the cells in the 400 microg/mL group showed the necrotic morphology changes including cytomembrane rupture and cytoplasm spillover. In addition, DNA Ladder could be demonstrated by DNA electrophoresis in each experiment group. Apoptosis peak was also evident in flow cytometry in each experiment group. After treating the HeLa cells with essential oil of Artemisia lavadulaefolia for 6 h, comet tail was detected by comet assay. Moreover, western blotting analysis showed that caspase-3 was activated and the cleavage of PARP was inactivated. Essential oil from Artemisia lavadulaefolia can inhibit the proliferation of HeLa cells in vitro. Low concentration of essential oil from Artemisia lavadulaefolia can induce apoptosis, whereas high concentration of the compounds result in necrosis of HeLa cells. And,the mechanism may be related to the caspase-3-mediated-PARP apoptotic signal pathway.

Enhanced electrochemical sensing of leukemia cells using drug/lipid co-immobilized on the conducting polymer layer.

PubMed

Gurudatt, N G; Naveen, M Halappa; Ban, Changill; Shim, Yoon-Bo

2016-12-15

Electrochemical biosensors using five anticancer drug and lipid molecules attached on the conducting polymer layer to obtain the orientation of drug molecules toward cancer cells, were evaluated as sensing materials and their performances were compared. Conjugation of the drug molecules with a lipid, phosphatidylcholine (PC) has enhanced the sensitivity towards leukemia cells and differentiates cancer cells from normal cells. The composition of each layer of sensor probe was confirmed by electrochemical and surface characterization experiments. Both impedance spectroscopy and voltammetry show the enhanced interaction of leukemia cells using the drug/lipid modified sensor probe. As the number of leukemia cells increased, the charge transfer resistance (Rct) in impedance spectra increased and the amine oxidation peak current of drug molecules in voltammograms decreased at around 0.7-1.0V. Of test drug molecules, raltitrexed (Rtx) showed the best performance for the cancer cells detection. Cancer and normal cell lines from different origins were examined to evaluate the degree of expression of folate receptors (FR) on cells surface, where cervical HeLa cell line was found to be shown the highest expression of the receptor. Impedance and chronoamperometric experiments for leukemia cell line (Jurkat E6-1) showed linear dynamic ranges of 1.0×10(3)-2.5×10(5) cells/mL and 1.0×10(3)-8.0×10(3) cells/mL with detection limits of 68±5 cells/mL and 21±3 cells/mL, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Final Technical Report: Hydrogen Energy in Engineering Education (H2E3)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lehman, Peter A.; Cashman, Eileen; Lipman, Timothy

2011-09-15

Schatz Energy Research Center's Hydrogen Energy in Engineering Education curriculum development project delivered hydrogen energy and fuel cell learning experiences to over 1,000 undergraduate engineering students at five California universities, provided follow-on internships for students at a fuel cell company; and developed commercializable hydrogen teaching tools including a fuel cell test station and a fuel cell/electrolyzer experiment kit. Monitoring and evaluation tracked student learning and faculty and student opinions of the curriculum, showing that use of the curriculum did advance student comprehension of hydrogen fundamentals. The project web site (hydrogencurriculum.org) provides more information.

Overexpression of sulfatase-1 in murine hepatocarcinoma Hca-F cell line downregulates mesothelin and leads to reduction in lymphatic metastasis, both in vitro and in vivo.

PubMed

Mahmoud, Salma; Ibrahim, Mohammed; Hago, Ahmed; Huang, Yuhong; Wei, Yuanyi; Zhang, Jun; Zhang, Qingqing; Xiao, Yu; Wang, Jingwen; Adam, Munkaila; Guo, Yu; Wang, Li; Zhou, Shuting; Xin, Boyi; Xuan, Wei; Tang, Jianwu

2016-11-15

Lymphatic vessels function as transport channels for tumor cells to metastasize from the primary site into the lymph nodes. In this experiment we evaluated the effect of Sulfatase-1 (Sulf-1) on metastasis by upregulating it in murine hepatocarcinoma cell line Hca-F with high lymph node metastatic rate of >75%. The study in vitro showed that up regulation of Sulf-1 in Hca-F cells significantly reduced cell proliferation, migration and invasion (p<0.05). Also, the forced expression of Sulf-1 down regulated Mesothelin (Msln) at both the protein and mRNA levels. The experiment in vivo further showed that up-regulation of Sulf-1 with the attendant downregulation of mesothelin delayed tumor growth and decreased lymph node metastasis. In conclusion, our findings show that Sulf-1 is an important tumor suppressor gene in hepatocellular carcinoma (HCC), and its over expression downregulates Msln and results in a decrease in HCC cell proliferation, migration, invasion, and lymphatic metastasis. This functional relationship between Sulf-1 and Msln could be exploited for the development of a novel liver cancer therapy.

Overexpression of sulfatase-1 in murine hepatocarcinoma Hca-F cell line downregulates mesothelin and leads to reduction in lymphatic metastasis, both in vitro and in vivo

PubMed Central

Mahmoud, Salma; Ibrahim, Mohammed; Hago, Ahmed; Huang, Yuhong; Wei, Yuanyi; Zhang, Jun; Zhang, Qingqing; Xiao, Yu; Wang, Jingwen; Adam, Munkaila; Guo, Yu; Wang, Li; Zhou, Shuting; Xin, Boyi; Xuan, Wei; Tang, Jianwu

2016-01-01

Lymphatic vessels function as transport channels for tumor cells to metastasize from the primary site into the lymph nodes. In this experiment we evaluated the effect of Sulfatase-1 (Sulf-1) on metastasis by upregulating it in murine hepatocarcinoma cell line Hca-F with high lymph node metastatic rate of >75%. The study in vitro showed that upregulation of Sulf-1 in Hca-F cells significantly reduced cell proliferation, migration and invasion (p<0.05). Also, the forced expression of Sulf-1 downregulated Mesothelin (Msln) at both the protein and mRNA levels. The experiment in vivo further showed that up-regulation of Sulf-1 with the attendant downregulation of mesothelin delayed tumor growth and decreased lymph node metastasis. In conclusion, our findings show that Sulf-1 is an important tumor suppressor gene in hepatocellular carcinoma (HCC), and its overexpression downregulates Msln and results in a decrease in HCC cell proliferation, migration, invasion, and lymphatic metastasis. This functional relationship between Sulf-1 and Msln could be exploited for the development of a novel liver cancer therapy. PMID:27626699

Use of piracetam improves sickle cell deformability in vitro and in vivo.

PubMed Central

Gini, E K; Sonnet, J

1987-01-01

Microsieving diluted suspensions of oxygenated sickle cell anaemia (HbSS) cells on polycarbonate filters shows that piracetam improves the red cell deformability in vitro. In vivo an oral intake of 160 mg/kg/day divided in four doses enhances the HbSS cell deformability as actively as it does in in vitro experiments. The drug is also able partially to restore the impaired deformability of physiologically deoxygenated HbSS cells. These findings are consistent with the results of clinical trials, which show that continuous treatment with piracetam reduces the incidence of vaso-occlusive crises in patients with sickle cell disease. PMID:3818978

Glial cell migration in the eye disc.

PubMed

Silies, Marion; Yuva, Yeliz; Engelen, Daniel; Aho, Annukka; Stork, Tobias; Klämbt, Christian

2007-11-28

Any complex nervous system is made out of two major cell types, neurons and glial cells. A hallmark of glial cells is their pronounced ability to migrate. En route to their final destinations, glial cells are generally guided by neuronal signals. Here we show that in the developing visual system of Drosophila glial cell migration is largely controlled by glial-glial interactions and occurs independently of axonal contact. Differentiation into wrapping glia is initiated close to the morphogenetic furrow. Using single cell labeling experiments we identified six distinct glial cell types in the eye disc. The migratory glial population is separated from the wrapping glial cells by the so-called carpet cells, extraordinary large glial cells, each covering a surface area of approximately 10,000 epithelial cells. Subsequent cell ablation experiments demonstrate that the carpet glia regulates glial migration in the eye disc epithelium and suggest a new model underlying glial migration and differentiation in the developing visual system.

GEMINI-TITAN (GT)-III - WEIGHTLESSNESS EXPERIMENT - AMES RESEARCH CENTER (ARC), CA

NASA Image and Video Library

1965-03-19

S65-18766 (March 1965) --- Diagram of experiment planned for the Gemini-Titan 3 mission scheduled on March 23, 1965, to find out if there are effects of weightlessness on individual living cells. The round canister (top) shows the experiment package. It will contain eight identical chambers, each with sections of sperm, eggs and fixative. Cells are eggs of the spiny, black sea animal, the sea urchin. Bottom panel shows the three stages of each chamber. From left in the first stage, sperm, eggs and fixative are separated. By turning the handle, astronauts will fertilize a certain portion of the eggs, which will begin to divide. At 20 minutes after launch, further turns of the handle will force fixative into two chambers and stop cell division. At 70 minutes after launch, cell division in four more chambers will be stopped, and just prior to re-entry, growth of the remaining two chambers will be terminated by a turn of the handle. This system will allow study after the flight of how cells divided after various time periods in weightlessness. Abnormalities would suggest weightlessness effects on living tissue and possible hazard to prolonged manned spaceflight.

Development of the Hippocampal Cognitive Map in Pre-weanling Rats

PubMed Central

Wills, Tom; Cacucci, Francesca; Burgess, Neil; O’Keefe, John

2011-01-01

Orienting in large-scale space depends on the interaction of environmental experience and pre-configured, possibly innate, constructs. Place, head-direction and grid cells in the hippocampal formation provide allocentric representations of space. Here we show how these cognitive representations emerge and develop as rat pups first begin to explore their environment. Directional, locational and rhythmic organization of firing are present during initial exploration, including adult-like directional firing. The stability and precision of place cell firing continues to develop throughout juvenility. Stable grid cell firing appears later but matures rapidly to adult levels. Our results demonstrate the presence of three neuronal representations of space prior to extensive experience, and show how they develop with age. PMID:20558720

Nocardia rubra cell-wall skeleton promotes CD4+ T cell activation and drives Th1 immune response.

PubMed

Wang, Guangchuan; Wu, Jie; Miao, Miao; Dou, Heng; Nan, Ning; Shi, Mingsheng; Yu, Guang; Shan, Fengping

2017-08-01

Several lines of evidences have shown that Nocardia rubra cell wall skeleton (Nr-CWS) has immunoregulatory and anti-tumor activities. However, there is no information about the effect of Nr-CWS on CD4 + T cells. The aim of this study was to explore the effect of Nr-CWS on the phenotype and function of CD4 + T cells. Our results of in vitro experiments showed that Nr-CWS could significantly up-regulate the expression of CD69 and CD25 on CD4 + T cells, promote the proliferation of CD4 + T cells, increase the production of IFN-γ, TNF-α and IL-2 in the supernatants, but has no significant effect on the apoptosis and death of CD4 + T cells. Results of in vivo experiments showed that Nr-CWS could promote the proliferation of CD4 + T cells, and increase the production of IL-2, IFN-γ and TNF-α (Th1 type cytokines). These data suggest that Nr-CWS can enhance the activation of CD4 + T cells, promote the proliferation of CD4 + T cells and the differentiation of CD4 + T cells to Th1 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Experiment on the factors for enhancing the susceptibility of cancer cells to chemotherapeutic drug by ultrasound microbubbles.

PubMed

Zhao, Ying-Zheng; Gao, Hui-Sheng; Zhou, Zhi-Cai; Tang, Qin-Qin; Lu, Cui-Tao; Jin, Zhuo; Tian, Ji-Lai; Xu, Yan-Yan; Tian, Xin-Qiao; Wang, Lee; Kong, Fan-Lei; Li, Xiao-Kun; Huang, Pin-Tong; He, Hui-Liao; Wu, Yan

2010-07-01

The objective of this study was to investigate the factors for enhancing the susceptibility of cancer cells to chemotherapeutic drug by ultrasound microbubbles. Ultrasound (US) combined with phospholipid-based microbubbles (MB) was used to enhance the susceptibility of colon cancer cell line SWD-620 to anticancer drugs Topotecan hydrochloride (TOP). Experiments were designed to investigate the influence of main factors on cell viability and cell inhibition, such as US intensity, MB concentration, drug combination with MB, asynchronous action between US triggered cavitation and drug entering cell, MB particle size. US exposure for 10 sec with US probe power at 0.6 W/cm(2) had satisfied cell viability. Treated with US combined with 15% MB, cell viability maintained more than 85% and cell inhibition 86.16%. Under optimal US combined with MB, TOP showed much higher cell inhibition than that of only TOP group. Cell inhibition under short delayed time (<2 h) for TOP addition did not show obvious difference. In terms of MB particle size, the order of cell inhibition was: Mixture > Micron bubble part > Nanometer bubble part. US combined with MB can enhance the susceptibility of cancer cells to chemotherapeutic drug, which may provide a potential method for US-mediated tumor chemotherapy.

Microgravity

NASA Image and Video Library

2001-01-24

This photo shows an individual cell from the Handheld Diffusion Test Cell (HH-DTC) apparatus flown on the Space Shuttle. Similar cells will be used in the Observable Protein Crystal Growth Apparatus (OPCGA) to be operated aboard the International Space Station (ISS). The principal investigator is Dr. Alex McPherson of the University of California, Irvine. Each individual cell comprises two sample chambers with a rotating center section that isolates the two from each other until the start of the experiment and after it is completed. The cells are made from optical-quality quartz glass to allow photography and interferometric observations. Each cell has a small light-emitting diode and lens to back-light the solution. In protein crystal growth experiments, a precipitating agent such as a salt solution is used to absorb and hold water but repel the protein molecules. This increases the concentration of protein until the molecules nucleate to form crystals. This cell is one of 96 that make up the experiment module portion of the OPCGA.

Handheld Diffusion Test Cells

NASA Technical Reports Server (NTRS)

2001-01-01

This photo shows the Handheld Diffusion Test Cell (HH-DTC) apparatus flown on the Space Shuttle. Similar cells (inside the plastic box) will be used in the Observable Protein Crystal Growth Apparatus (OPCGA) to be operated aboard the International Space Station (ISS). The principal investigator is Dr. Alex McPherson of the University of California, Irvine. Each individual cell comprises two sample chambers with a rotating center section that isolates the two from each other until the start of the experiment and after it is completed. The cells are made from optical-quality quartz glass to allow photography and interferometric observations. Each cell has a small light-emitting diode and lens to back-light the solution. In protein crystal growth experiments, a precipitating agent such as a salt solution is used to absorb and hold water but repel the protein molecules. This increases the concentration of protein until the molecules nucleate to form crystals. This cell is one of 96 that make up the experiment module portion of the OPCGA.

Multi-Scale Modeling to Improve Single-Molecule, Single-Cell Experiments

NASA Astrophysics Data System (ADS)

Munsky, Brian; Shepherd, Douglas

2014-03-01

Single-cell, single-molecule experiments are producing an unprecedented amount of data to capture the dynamics of biological systems. When integrated with computational models, observations of spatial, temporal and stochastic fluctuations can yield powerful quantitative insight. We concentrate on experiments that localize and count individual molecules of mRNA. These high precision experiments have large imaging and computational processing costs, and we explore how improved computational analyses can dramatically reduce overall data requirements. In particular, we show how analyses of spatial, temporal and stochastic fluctuations can significantly enhance parameter estimation results for small, noisy data sets. We also show how full probability distribution analyses can constrain parameters with far less data than bulk analyses or statistical moment closures. Finally, we discuss how a systematic modeling progression from simple to more complex analyses can reduce total computational costs by orders of magnitude. We illustrate our approach using single-molecule, spatial mRNA measurements of Interleukin 1-alpha mRNA induction in human THP1 cells following stimulation. Our approach could improve the effectiveness of single-molecule gene regulation analyses for many other process.

Experiment on aggregation of red cells under microgravity on STS 51-C

NASA Astrophysics Data System (ADS)

Dintenfass, L.; Osman, P.; Maguire, B.; Jedrzejczyk, H.

Kinetics and morphology of aggregation of red cells were studied using automatic slit-capillary photo-viscometers, one situated on the middeck of the space shuttle `Discovery', and the other in the ground laboratory at KSC. Experiments were run simultaneously, blood samples being adjusted to haematocrit of 0.30 using native plasma, at temp. of 25°C, and anticoagulated by EDTA. Donors included patients with myocardial infarction, insulin-dependent diabetes, hyperlipidaemia and hypertension. Macro and microphotographs were obtained during flow and statis. There was a striking difference in the morphology of aggregates formed in space and on the ground. Aggregates formed under zero gravity showed rouleaux formation, while the same blood samples showed severe clumping on the ground, in all patients blood. Normal blood showed rouleaux on the ground, but a random swarm-like pattern in space. The shape of the red cells remained normal under zero gravity.

Research on equipment of micro-pressure measure and control in loading experiment of plant cell mechanics

NASA Astrophysics Data System (ADS)

Zhang, Lian; Yu, Chengbo; Tao, Hongyan; Chen, Xuejun; Zhai, Feng

2005-12-01

The equipment is developed to measure and control micro-pressure in loading experiment of plant cell mechanics. The motivation for the development of this equipment was to maintain a stationary micro-pressure on the agar of culturing cells to keep cytoactive in biology experiments. A singlechip controls the stepping motor of this equipment to drive loading equipment in the system, in order to load between 50mN and 250mN under a constant voltage. The accuracy is estimated to be +/-0.4 mN. The structure and control system of this equipment is introduced and described in detail. The experimental results show that the equipment is capable of maintaining a constant, stationary micropressure in cell culturing application and is worth of extending and applying.

Role of 5-aminolevulinic acid-conjugated gold nanoparticles for photodynamic therapy of cancer

NASA Astrophysics Data System (ADS)

Zhang, Zhenxi; Wang, Sijia; Xu, Hao; Wang, Bo; Yao, Cuiping

2015-05-01

There are three possible mechanisms for 5-aminolevulinic acid (5-ALA) conjugated gold nanoparticles (GNPs) through electrostatic bonding for photodynamic therapy (PDT) of cancer: GNPs delivery function, singlet oxygen generation (SOG) by GNPs irradiated by light, and surface resonance enhancement (SRE) of SOG. Figuring out the exact mechanism is important for further clinical treatment. 5-ALA-GNPs and human chronic myeloid leukemia K562 cells were used to study delivery function and SOG by GNPs. The SRE of SOG enabled by GNPs was explored by protoporphyrin IX (PpIX)-GNPs conjugate through electrostatic bonding. Cell experiments show that the GNPs can improve the efficiency of PDT, which is due to the vehicle effect of GNPs. PpIX-GNPs conjugate experiments demonstrated that SOG can be improved about 2.5 times over PpIX alone. The experiments and theoretical results show that the local field enhancement (LFE) via localized surface plasmon resonance (LSPR) of GNPs is the major role; the LFE was dependent on the irradiation wavelength and the GNP's size. The LFE increased with an increase of the GNP size (2R ≤50 nm). However, the LSPR function of the GNPs was not found in cell experiments. Our study shows that in 5-ALA-conjugated GNPs PDT, the delivery function of GNPs is the major role.

Screening of soy protein-derived hypotriglyceridemic di-peptides in vitro and in vivo

PubMed Central

2011-01-01

Background Soy protein and soy peptides have attracted considerable attention because of their potentially beneficial biological properties, including antihypertensive, anticarcinogenic, and hypolipidemic effects. Although soy protein isolate contains several bioactive peptides that have distinct physiological activities in lipid metabolism, it is not clear which peptide sequences are responsible for the triglyceride (TG)-lowering effects. In the present study, we investigated the effects of soy protein-derived peptides on lipid metabolism, especially TG metabolism, in HepG2 cells and obese Otsuka Long-Evans Tokushima fatty (OLETF) rats. Results In the first experiment, we found that soy crude peptide (SCP)-LD3, which was prepared by hydrolyze of soy protein isolate with endo-type protease, showed hypolipidemic effects in HepG2 cells and OLETF rats. In the second experiment, we found that hydrophilic fraction, separated from SCP-LD3 with hydrophobic synthetic absorbent, revealed lipid-lowering effects in HepG2 cells and OLETF rats. In the third experiment, we found that Fraction-C (Frc-C) peptides, fractionated from hydrophilic peptides by gel permeation chromatography-high performance liquid chromatography, significantly reduced TG synthesis and apolipoprotein B (apoB) secretion in HepG2 cells. In the fourth experiment, we found that the fraction with 0.1% trifluoroacetic acid, isolated from Frc-C peptides by octadecylsilyl column chromatography, showed hypolipidemic effects in HepG2 cells. In the final experiment, we found that 3 di-peptides, Lys-Ala, Val-Lys, and Ser-Tyr, reduced TG synthesis, and Ser-Tyr additionally reduced apoB secretion in HepG2 cells. Conclusion Novel active peptides with TG-lowering effects from soy protein have been isolated. PMID:21600040

Spin glass model for dynamics of cell reprogramming

NASA Astrophysics Data System (ADS)

Pusuluri, Sai Teja; Lang, Alex H.; Mehta, Pankaj; Castillo, Horacio E.

2015-03-01

Recent experiments show that differentiated cells can be reprogrammed to become pluripotent stem cells. The possible cell fates can be modeled as attractors in a dynamical system, the ``epigenetic landscape.'' Both cellular differentiation and reprogramming can be described in the landscape picture as motion from one attractor to another attractor. We perform Monte Carlo simulations in a simple model of the landscape. This model is based on spin glass theory and it can be used to construct a simulated epigenetic landscape starting from the experimental genomic data. We re-analyse data from several cell reprogramming experiments and compare with our simulation results. We find that the model can reproduce some of the main features of the dynamics of cell reprogramming.

X ray sensitivity of diploid skin fibroblasts from patients with Fanconi's anemia

NASA Technical Reports Server (NTRS)

Kale, Ranjini

1989-01-01

Experiments were performed on Fanconi's anemia and normal human fibroblast cell lines growing in culture in an attempt to correlate cell cycle kinetics with genomic damage and determine their bearing on the mechanism of chromosome aberration induction. FA fibroblasts showed a significantly increased susceptibility to chromosomal breakage by x rays in the G2 phase of the cell cycle. No such response was observed in fibroblasts irradiated in the G0 phase. The observed increases in achromatic lesions and in chromatid deletions in FA cells as compared with normal cells appear to indicate that FA cells are deficient in strand break repair and also possibly in base damage excision repair. Experiments are now in progress to further elucidate the mechanisms involved.

Normal Untreated Jurkat Cells

NASA Technical Reports Server (NTRS)

2004-01-01

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. The objective of the research was to define a way to differentiate between effects due to microgravity and those due to possible stress from non-optimal spaceflight conditions. These Jurkat cells, a human acute T-cell leukemia was obtained to evaluate three types of potential experimental stressors: a) Temperature elevation; b) Serum starvation; and c) Centrifugal force. The data from previous spaceflight experiments showed that actin filaments and cell shape are significantly different for the control. These normal cells serve as the baseline for future spaceflight experiments.

Androgens Exert a Cysticidal Effect upon Taenia crassiceps by Disrupting Flame Cell Morphology and Function

PubMed Central

Ambrosio, Javier R.; Valverde-Islas, Laura; Nava-Castro, Karen E.; Palacios- Arreola, M. Isabel; Ostoa-Saloma, Pedro; Reynoso-Ducoing, Olivia; Escobedo, Galileo; Ruíz-Rosado, Azucena; Dominguez-Ramírez, Lenin; Morales-Montor, Jorge

2015-01-01

The effects of testosterone (T4) and dihydrotestosterone (DHT) on the survival of the helminth cestode parasite Taenia crassiceps, as well as their effects on actin, tubulin and myosin expression and their assembly into the excretory system of flame cells are described in this paper. In vitro evaluations on parasite viability, flow cytometry, confocal microscopy, video-microscopy of live flame cells, and docking experiments of androgens interacting with actin, tubulin, and myosin were conducted. Our results show that T4 and DHT reduce T. crassiceps viability in a dose- and time-dependent fashion, reaching 90% of mortality at the highest dose used (40 ng/ml) and time exposed (10 days) in culture. Androgen treatment does not induce differences in the specific expression pattern of actin, tubulin, and myosin isoforms as compared with control parasites. Confocal microscopy demonstrated a strong disruption of the parasite tegument, with reduced assembly, shape, and motion of flame cells. Docking experiments show that androgens are capable of affecting parasite survival and flame cell morphology by directly interacting with actin, tubulin and myosin without altering their protein expression pattern. We show that both T4 and DHT are able to bind actin, tubulin, and myosin affecting their assembly and causing parasite intoxication due to impairment of flame cell function. Live flame cell video microscopy showing a reduced motion as well changes in the shape of flame cells are also shown. In summary, T4 and DHT directly act on T. crassiceps cysticerci through altering parasite survival as well as the assembly and function of flame cells. PMID:26076446

[Biological experiments on "Kosmos-1887"].

PubMed

Alpatov, A M; I'lin, E A; Antipov, V V; Tairbekov, M G

1989-01-01

In the 13-ray space flight on Kosmos-1887 various experiments in the field of cell biology, genetics, biorhythm, developmental biology and regeneration were performed using bacteria, protozoa, plants, worms, insects, fish and amphibia. Paramecia showed enhanced cell proliferation, spheroidization and diminished protein content. Experiments on fruit-flies, newt oocytes and primate lymphocytes confirmed involvement of the cell genetic apparatus in responses to microgravity. Beetles exhibited a reduction of the length of the spontaneous period of freely running circadian rhythms. Carausius morosus developed latent changes in early embryogenesis which manifested at later stages of ontogenesis. Exposure to microgravity did not prevent recovery of injured tissues; moreover their regeneration may be accelerated after recovery. Biology research programs in future biosatellite flights are discussed.

Proton pump inhibitors while belonging to the same family of generic drugs show different anti-tumor effect.

PubMed

Lugini, Luana; Federici, Cristina; Borghi, Martina; Azzarito, Tommaso; Marino, Maria Lucia; Cesolini, Albino; Spugnini, Enrico Pierluigi; Fais, Stefano

2016-08-01

Tumor acidity represents a major cause of chemoresistance. Proton pump inhibitors (PPIs) can neutralize tumor acidity, sensitizing cancer cells to chemotherapy. To compare the anti-tumor efficacy of different PPIs in vitro and in vivo. In vitro experiments PPIs anti-tumor efficacy in terms of cell proliferation and cell death/apoptosis/necrosis evaluation were performed. In vivo PPIs efficacy experiments were carried out using melanoma xenograft model in SCID mice. Lansoprazole showed higher anti-tumor effect when compared to the other PPIs. The lansoprazole effect lasted even upon drug removal from the cell culture medium and it was independent from the lipophilicity of the PPIs formulation. These PPIs have shown different anti-tumoral efficacy, and the most effective at low dose was lansoprazole. The possibility to contrast tumor acidity by off-label using PPIs opens a new field of oncology investigation.

Observable Protein Crystal Growth Apparatus

NASA Technical Reports Server (NTRS)

2001-01-01

This diagram shows a cross sectrion of the fluid volume of an individual cell in the Observable Protein Crystal Growth Apparatus (OPCGA) to be operated aboard the International Space Station (ISS). The principal investigator is Dr. Alex McPherson of the University of California, Irvine. Each individual cell comprises two sample chambers with a rotating center section that isolates the two from each other until the start of the experiment and after it is completed. The cells are made from optical-quality quartz glass to allow photography and interferometric observations. Each cell has a small light-emitting diode and lens to back-light the solution. In protein crystal growth experiments, a precipitating agent such as a salt solution is used to absorb and hold water but repel the protein molecules. This increases the concentration of protein until the molecules nucleate to form crystals. This cell is one of 96 that make up the experiment module portion of the OPCGA.

Non-linear Min protein interactions generate harmonics that signal mid-cell division in Escherichia coli

PubMed Central

Walsh, James C.; Angstmann, Christopher N.; Duggin, Iain G.

2017-01-01

The Min protein system creates a dynamic spatial pattern in Escherichia coli cells where the proteins MinD and MinE oscillate from pole to pole. MinD positions MinC, an inhibitor of FtsZ ring formation, contributing to the mid-cell localization of cell division. In this paper, Fourier analysis is used to decompose experimental and model MinD spatial distributions into time-dependent harmonic components. In both experiment and model, the second harmonic component is responsible for producing a mid-cell minimum in MinD concentration. The features of this harmonic are robust in both experiment and model. Fourier analysis reveals a close correspondence between the time-dependent behaviour of the harmonic components in the experimental data and model. Given this, each molecular species in the model was analysed individually. This analysis revealed that membrane-bound MinD dimer shows the mid-cell minimum with the highest contrast when averaged over time, carrying the strongest signal for positioning the cell division ring. This concurs with previous data showing that the MinD dimer binds to MinC inhibiting FtsZ ring formation. These results show that non-linear interactions of Min proteins are essential for producing the mid-cell positioning signal via the generation of second-order harmonic components in the time-dependent spatial protein distribution. PMID:29040283

Cytotoxic activity of natural killer cells in vitro under microgravity

NASA Astrophysics Data System (ADS)

Grigorieva, O. V.; Buravkova, L. B.; Rykova, M. P.

2005-08-01

Changes in the immune response during space flight are close relation to functions of NK lymphocytes and their ability to interact with target cells. The aim of this research was to study NK cells cytotoxic activity and their ability to produce cytokines under microgravity in vitro. The modification of the method to study NK cells cytotoxic activity with the use of human peripheral blood mononuclear cells and myeloblasts K-562 (as target cells) proved highly effective (Buravkova et al., 2004). The flight experiment "Cell-to-cell interaction" with the use of the special device "Fibroblast-1" was carried out by Russian cosmonauts within the first two days after the docking when a new crew was taking over on International Space Station (ISS 8 - 10). The data collected on board ISS revealed that NK lymphocytes cytotoxic activity in vitro can increase under microgravity. The ground-based simulation experiments showed that long-term changes in gravity vector direction clinorotation resulted in a smaller increase of NK cells cytotoxic activity than it did in microgravity. As lymphocytes produce cytokines while interacting with target cells, the levels of TNF-α, IL-1α, IL- 2, IL-6 in cell-conditioned medium were assessed. The data showed that microgravity has varied effects on cytokines production level.

Experiment on ``discovery'' STS 51-C: Aggregation of red cells and thrombocytes in heart disease, hyperlipidaemia and other conditions

NASA Astrophysics Data System (ADS)

Dintenfass, L.

The aim of this experiment was to study aggregation of red cells in the blood of patients with ischaemic heart disease, diabetes, hyperlipidaemia, and (silent) cancer, and in two normal donors. Reconstituted blood using IgG was also used. The instrument, the automated slit-capillary photo-viscometer (100 kg weight) was set on the middeck of the Space Shuttle. An analogous instrument was at the Kennedy Space Center. Blood was obtained from donors, anticoagulated, and adjusted to haematocrit of 30% using native plasma. Experiments took place at 25°C, during which blood was forced to flow in the slit formed by two parallel glass plates. Macro and microphotography was carried out at specific intervals controlled by a computer. During stasis, lasting 6 minutes, aggregates (or clumps) of the red cells were formed. Results indicated that red cell aggregates do form under zero-G; that such aggregates are smaller than the ones obtained at one-G; that morphology is different, the zero-G showing rouleaux while one-G showing usual sludge-like clumps of red cells in all severe disorders. Platelets appeared to remain monodisperse under zero-G. Assuming that these data can be confirmed, one could suggest that zero-G affects cell-cell interaction, and may consequently influence the internal microstructure of the cell membrane and of the receptors, as well as their activity. Gravitational studies may thus open a new door on immunology and haematology in general.

Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.

PubMed

Sørensen, M S; Duch, M; Paludan, K; Jørgensen, P; Pedersen, F S

1992-03-15

Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.

GEMINI-TITAN (GT)-3 - WEIGHTLESSNESS EXPERIMENT - AMES RESEARCH CENTER (ARC), CA

NASA Image and Video Library

1965-03-01

S65-18762 (March 1965) --- Effects of the weightless environment on cell division, the basic growth process for living tissue, will be studied during the Gemini-Titan 3 flight scheduled for March 23, 1965. A spiny black sea urchin (upper left) is stimulated by mild electric shock or potassium chloride. As a result it sheds many thousands of eggs. When fertilized, these eggs become actively dividing cells very similar in basic processes to cells of other animals, including humans. These pictures show stages of cell division. At upper right is a single cell; at lower right cell divisions have produced many cells. Cell photos are magnified about 700 times, and all cells shown are too small to be seen by the naked eye. (Photos at upper right and lower left are of sea urchin eggs. Group of cells at lower right are from a sand dollar, which like the sea urchin, is an Echinoderm. Its eggs are virtually identical and are used interchangeably with those of the sea urchin in NASA Ames Center weightlessness experiments.) The Gemini experiment will involve cell division like that shown here. This will take place during several hours of weightlessness aboard the Gemini spacecraft. The experiment will be flown back to laboratories at Cape Kennedy after spacecraft recovery. It has been designed so that any abnormal cell division found by postflight analysis should suggest that the weightless environment has effects on individual cells. This might mean hazards for prolonged periods of manned spaceflight.

Microgravity

NASA Image and Video Library

2001-01-24

This photo shows the Handheld Diffusion Test Cell (HH-DTC) apparatus flown on the Space Shuttle. Similar cells (inside the plastic box) will be used in the Observable Protein Crystal Growth Apparatus (OPCGA) to be operated aboard the International Space Station (ISS). The principal investigator is Dr. Alex McPherson of the University of California, Irvine. Each individual cell comprises two sample chambers with a rotating center section that isolates the two from each other until the start of the experiment and after it is completed. The cells are made from optical-quality quartz glass to allow photography and interferometric observations. Each cell has a small light-emitting diode and lens to back-light the solution. In protein crystal growth experiments, a precipitating agent such as a salt solution is used to absorb and hold water but repel the protein molecules. This increases the concentration of protein until the molecules nucleate to form crystals. This cell is one of 96 that make up the experiment module portion of the OPCGA.

Transport of polyamines in Drosophila S2 cells: kinetics, pharmacology and dependence on the plasma membrane proton gradient

PubMed Central

Romero-Calderón, Rafael; Krantz, David E.

2005-01-01

Polyamine transport activities have been described in diverse multicellular systems, but their bioenergetic mechanisms and molecular identity remain unclear. In the present paper, we describe a high-affinity spermine/spermidine transport activity expressed in Drosophila S2 cells. Ion-replacement experiments indicate that polyamine uptake across the cell membrane is Na+-, K+-, Cl−- and Ca2+-independent, but pH-sensitive. Additional experiments using ionophores suggest that polyamine uptake may be H+-coupled. Pharmacological experiments show that polyamine uptake in S2 cells is selectively blocked by MGBG {methylglyoxal bis(guanylhydrazone) or 1,1′-[(methylethanediylidine)-dinitrilo]diguanidine} and paraquat (N,N-dimethyl-4,4′-bipyridylium), two known inhibitors of polyamine uptake in mammalian cells. In addition, inhibitors known to block the Slc22 (solute carrier 22) family of organic anion/cation transporters inhibit spermine uptake in S2 cells. These data and the genetic tools available in Drosophila will facilitate the molecular identification and further characterization of this activity. PMID:16248856

Transport of polyamines in Drosophila S2 cells: kinetics, pharmacology and dependence on the plasma membrane proton gradient.

PubMed

Romero-Calderón, Rafael; Krantz, David E

2006-01-15

Polyamine transport activities have been described in diverse multicellular systems, but their bioenergetic mechanisms and molecular identity remain unclear. In the present paper, we describe a high-affinity spermine/spermidine transport activity expressed in Drosophila S2 cells. Ion-replacement experiments indicate that polyamine uptake across the cell membrane is Na+-, K+-, Cl-- and Ca2+-independent, but pH-sensitive. Additional experiments using ionophores suggest that polyamine uptake may be H+-coupled. Pharmacological experiments show that polyamine uptake in S2 cells is selectively blocked by MGBG {methylglyoxal bis(guanylhydrazone) or 1,1'-[(methylethanediylidine)-dinitrilo]diguanidine} and paraquat (N,N-dimethyl-4,4'-bipyridylium), two known inhibitors of polyamine uptake in mammalian cells. In addition, inhibitors known to block the Slc22 (solute carrier 22) family of organic anion/cation transporters inhibit spermine uptake in S2 cells. These data and the genetic tools available in Drosophila will facilitate the molecular identification and further characterization of this activity.

Evaluation of the anti-proliferative and cytostatic effect of Citrus sinensis (orange) fruit juice.

PubMed

Chinedu, Enegide; Arome, David; Ameh, Solomon F; Ameh, Gift E

2014-09-01

This work has been designed to evaluate the anti-proliferative and cytostatic effects of Citrus sinensis (orange) fruit juice on rapidly proliferating cells. The study was carried out on the seeds of Sorghum bicolor for 72 h. The mean radicle length (mm) of the seeds was taken at 48 and 72 h. The result showed that when compared with the control, methotrexate, the standard drug showed a significant (P < 0.001) anti-proliferative effect throughout the experiment. The inhibition of the radicle growth was more after 72 h (87.42%). At a dose of 5% (v/v), the juice showed a slightly significant (P < 0.05) effect affect after 72 h; however, there was no significant effect at 48 h. The juice at doses of 10% and 20% (v/v) showed a highly significant (P < 0.001) anti-proliferative effect throughout the experiment; however, the percentage inhibitions were higher at 72 h. At 72 h, the percentage inhibition for juice at 10% (v/v) was 72.37% and at 20% (v/v) was 91.96%. The concentrations of 40% and 60% (v/v) showed cytostatic effects as no appreciable growth of the radicles of the seeds was observed throughout the experiment. The percentage inhibition for 40% (v/v) was 100% and 99.72% for 48 and 72 h, respectively, while that for the juice concentration of 60% (v/v) was 100% throughout the study. The experiment has shown that C. sinensis fruit juice has a potential for causing both anti-proliferative and cytostatic effects on fast proliferating cells and hence cancerous cells.

[CONDITIONS OF SYNOVIAL MESENCHYMAL STEM CELLS DIFFERENTIATING INTO FIBROCARTILAGE CELLS].

PubMed

Fu, Peiliang; Cong, Ruijun; Chen, Song; Zhang, Lei; Ding, Zheru; Zhou, Qi; Li, Lintao; Xu, Zhenyu; Wu, Yuli; Wu, Haishan

2015-01-01

To explore the conditions of synovial derived mesenchymal stem cells (SMSCs) differentiating into the fibrocartilage cells by using the orthogonal experiment. The synovium was harvested from 5 adult New Zealand white rabbits, and SMSCs were separated by adherence method. The flow cytometry and multi-directional differentiation method were used to identify the SMSCs. The conditions were found from the preliminary experiment and literature review. The missing test was carried out to screen the conditions and then 12 conditions were used for the orthogonal experiment, including transforming growth factor β1 (TGF-β1), bone morphogenic protein 2 (BMP-2), dexamethasone (DEX), proline, ascorbic acid (ASA), pyruvic acid, insulin + transferrin + selenious acid pre-mixed solution (ITS), bovin serum albumin (BSA), basic fibroblast growth factor (bFGF), intermittent hydraulic pressure (IHP), bone morphogenic protein 7 (BMP-7), and insulin-like growth factor (IGF). The L60 (212) orthogonal experiment was designed using the SPSS 18.0 with 2 level conditions and the cells were induced to differentiate on the small intestinal submucosa (SIS)-3D scaffold. The CD151+/CD44+ cells were detected with the flow cytometry and then the differentiation rate was recorded. The immumohistochemical staining, cellular morphology, toluidine blue staining, and semi-quantitative RT-PCR examination for the gene expressions of sex determining region Y (SRY)-box 9 gene (Sox9), aggrecan gene (AGN), collagen type I gene (Col I), collagen type II gene (Col II), collagen type IX gene (Col IX) were used for result confirmation. The differentiation rate was calculated as the product of CD151/CD44+ cells and cells with Col I high expression. The grow curve was detected with the DNA abundance using the PicoGreen Assay. The visual observation and the variances analysis among the variable were used to evaluate the result of the orthogonal experiment, 1 level interaction was considered. The q-test and the least significant difference (LDS) were used for the variance analysis with a type III calibration model. The test criteria (a) was 0.05. The cells were certified as SMSCs, the double-time of the cells was 28 hours. During the differentiation into the fibrocartilage, the volume of the SIS-3D scaffold enlarged double every 5 days. The scaffolds were positively stained by toluidine blue at 14 days. The visual observation showed that high levels of TGF-β1 and BMP-7 were optimum for the differentiation, and BMP-7 showed the interaction with BMP-2. The conditions of DEX, ASA, ITS, transferrin, bFGF showed decreasing promotional function by degrees, and the model showed the perfect relevance. P value was 0.000 according to the variance analysis. The intercept analysis showed different independent variables brought about variant contribution; the TGF-β1, ASA, bFGF, IGF, and BMP-7 were more remarkable, which were similar to the visual observation. In the process of the SMSCs differentiation into the fibrocartilage, the concentrations of TGF-β1, ASA, bFGF, and IGF reasonably can improve the conversion rate of the fibrocartilage cells. The accurate conditions of the reaulatory factor should be explored further.

Effects of peptides on proliferative activity of retinal and pigmented epithelial cells.

PubMed

Khavinson, V Kh; Zemchikhina, V N; Trofimova, S V; Malinin, V V

2003-06-01

We studied the effects of Retinalamin (polypeptide preparation isolated from the retina) and a synthetic peptide Epithalon (Ala-Glu-Asp-Gly) on proliferative activity of retinal and pigmented epithelial cells. Experiments showed that Retinalamin and Epithalon (in certain concentrations) tissue-specifically stimulated proliferation of retinal and pigmented epithelial cell in culture.

Fabrication of Biocompatible Potassium Sodium Niobate Piezoelectric Ceramic as an Electroactive Implant

PubMed Central

Chen, Wei; Yu, Zunxiong; Pang, Jinshan; Yu, Peng; Tan, Guoxin; Ning, Chengyun

2017-01-01

The discovery of piezoelectricity in natural bone has attracted extensive research in emulating biological electricity for various tissue regeneration. Here, we carried out experiments to build biocompatible potassium sodium niobate (KNN) ceramics. Then, influence substrate surface charges on bovine serum albumin (BSA) protein adsorption and cell proliferation on KNN ceramics surfaces was investigated. KNN ceramics with piezoelectric constant of ~93 pC/N and relative density of ~93% were fabricated. The adsorption of protein on the positive surfaces (Ps) and negative surfaces (Ns) of KNN ceramics with piezoelectric constant of ~93 pC/N showed greater protein adsorption capacity than that on non-polarized surfaces (NPs). Biocompatibility of KNN ceramics was verified through cell culturing and live/dead cell staining of MC3T3. The cells experiment showed enhanced cell growth on the positive surfaces (Ps) and negative surfaces (Ns) compared to non-polarized surfaces (NPs). These results revealed that KNN ceramics had great potential to be used to understand the effect of surface potential on cells processes and would benefit future research in designing piezoelectric materials for tissue regeneration. PMID:28772704

Fabrication of Biocompatible Potassium Sodium Niobate Piezoelectric Ceramic as an Electroactive Implant.

PubMed

Chen, Wei; Yu, Zunxiong; Pang, Jinshan; Yu, Peng; Tan, Guoxin; Ning, Chengyun

2017-03-26

The discovery of piezoelectricity in natural bone has attracted extensive research in emulating biological electricity for various tissue regeneration. Here, we carried out experiments to build biocompatible potassium sodium niobate (KNN) ceramics. Then, influence substrate surface charges on bovine serum albumin (BSA) protein adsorption and cell proliferation on KNN ceramics surfaces was investigated. KNN ceramics with piezoelectric constant of ~93 pC/N and relative density of ~93% were fabricated. The adsorption of protein on the positive surfaces (Ps) and negative surfaces (Ns) of KNN ceramics with piezoelectric constant of ~93 pC/N showed greater protein adsorption capacity than that on non-polarized surfaces (NPs). Biocompatibility of KNN ceramics was verified through cell culturing and live/dead cell staining of MC3T3. The cells experiment showed enhanced cell growth on the positive surfaces (Ps) and negative surfaces (Ns) compared to non-polarized surfaces (NPs). These results revealed that KNN ceramics had great potential to be used to understand the effect of surface potential on cells processes and would benefit future research in designing piezoelectric materials for tissue regeneration.

The citrus methoxyflavone tangeretin affects human cell-cell interactions.

PubMed

Brack, Marc E; Boterberg, Tom; Depypere, Herman T; Stove, Christophe; Leclercq, Georges; Mareel, Marc M

2002-01-01

Two effects of the citrus methoxyflavone tangeretin on cell-cell interactions are biologically relevant. Firstly, tangeretin upregulates the function of the E-cadherin/catenin complex in human MCF-7/6 breast carcinoma cells. This leads to firm cell-cell adhesion and inhibition of invasion in vitro. Secondly, tangeretin downregulates the interleukin-2 receptor on T-lymphocytes and natural killer cells. This leads to a decrease in the cytotoxic competence of these immunocytes against cancer cells. The second effect can become important when high doses of tangeretin are combined with adjuvant tamoxifen treatment for breast cancer. Experiments with nude mice bearing MCF-7/6 tumors showed that tangeretin given orally at high doses, abrogated the therapeutic suppression of tumor growth exerted by tamoxifen. No evidence for a tumor promoting effect of tangeretin by itself was found in these experiments. Tangeretin may be an interesting molecule for application in cases where immunosuppression could be clinically beneficial.

Space environment effect on cell cycle of proliferating FRTL-5 cells

NASA Astrophysics Data System (ADS)

Curcio, Francesco; Saverio Ambesi-Impiombato, Francesco; Meli, Antonella; Perrella, Giuseppina; Spelat, Renza; Zambito, Anna Maria

The space environment is a unique laboratory to study the response of living organisms to microgravity and cosmic radiation at the molecular and cellular levels. Significant results obtained by us during the Eneide Mission (Soyuz 9S and 10S 2005) showed a different sensitivity to space environment of cells in proliferative state as compared to those in physiological stand-by. The main object of our investigation was to validate these important findings and to study the molecular mechanisms underlying the phenomenon. To this purpose, a cell model of normal cells derived from rat thyroids which can be kept unattended for up to 20 days in a proliferative medium and at room temperature (FRTL-5) were used in a 10 days experiment on a FOTON satellite and in a 15 days experiment in the STS-120 shuttle mission. Experimental design for both flights was planned on the basis of the "ENEIDE" mission results. Microarray analysis has been performed on the samples from Foton M3 and STS-120. Background subtraction, quality assessment and normalization as well as the definition of specific evaluation algorithms have been performed. Based on the hyper G Test function we computed the Hyper geometric p-values for over representation of genes at all Gene Ontology (GO) terms in the induced GO graphs; this test was performed for each GO category and applied also to KEGG pathways. Results show the good quality of the experiment and our data show that the pathways mostly affected by the flight are: a) the cell cycle, b) the ubiquitin mediated proteolysis, c) the repair mechanisms, d) the adherens junction and e) the pyrimidine metabolism. The patways studied indicate that the cells suffer a slowing of cell cycle as well as upregulation of the DNA and RNA repair processes and even further corroborate the validity of using the FRTL5 cells as biosensors for monitoring the effectiveness of countermeasures to damage caused by the Space.

Synthesis and anticancer activity of novel curcumin-quinolone hybrids.

PubMed

Raghavan, Saiharish; Manogaran, Prasath; Gadepalli Narasimha, Krishna Kumari; Kalpattu Kuppusami, Balasubramanian; Mariyappan, Palanivelu; Gopalakrishnan, Anjana; Venkatraman, Ganesh

2015-09-01

A number of new curcumin-quinolone hybrids were synthesised from differently substituted 3-formyl-2-quinolones and vanillin and their in vitro cytotoxicity was determined on a panel of representative cell lines (A549, MCF7, SKOV3 and H460) using MTT assay. The most potent compound 14, was analysed for its mode of action using various cell biology experiments. SKOV3 cells treated with compound 14 showed distorted cell morphology under phase contrast imaging and induction of apoptosis was confirmed by Annexin V/PE assay. Further experiments on generation of reactive oxygen species (ROS) and cell cycle analysis revealed that these hybrids induce apoptosis by ROS generation and arrest cell cycle progression in S and G2/M phase. Copyright © 2015 Elsevier Ltd. All rights reserved.

Microgravity and bone cell mechanosensitivity: FLOW experiment during the DELTA mission

NASA Astrophysics Data System (ADS)

Bacabac, Rommel G.; Van Loon, Jack J. W. A.; de Blieck-Hogervorst, Jolanda M. A.; Semeins, Cor M.; Zandieh-Doulabi, Behrouz; Helder, Marco N.; Smit, Theo H.; Klein-Nulend, Jenneke

2007-09-01

The catabolic effects of microgravity on mineral metabolism in bone organ cultures might be explained as resulting from an exceptional form of disuse. It is possible that the mechanosensitivity of bone cells is altered under near weightlessness conditions, which likely contributes to disturbed bone metabolism observed in astronauts. In the experiment "FLOW", we tested whether the production of early signaling molecules that are involved in the mechanical load-induced osteogenic response by bone cells is changed under microgravity conditions. FLOW was one of the Biological experiment entries to the Dutch Soyuz Mission "DELTA" (Dutch Expedition for Life Science, Technology and Atmospheric Research). FLOW was flown by the Soyuz craft, launched on April 19, 2004, on its way to the International Space Station. Primary osteocytes, osteoblasts, and periosteal fibroblasts were incubated in plunger boxes, developed by Centre for Concepts in Mechatronics, using plunger activation events for single pulse fluid shear stress stimulations. Due to unforeseen hardware complications, results from in-flight cultures are considered lost. Ground control experiments showed an accumulative increase of NO in medium for osteocytes (as well as for osteoblasts and periosteal fibroblasts). Data from the online-NO sensor showed that the NO produced in medium by osteocytes increased sharply after pulse shear stress stimulations. COX-2 mRNA expression revealed high levels in osteoblasts compared to the other cell types tested. In conclusion, preparations for the FLOW experiment and preliminary ground results indicate that the FLOW setup is viable for a future flight opportunity.

The illness of women and men with sickle cell disease: a Grounded Theory study.

PubMed

Cordeiro, Rosa Cândida; Ferreira, Silvia Lúcia; Santos, Ane Caroline da Cruz

2015-01-01

To understand the meanings given by women and men with sickle cell disease on the illness experience. Analytical study with a qualitative approach, conducted with 17 adults with sickle cell disease using the Theory Based on Data, or Grounded Theory, as theoretical-methodological referential. Data were collected between the years of 2012 and 2013, in an individual in-depth interview. All the interviews were recorded and analyzed according to the Grounded Theory comparative analysis technique. Data show four categories which group the experience of illness, the feelings experienced and the path to living with sickle cell disease. It was possible to understand that the experience was built by a process in which these people redefined the meaning of their lives, applying new directions to life and to care regarding the experience of the illness. In the context of chronic disease, the nurse's care is also seen in this study as a foundation, providing attention, directions, and guidance through the required confrontations. Understanding the experience lived by these people, it is possible to enlarge the dimensions and the essence of nursing care required throughout life.

Microgravity

NASA Image and Video Library

2004-04-15

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. The objective of the research was to define a way to differentiate between effects due to microgravity and those due to possible stress from non-optimal spaceflight conditions. These Jurkat cells, a human acute T-cell leukemia was obtained to evaluate three types of potential experimental stressors: a) Temperature elevation; b) Serum starvation; and c) Centrifugal force. The data from previous spaceflight experiments showed that actin filaments and cell shape are significantly different for the control. These normal cells serve as the baseline for future spaceflight experiments.

High-content image analysis (HCIA) assay has the highest correlation with direct counting cell suspension compared to the ATP, WST-8 and Alamar blue assays for measurement of cytotoxicity.

PubMed

Tahara, Haruna; Matsuda, Shun; Yamamoto, Yusuke; Yoshizawa, Hiroe; Fujita, Masaharu; Katsuoka, Yasuhiro; Kasahara, Toshihiko

2017-11-01

Various cytotoxicity assays measuring indicators such as enzyme activity, dye uptake, or cellular ATP content are often performed using 96-well microplates. However, recent reports show that cytotoxicity assays such as the ATP assay and MTS assay underestimate cytotoxicity when compounds such as anti-cancer drugs or mutagens induce cell hypertrophy whilst increasing intracellular ATP content. Therefore, we attempted to evaluate the reliability of a high-content image analysis (HCIA) assay to count cell number in a 96-well microplate automatically without using a cell-number indicator. We compared cytotoxicity results of 25 compounds obtained from ATP, WST-8, Alamar blue, and HCIA assays with those directly measured using an automatic cell counter, and repeating individual experiments thrice. The number of compounds showing low correlation in cell viability measured using cytotoxicity assays compared to automatic cell counting (r 2 <0.8, at least 2 of 3 experiments) were follows: ATP assay; 7; WST-8 assay, 2; Alamar blue assay, 3; HCIA cytotoxicity assay, 0. Compounds for which correlation was poor in 3 assays, except the HCIA assay, induced an increase in nuclear and cell size. However, correlation between cell viability measured by automatic cell counter and the HCIA assay was strong regardless of nuclear and cell size. Additionally, correlation coefficients between IC 50 values obtained from automatic cell counter and from cytotoxicity assays were as follows: ATP assay, 0.80; WST-8 assay, 0.84; Alamar blue assay, 0.84; and HCIA assay, 0.98. From the above, we showed that the HCIA cytotoxicity assay produces similar data to the automatic cell counter and is highly accurate in measuring cytotoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

Cell therapy: a therapeutic alternative to treat focal cartilage lesions.

PubMed

Gimeno, M J; Maneiro, E; Rendal, E; Ramallal, M; Sanjurjo, L; Blanco, F J

2005-11-01

Human mesenchymal stem cells (MSCs) are present in most of the tissue matrix, taking part in their regeneration when injury or damage occurs. The aim of this study was to investigate the presence of cells with pluripotential characteristics in synovial membranes from osteoarthritic (OA) patients and the capacity of these cells to differentiate to chondrocytes. Synovial membranes (n = 8) from OA patients were digested with collagenase. Isolated cells were cultured with DMEM, 20% FBS, and FGFb10 ng/mL. Cells from second subculture were used to carry out phenotypic characterization experiments (flow cytometry analysis with 11 monoclonal antibodies) and chondrogenic differentiation experiments(micropellet cultured in chondrogenic medium). Chondrogenic differentiation of cells was assessment by quantification of cartilage extracellular matrix components by following techniques: Safranin O, Toluidine Blue, and Alcian Blue stains to detect proteoglycans and immunohistochemistry to detect type I and II collagen. Flow cytometry analyses showed that in our population more than 90% of cells were positive for MSC markers: CD29 (95%), CD44 (90%), CD73 (95%), CD90 (98%). Cells were negative for hematopoietic markers (CD11b, CD34, and CD45). Furthermore, cells showed positive stain to multipotent markers such as CD117 (c-kit) (98%), CD166 (74%), and STRO-1 (88%) and to quiescent satellite cells like PAX-7 (35%). The micropellet analyses showed that the culture of these cells with TGFbeta-3 for 2 and 3 weeks stimulates proteoglycan and collagen type II synthesis. Both molecules are characteristic of hyaline articular cartilage. In this work, we demonstrate the presence of a cellular population with MSC characteristics in synovial tissue from OA patients. As MSC takes part in reparative processes of adult tissues, these cells could play an important role in OA pathogenesis and treatment.

Hematopoietic Stem Cell Transplantation in Thalassemia and Sickle Cell Disease. Unicenter Experience in a Multi-Ethnic Population.

PubMed Central

Marziali, Marco; Isgrò, Antonella; Gaziev, Javid; Lucarelli, Guido

2009-01-01

Hematopoietic stem cell transplantation (HSCT) still remains the only definitive cure currently available for patients with thalassemia and sickle cell anemia. Results of transplant in thalassemia and in sickle cell anemia have steadily improved over the last two decades due to improvements in preventive strategies, and effective control of transplant-related complications. From 2004 through 2009, 145 consecutive patients with thalassemia and sickle cell anemia, ethnically heterogeneous from Mediterranean and Middle East countries, were given HSCT in the International Center for Transplantation in Thalassemia and Sickle Cella Anemia in Rome. This experience is characterized by two peculiarities: patients were ethnically very heterogeneous and the vast majority of these patients were not regularly transfesed/chelated and therefore were highly sensitized due to RBC transfusions without leukodepletion filters. Consequently, they could have a high risk of graft rejection as a result of sensitization to HLA antigens. The Rome experience of SCT in patients with thalassemia and sickle cell anemia confirmed the results obtained in Pesaro, and most importantly showed the reproducibility of these results in other centers. PMID:21415995

Solar Cells in the School Physics Laboratory.

ERIC Educational Resources Information Center

Mikulski, Kazimeirz

1996-01-01

Discusses the goals of experiments which show examples of the use of solar energy on a scale suitable for a school laboratory. Highlights the history of discoveries and developments in photoelectricity. Presents investigations and experiments, that can be performed by students. (JRH)

Contribution of macrophages in the contrast loss in iron oxide-based MRI cancer cell tracking studies

PubMed Central

Danhier, Pierre; Deumer, Gladys; Joudiou, Nicolas; Bouzin, Caroline; Levêque, Philippe; Haufroid, Vincent; Jordan, Bénédicte F.; Feron, Olivier; Sonveaux, Pierre; Gallez, Bernard

2017-01-01

Magnetic resonance imaging (MRI) cell tracking of cancer cells labeled with superparamagnetic iron oxides (SPIO) allows visualizing metastatic cells in preclinical models. However, previous works showed that the signal void induced by SPIO on T2(*)-weighted images decreased over time. Here, we aim at characterizing the fate of iron oxide nanoparticles used in cell tracking studies and the role of macrophages in SPIO metabolism. In vivo MRI cell tracking of SPIO positive 4T1 breast cancer cells revealed a quick loss of T2* contrast after injection. We next took advantage of electron paramagnetic resonance (EPR) spectroscopy and inductively coupled plasma mass spectroscopy (ICP-MS) for characterizing the evolution of superparamagnetic and non-superparamagnetic iron pools in 4T1 breast cancer cells and J774 macrophages after SPIO labeling. These in vitro experiments and histology studies performed on 4T1 tumors highlighted the quick degradation of iron oxides by macrophages in SPIO-based cell tracking experiments. In conclusion, the release of SPIO by dying cancer cells and the subsequent uptake of iron oxides by tumor macrophages are limiting factors in MRI cell tracking experiments that plead for the use of (MR) reporter-gene based imaging methods for the long-term tracking of metastatic cells. PMID:28467814

Multiple P2Y receptor subtypes in the apical membranes of polarized epithelial cells

PubMed Central

McAlroy, H L; Ahmed, S; Day, S M; Baines, D L; Wong, H Y; Yip, C Y; Ko, W H; Wilson, S M; Collett, A

2000-01-01

Apical ATP, ATP, UTP and UDP evoked transient increases in short circuit current (ISC, a direct measure of transepithelial ion transport) in confluent Caco-2 cells grown on permeable supports. These responses were mediated by a population of at least three pharmacologically distinct receptors. Experiments using cells grown on glass coverslips showed that ATP and UTP consistently increased intracellular free calcium ([Ca2+]i) whilst sensitivity to UDP was variable. Cross desensitization experiments suggested that the responses to UTP and ATP were mediated by a common receptor population. Messenger RNA transcripts corresponding to the P2Y2, P2Y4 and P2Y6 receptors genes were detected in cells grown on Transwell membranes by the reverse transcriptase–polymerase chain reaction. Identical results were obtained for cells grown on glass. Experiments in which ISC and [Ca2+]i were monitored simultaneously in cells on Transwell membranes, confirmed that apical ATP and UTP increased both parameters and showed that the UDP-evoked increase in ISC was accompanied by a [Ca2+]i-signal. Ionomycin consistently increased [Ca2+]i in such polarized cells but caused no discernible change in ISC. However, subsequent application of apical ATP or UTP evoked a small rise in ISC but no rise in [Ca2+]i. UDP evoked no such response. As well as evoking increases in [Ca2+]i, the ATP/UTP-sensitive receptors present in Caco-2 cells thus allow direct control over ion channels in the apical membrane. The UDP-sensitive receptors, however, appear to simply evoke a rise in [Ca2+]i. PMID:11139443

Drosophila glypican Dally-like acts in FGF-receiving cells to modulate FGF signaling during tracheal morphogenesis

PubMed Central

Yan, Dong; Lin, Xinhua

2007-01-01

Summary Previous studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are involved in both breathless (btl)- and heartless (htl)-mediated FGF signaling during embryogenesis. However, the mechanism(s) by which HSPGs control Btl and Htl signaling is unknown. Here we show that dally-like (dlp, a Drosophila glypican) mutant embryos exhibit severe defects in tracheal morphogenesis and show a reduction in btl-mediated FGF signaling activity. However, htl-dependent mesodermal cell migration is not affected in dlp mutant embryos. Furthermore, expression of Dlp, but not other Drosophila HSPGs, can restore effectively the tracheal morphogenesis in dlp embryos. Rescue experiments in dlp embryos demonstrate that Dlp functions only in Bnl/FGF receiving cells in a cell-autonomous manner, but is not essential for Bnl/FGF expression cells. To further dissect the mechanism(s) of Dlp in Btl signaling, we analyzed the role of Dlp in Btl-mediated air sac tracheoblast formation in wing discs. Mosaic analysis experiments show that removal of HSPG activity in FGF-producing or other surrounding cells does not affect tracheoblasts migration, while HSPG mutant tracheoblast cells fail to receive FGF signaling. Together, our results argue strongly that HSPGs regulate Btl signaling exclusively in FGF-receiving cells as co-receptors, but are not essential for the secretion and distribution of the FGF ligand. This mechanism is distinct from HSPG functions in morphogen distribution, and is likely a general paradigm for HSPG functions in FGF signaling in Drosophila. PMID:17959166

Loss of Citron Kinase Affects a Subset of Progenitor Cells That Alters Late but Not Early Neurogenesis in the Developing Rat Retina

PubMed Central

Karunakaran, Devi Krishna Priya; Chhaya, Nisarg; Lemoine, Christopher; Congdon, Sean; Black, Amye; Kanadia, Rahul

2015-01-01

Purpose. To understand how loss of citron kinase (CitK) affects retinal progenitor cells (RPCs) in the developing rat retina. Methods. We compared knockout (KO) and wild-type (WT) retinae by immunohistochemistry. The TdT-mediated dUTP terminal nick-end labeling (TUNEL) assay was performed to determine cell death. Pulse-chase experiments using 5-ethynyl-2’-deoxyuridine (EdU) were carried out to interrogate RPC behavior and in turn neurogenesis. Results. Reverse transcription–polymerase chain reaction analysis showed that CitK was expressed at embryonic day (E)12 and was turned off at approximately postnatal day (P)4. Immunohistochemistry showed CitK being localized as puncta at the apical end of the outer neuroblastic layer (ONBL). Analyses during embryonic development showed that the KO retina was of comparable size to that of WT until E13. However, by E14, there was a reduction in the number of S-phase RPCs with a concomitant increase in TUNEL+ cells in the KO retina. Moreover, early neurogenesis, as reflected by retinal ganglion cell production, was not affected. Postnatal analysis of the retina showed that ONBL in the KO retina was reduced to half the size of that in WT and showed further degeneration. Immunohistochemistry revealed absence of Islet1+ bipolar cells at P2, which was further confirmed by EdU pulse-chase experiments. The CitK KO retinae underwent complete degeneration by P14. Conclusions. Our study showed that CitK is not required for a subset of RPCs before E14, but is necessary for RPC survival post E14. This in turn results in normal early embryonic neurogenesis, but severely compromised later embryonic and postnatal neurogenesis. PMID:25593024

Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

PubMed

Brewer, B J; Chlebowicz-Sledziewska, E; Fangman, W L

1984-11-01

During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.

Formation of a cylindrical bridge in cell division

NASA Astrophysics Data System (ADS)

Citron, Daniel; Schmidt, Laura E.; Reichl, Elizabeth; Ren, Yixin; Robinson, Douglas; Zhang, Wendy W.

2007-11-01

In nature, the shape transition associated with the division of a mother cell into two daughter cells proceeds via a variety of routes. In the cylinder-thinning route, which has been observed in Dictyostelium and most animal cells, the mother cell first forms a broad bridge-like region, also known as a furrow, between two daughter cells. The furrow then rapidly evolves into a cylindrical bridge, which thins and eventually severs the mother cell into two. The fundamental mechanism underlying this division route is not understood. Recent experiments on Dictyostelium found that, while the cylinder-thinning route persists even when key actin cross-linking proteins are missing, it is disrupted by the removal of force-generating myosin-II proteins. Other measurements revealed that mutant cells lacking myosin-II have a much more uniform tension over the cell surface than wild-type cells. This suggests that tension variation may be important. Here we use a fluid model, previously shown to reproduce the thinning dynamics [Zhang & Robinson, PNAS 102, 7186 (2005)], to test this idea. Consistent with the experiments, the model shows that the cylinder formation process occurs regardless of the exact viscoelastic properties of the cell. In contrast to the experiments, a tension variation in the model hinders, rather then expedites, the cylinder formation.

Nanocomposite (Janus) paper as 3D cell culture system.

PubMed

Kehr, N S; Motealleh, A

2017-08-01

Nanocomposite (NC) papers using bifunctional nanoparticles are prepared for 3D cell growth experiments. Cells show better affinity to NC papers than to paper itself and differentiate the chemical group on the external surface of the nanoparticles. Cells possess spherical shaped morphology and form agglomerates onto the (NC) papers like as observed by cell growth in 3D materials. Furthermore, the preparation of "Janus" paper and the differentiation of its two distinct faces by cells is presented. Copyright © 2017 Elsevier B.V. All rights reserved.

PR01 Molecular Pathogenesis of Rickettsioses and Development of Anti-Rickettsial Treatment by Combinatorial Peptide-Based Libraries

DTIC Science & Technology

2006-02-01

likely reflecting similar cell death rates in all monolayers at late time points. By the end of the experiment at 120 hours, all monolayers showed a...50-55% increase in permeability when compared to the controls. 2. Cell death rates in rickettsiae-infected SV-HCEC monolayers In order to...necrotic cell death. Quantification of cell death was performed by determining the percent of total cells staining positive for PI. Cell death rates did

Spaceflight alters immune cell function and distribution

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald; Mandel, Adrian D.; Konstantinova, Irina V.; Berry, Wallace D.; Taylor, Gerald R.; Lesniak, A. T.; Fuchs, Boris B.; Rakhmilevich, Alexander L.

1992-01-01

Experiments are described which were performed onboard Cosmos 2044 to determine spaceflight effects on immunologically important cell function and distribution. Results indicate that bone marrow cells from flown and suspended rats exhibited a decreased response to a granulocyte/monocyte colony-stimulating factor compared with the bone marrow cells from control rats. Bone marrow cells showed an increase in the percentage of cells expressing markers for helper T-cells in the myelogenous population and increased percentages of anti-asialo granulocyte/monocyte-1-bearing interleulin-2 receptor bearing pan T- and helper T-cells in the lymphocytic population.

Cutaway line drawing of STS-34 middeck experiment Polymer Morphology (PM)

NASA Technical Reports Server (NTRS)

1989-01-01

Cutaway line drawing shows components of STS-34 middeck experiment Polymer Morphology (PM). Components include the EAC, heat exchanger, sample cell control (SCC), sample cells, source, interferometer, electronics, carousel drive, infrared (IR) beam, and carousel. PM, a 3M-developed organic materials processing experiment, is designed to explore the effects of microgravity on polymeric materials as they are processed in space. The samples of polymeric materials being studied in the PM experiment are thin films (25 microns or less) approximately 25mm in diameter. The samples are mounted between two infrared transparent windows in a specially designed infrared cell that provides the capability of thermally processing the samples to 200 degrees Celsius with a high degree of thermal control. The samples are mounted on a carousel that allows them to be positioned, one at a time, in the infrared beam where spectra may be acquired. The Generic Electronics Module (GEM) provides all carousel and

Analysis of results of ASTP experiment in electrophoresis

NASA Technical Reports Server (NTRS)

Vanderhoff, J. W.; Micale, F. J.; Krumrine, P. H.

1977-01-01

The Apollo-Soyuz Test Project (ASTP) included an electrophoretic separation experiment of biological cells. The nature separation results of aldehyde-fixed rabbit, human and horse red blood cells, which were taken in the form of photographs taken at three-minute intervals, are the subject of this report. The electrophoretic separation was successful in that fractionation according to mobility did occur and was found in the sliced samples. Photographic evidence indicates that the low electroosmotic methylcellulose coating was successful in reducing the electroosmosis to a near zero value. Also, the flight film shows that the bands migrated down the column as theory would predict, producing two bands of high cell concentration separated and surrounded by regions of lower cell concentration. However, most likely some clumping of cells occurred to cause the trailing band to be larger than expected from theory. Overall, the experiment was a success in demonstrating a static electrophoresis separation under microgravity conditions with a resolution not possible on earth.

Spaceflight Effects on the Hematopoietic Tissue of Ribbed Newts

NASA Astrophysics Data System (ADS)

Domaratskaya, E. I.; Almeida, E. A. C.; Butorina, N. N.; Nikonova, T. M.; Grigoryan, E. N.; Poplinskaya, V. A.

2008-06-01

The newts Pleurodeles waltl flown on Foton-M2 for 12 days were used for studying the effects of spaceflight on hematopoiesis in lower vertebrates. Prior to the flight, all the animals underwent to removal their lenses and tail tips for regeneration studies. No significant differences in blood cell contents were detected between flight and control animals. Morphological examination of hematopoietic areas of the liver in both groups also showed no significant differences. Experiments with BrdU incorporation revealed labeled cells in the hemopoietic area of the liver as well as in blood. The blood cell composition of newts flown on Foton-M3 was similar to that in intact (nonoperated) newts used in Bion-11 and Foton-M2 experiments. The lack of blood changes in newts during the current experiments distinguishes them from mammals flown in space (rats and mice), which developed significant changes in both blood cell counts, stem and committed cells in the blood-forming tissues.

Lipopolysaccharide-induced inflammation attenuates taste progenitor cell proliferation and shortens the life span of taste bud cells.

PubMed

Cohn, Zachary J; Kim, Agnes; Huang, Liquan; Brand, Joseph; Wang, Hong

2010-06-10

The mammalian taste bud, a complex collection of taste sensory cells, supporting cells, and immature basal cells, is the structural unit for detecting taste stimuli in the oral cavity. Even though the cells of the taste bud undergo constant turnover, the structural homeostasis of the bud is maintained by balancing cell proliferation and cell death. Compared with nongustatory lingual epithelial cells, taste cells express higher levels of several inflammatory receptors and signalling proteins. Whether inflammation, an underlying condition in some diseases associated with taste disorders, interferes with taste cell renewal and turnover is unknown. Here we report the effects of lipopolysaccharide (LPS)-induced inflammation on taste progenitor cell proliferation and taste bud cell turnover in mouse taste tissues. Intraperitoneal injection of LPS rapidly induced expression of several inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-6, in mouse circumvallate and foliate papillae. TNF-alpha and IFN-gamma immunoreactivities were preferentially localized to subsets of cells in taste buds. LPS-induced inflammation significantly reduced the number of 5-bromo-2'-deoxyuridine (BrdU)-labeled newborn taste bud cells 1-3 days after LPS injection, suggesting an inhibition of taste bud cell renewal. BrdU pulse-chase experiments showed that BrdU-labeled taste cells had a shorter average life span in LPS-treated mice than in controls. To investigate whether LPS inhibits taste cell renewal by suppressing taste progenitor cell proliferation, we studied the expression of Ki67, a cell proliferation marker. Quantitative real-time RT-PCR revealed that LPS markedly reduced Ki67 mRNA levels in circumvallate and foliate epithelia. Immunofluorescent staining using anti-Ki67 antibodies showed that LPS decreased the number of Ki67-positive cells in the basal regions surrounding circumvallate taste buds, the niche for taste progenitor cells. PCR array experiments showed that the expression of cyclin B2 and E2F1, two key cell cycle regulators, was markedly downregulated by LPS in the circumvallate and foliate epithelia. Our results show that LPS-induced inflammation inhibits taste progenitor cell proliferation and interferes with taste cell renewal. LPS accelerates cell turnover and modestly shortens the average life span of taste cells. These effects of inflammation may contribute to the development of taste disorders associated with infections.

Gene transfer preferentially selects MHC class I positive tumour cells and enhances tumour immunogenicity.

PubMed

Hacker, Ulrich T; Schildhauer, Ines; Barroso, Margarita Céspedes; Kofler, David M; Gerner, Franz M; Mysliwietz, Josef; Buening, Hildegard; Hallek, Michael; King, Susan B S

2006-05-01

The modulated expression of MHC class I on tumour tissue is well documented. Although the effect of MHC class I expression on the tumorigenicity and immunogenicity of MHC class I negative tumour cell lines has been rigorously studied, less is known about the validity of gene transfer and selection in cell lines with a mixed MHC class I phenotype. To address this issue we identified a C26 cell subline that consists of distinct populations of MHC class I (H-2D/K) positive and negative cells. Transient transfection experiments using liposome-based transfer showed a lower transgene expression in MHC class I negative cells. In addition, MHC class I negative cells were more sensitive to antibiotic selection. This led to the generation of fully MHC class I positive cell lines. In contrast to C26 cells, all transfectants were rejected in vivo and induced protection against the parental tumour cells in rechallenge experiments. Tumour cell specificity of the immune response was demonstrated in in vitro cytokine secretion and cytotoxicity assays. Transfectants expressing CD40 ligand and hygromycin phosphotransferase were not more immunogenic than cells expressing hygromycin resistance alone. We suggest that the MHC class I positive phenotype of the C26 transfectants had a bearing on their immunogenicity, because selected MHC class I positive cells were more immunogenic than parental C26 cells and could induce specific anti-tumour immune responses. These data demonstrate that the generation of tumour cell transfectants can lead to the selection of subpopulations that show an altered phenotype compared to the parental cell line and display altered immunogenicity independent of selection marker genes or other immune modulatory genes. Our results show the importance of monitoring gene transfer in the whole tumour cell population, especially for the evaluation of in vivo therapies targeted to heterogeneous tumour cell populations.

Evaluation of the anti-proliferative and cytostatic effect of Citrus sinensis (orange) fruit juice

PubMed Central

Chinedu, Enegide; Arome, David; Ameh, Solomon F; Ameh, Gift E

2014-01-01

Aim: This work has been designed to evaluate the anti-proliferative and cytostatic effects of Citrus sinensis (orange) fruit juice on rapidly proliferating cells. Materials and Methods: The study was carried out on the seeds of Sorghum bicolor for 72 h. The mean radicle length (mm) of the seeds was taken at 48 and 72 h. Result: The result showed that when compared with the control, methotrexate, the standard drug showed a significant (P < 0.001) anti-proliferative effect throughout the experiment. The inhibition of the radicle growth was more after 72 h (87.42%). At a dose of 5% (v/v), the juice showed a slightly significant (P < 0.05) effect affect after 72 h; however, there was no significant effect at 48 h. The juice at doses of 10% and 20% (v/v) showed a highly significant (P < 0.001) anti-proliferative effect throughout the experiment; however, the percentage inhibitions were higher at 72 h. At 72 h, the percentage inhibition for juice at 10% (v/v) was 72.37% and at 20% (v/v) was 91.96%. The concentrations of 40% and 60% (v/v) showed cytostatic effects as no appreciable growth of the radicles of the seeds was observed throughout the experiment. The percentage inhibition for 40% (v/v) was 100% and 99.72% for 48 and 72 h, respectively, while that for the juice concentration of 60% (v/v) was 100% throughout the study. Conclusion: The experiment has shown that C. sinensis fruit juice has a potential for causing both anti-proliferative and cytostatic effects on fast proliferating cells and hence cancerous cells. PMID:25298937

Improving cell therapy – experiments using transplanted telomerase-immortalized cells in immunodeficient mice

PubMed Central

Huang, Qin; Chen, Meizhen; Liang, Sitai; Acha, Victor; Liu, Dan; Yuan, Furong; Hawks, Christina L.; Hornsby, Peter J.

2007-01-01

Cell therapy is the use of stem cells and other types of cells in various therapies for age-related diseases. Two issues that must be addressed before cell therapy could be used routinely in medicine are improved efficacy of the transplanted cells and demonstrated long-term safety. Desirable genetic modifications that could be made to cells to be used for cell therapy include immortalization with hTERT (human telomerase reverse transcriptase). We have used a model for cell therapy in which transplantation of adrenocortical cells restores glucocorticoid and mineralocorticoid hormone levels in adrenalectomized immunodeficient mice. In this model, clones of cells that had been immortalized with hTERT were shown to be able to replace the function of the animals'adrenal glands by forming vascularized tissue structures when cells were transplanted beneath the capsule of the kidney. hTERT-modified cells showed no tendency for neoplastic changes. Moreover, a series of experiments showed that hTERT does not cooperate with known oncoproteins in tumorigenesis either in adrenocortical cells or in human fibroblasts. Nevertheless, hTERT was required for tumorigenesis when cells were implanted subcutaneously rather than in the subrenal capsule space. Changes in gene expression make hTERT-modified cells more robust. Understanding these changes is important so as to be able to separately control immortalization and other desirable properties of cells that could be used in cell therapy. Alternatively, desirable properties of transplants might be provided by co-transplanted mesenchymal cells: mesenchymal cell-assisted cell therapy. For both hTERT modification and mesenchymal cell-assisted cell therapy, genomics approaches will be needed to define what genetic modifications are desirable and safe in cells used in cell therapy. PMID:17123586

A temperature-controlled photoelectrochemical cell for quantitative product analysis.

PubMed

Corson, Elizabeth R; Creel, Erin B; Kim, Youngsang; Urban, Jeffrey J; Kostecki, Robert; McCloskey, Bryan D

2018-05-01

In this study, we describe the design and operation of a temperature-controlled photoelectrochemical cell for analysis of gaseous and liquid products formed at an illuminated working electrode. This cell is specifically designed to quantitatively analyze photoelectrochemical processes that yield multiple gas and liquid products at low current densities and exhibit limiting reactant concentrations that prevent these processes from being studied in traditional single chamber electrolytic cells. The geometry of the cell presented in this paper enables front-illumination of the photoelectrode and maximizes the electrode surface area to electrolyte volume ratio to increase liquid product concentration and hence enhances ex situ spectroscopic sensitivity toward them. Gas is bubbled through the electrolyte in the working electrode chamber during operation to maintain a saturated reactant concentration and to continuously mix the electrolyte. Gaseous products are detected by an in-line gas chromatograph, and liquid products are analyzed ex situ by nuclear magnetic resonance. Cell performance was validated by examining carbon dioxide reduction on a silver foil electrode, showing comparable results both to those reported in the literature and identical experiments performed in a standard parallel-electrode electrochemical cell. To demonstrate a photoelectrochemical application of the cell, CO 2 reduction experiments were carried out on a plasmonic nanostructured silver photocathode and showed different product distributions under dark and illuminated conditions.

Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization

PubMed Central

Komor, Alexis C.; Schneider, Curtis J.; Weidmann, Alyson G.; Barton, Jacqueline K.

2013-01-01

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells. Ten distinct metalloinsertors with varying lipophilicities have been synthesized and their mismatch binding affinities and biological activities determined. Although DNA photocleavage experiments demonstrate that their binding affinities are quite similar, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments have uncovered a relationship between the subcellular distribution of these metalloinsertors and their biological activities. Specifically, we find that all of our metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. However, the metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells, whereas metalloinsertors with less mitochondrial rhodium show activity that is highly selective for MMR-deficient versus proficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cell-selective cytotoxic and antiproliferative activities. The selectivity in cellular targeting depends upon binding to mismatches in genomic DNA. PMID:23137296

A temperature-controlled photoelectrochemical cell for quantitative product analysis

NASA Astrophysics Data System (ADS)

Corson, Elizabeth R.; Creel, Erin B.; Kim, Youngsang; Urban, Jeffrey J.; Kostecki, Robert; McCloskey, Bryan D.

2018-05-01

In this study, we describe the design and operation of a temperature-controlled photoelectrochemical cell for analysis of gaseous and liquid products formed at an illuminated working electrode. This cell is specifically designed to quantitatively analyze photoelectrochemical processes that yield multiple gas and liquid products at low current densities and exhibit limiting reactant concentrations that prevent these processes from being studied in traditional single chamber electrolytic cells. The geometry of the cell presented in this paper enables front-illumination of the photoelectrode and maximizes the electrode surface area to electrolyte volume ratio to increase liquid product concentration and hence enhances ex situ spectroscopic sensitivity toward them. Gas is bubbled through the electrolyte in the working electrode chamber during operation to maintain a saturated reactant concentration and to continuously mix the electrolyte. Gaseous products are detected by an in-line gas chromatograph, and liquid products are analyzed ex situ by nuclear magnetic resonance. Cell performance was validated by examining carbon dioxide reduction on a silver foil electrode, showing comparable results both to those reported in the literature and identical experiments performed in a standard parallel-electrode electrochemical cell. To demonstrate a photoelectrochemical application of the cell, CO2 reduction experiments were carried out on a plasmonic nanostructured silver photocathode and showed different product distributions under dark and illuminated conditions.

Exosome secretion promotes chemotaxis of cancer cells.

PubMed

Sung, Bong Hwan; Weaver, Alissa M

2017-03-04

Migration of cells toward chemical cues, or chemotaxis, is important for many biologic processes such as immune defense, wound healing and cancer metastasis. Although chemotaxis is thought to occur in cancer cells, it is less well characterized than chemotaxis of professional immune cells such as neutrophils. Here, we show that cancer cell chemotaxis relies on secretion of exosome-type extracellular vesicles. Migration of fibrosarcoma cells toward a gradient of exosome-depleted serum was diminished by knockdown of the exosome secretion regulator Rab27a. Rescue experiments in which chemotaxis chambers were coated with purified extracellular vesicles demonstrate that exosomes but not microvesicles affect both speed and directionality of migrating cells. Chamber coating with purified fibronectin and fibronectin-depleted exosomes demonstrates that the exosome cargo fibronectin promotes cell speed but cannot account for the role of exosomes in promoting directionality of fibrosarcoma cell movement during chemotaxis. These experiments indicate that exosomes contain multiple motility-promoting cargoes that contribute to different aspects of cell motility.

Spatially variant red blood cell crenation in alternating current non-uniform fields.

PubMed

An, Ran; Wipf, David O; Minerick, Adrienne R

2014-03-01

Alternating-current (AC) electrokinetics involve the movement and behaviors of particles or cells. Many applications, including dielectrophoretic manipulations, are dependent upon charge interactions between the cell or particle and the surrounding medium. Medium concentrations are traditionally treated as spatially uniform in both theoretical models and experiments. Human red blood cells (RBCs) are observed to crenate, or shrink due to changing osmotic pressure, over 10 min experiments in non-uniform AC electric fields. Cell crenation magnitude is examined as functions of frequency from 250 kHz to 1 MHz and potential from 10 Vpp to 17.5 Vpp over a 100 μm perpendicular electrode gap. Experimental results show higher peak to peak potential and lower frequency lead to greater cell volume crenation up to a maximum volume loss of 20%. A series of experiments are conducted to elucidate the physical mechanisms behind the red blood cell crenation. Non-uniform and uniform electrode systems as well as high and low ion concentration experiments are compared and illustrate that AC electroporation, system temperature, rapid temperature changes, medium pH, electrode reactions, and convection do not account for the crenation behaviors observed. AC electroosmotic was found to be negligible at these conditions and AC electrothermal fluid flows were found to reduce RBC crenation behaviors. These cell deformations were attributed to medium hypertonicity induced by ion concentration gradients in the spatially nonuniform AC electric fields.

Mechanical-Electrochemical-Thermal Simulation of Lithium-Ion Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santhanagopalan, Shriram; Zhang, Chao; Sprague, Michael A.

2016-06-01

Models capture the force response for single-cell and cell-string levels to within 15%-20% accuracy and predict the location for the origin of failure based on the deformation data from the experiments. At the module level, there is some discrepancy due to poor mechanical characterization of the packaging material between the cells. The thermal response (location and value of maximum temperature) agrees qualitatively with experimental data. In general, the X-plane results agree with model predictions to within 20% (pending faulty thermocouples, etc.); the Z-plane results show a bigger variability both between the models and test-results, as well as among multiple repeatsmore » of the tests. The models are able to capture the timing and sequence in voltage drop observed in the multi-cell experiments; the shapes of the current and temperature profiles need more work to better characterize propagation. The cells within packaging experience about 60% less force under identical impact test conditions, so the packaging on the test articles is robust. However, under slow-crush simulations, the maximum deformation of the cell strings with packaging is about twice that of cell strings without packaging.« less

The lived experience of autologous stem cell-transplanted patients: Post-transplantation and before discharge.

PubMed

Alnasser, Qasem; Abu Kharmah, Salahel Deen; Attia, Manal; Aljafari, Akram; Agyekum, Felicia; Ahmed, Falak Aftab

2018-04-01

To explore the lived experience of the patients post-haematopoietic stem cell transplantation and specifically after engraftment and before discharge. Patients post-stem cell transplantation experience significant changes in all life aspects. Previous studies carried out by other researchers focused mainly on the postdischarge experience, where patients reported their perceptions that have always been affected by the life post-transplantation and influenced by their surroundings. The lived experience of patients, specifically after engraftment and prior to discharge (the "transition" phase), has not been adequately explored in the literature. Doing so might provide greater insight into the cause of change post-haematopoietic stem cell transplantation. This study is a phenomenological description of the participants' perception about their lived experience post-haematopoietic stem cell transplantation. The study used Giorgi's method of analysis. Through purposive sampling, 15 post-haematopoietic stem cell transplantation patients were recruited. Data were collected by individual interviews. Data were then analysed based on Giorgi's method of analysis to reveal the meaning of a phenomenon as experienced through the identification of essential themes. The analysis process revealed 12 core themes covered by four categories that detailed patients lived experience post-haematopoietic stem cell transplantation. The four categories were general transplant experience, effects of transplantation, factors of stress alleviation and finally life post-transplantation. This study showed how the haematopoietic stem cell transplantation affected the patients' physical, psychological and spiritual well-being. Transplantation also impacted on the patients' way of thinking and perception of life. Attending to patients' needs during transplantation might help to alleviate the severity of the effects and therefore improve experience. Comprehensive information about transplantation needs to be provided over different intervals and at different occasions. The role of the haematopoietic stem cell transplantation coordinators is important, and their communication skills and knowledge were found to be significant in patients' preparation and decision-making. As healthcare providers usually attend to only the patients' physical and psychological needs, spirituality was found to play an important role in maintaining morale and making sense of the meaning of life. © 2018 John Wiley & Sons Ltd.

Distinct constrictive processes, separated in time and space, divide caulobacter inner and outer membranes.

PubMed

Judd, Ellen M; Comolli, Luis R; Chen, Joseph C; Downing, Kenneth H; Moerner, W E; McAdams, Harley H

2005-10-01

Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed pre-fission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.

Design of a microfluidic cell using microstereolithography for electronic tongue applications

NASA Astrophysics Data System (ADS)

Jacesko, Stefany L.; Ji, Taeksoo; Abraham, Jose K.; Varadan, Vijay K.; Gardner, Julian W.

2003-07-01

In this paper we present design, fabrication and integration of a micro fluidic cell for use with the electronic tongue. The cell was machined using microstereo lithography on a Hexanediol Diacrylate (HDDA) liquid monomer. The wet cell was designed to confine the liquid under test to the sensing area and insure complete isolation of the interdigital transducers (IDTs). The electronic tongue is a shear horizontal surface acoustic wave (SH-SAW) device. Shear horizontally polarized Love-waves are guided between transmitting and receiving IDTs, over a piezoelectric substrate, which creates an electronic oscillator effect. This device has a dual delay line configuration, which accounts for the measuring of both mechanical and electrical properties of a liquid, simultaneously, with the ability to eliminate environmental factors. The data collected is distinguished using principal components analysis in conjunction with pre-processing parameters. The experiments show that the micro fluidic cell for this electronic tongue does not affect the losses or phase of the device to any extent of concern. Experiments also show that liquids such as Strawberry Hi-C, Teriyaki Sauce, DI Water, Coca Cola, and Pepsi are distinguishable using these methods.

A Novel Photosensitizer 3¹,13¹-phenylhydrazine -Mppa (BPHM) and Its in Vitro Photodynamic Therapy against HeLa Cells.

PubMed

Li, Wenting; Tan, Guanghui; Cheng, Jianjun; Zhao, Lishuang; Wang, Zhiqiang; Jin, Yingxue

2016-04-29

Photodynamic therapy (PDT) has attracted widespread attention due to its potential in the treatment of various cancers. Porphyrinic pyropheophorbide-a (PPa) has been shown to be a potent photosensitizer in PDT experiments. In this paper, a C-3¹,13¹ bisphenylhydrazone modified methyl pyropheophorbide-a (BPHM) was designed and synthesized with the consideration that phenylhydrazone structure may extend absorption wavelength of methyl pyro-pheophorbide-a (Mppa), and make the photosensitizer potential in deep tumor treatment. The synthesis, spectral properties and in vitro photodynamic therapy (PDT) against human HeLa cervical cancer cell line was studied. Methyl thiazolyl tetrazolium (MTT) assay showed the title compound could achieve strong inhibition of cervical cancer cell viability under visible light (675 nm, 25 J/cm²). Cell uptake experiments were performed on HeLa cells. Morphological changes were examined and analyzed by fluorescent inverted microscope. In addition, the mechanism of the photochemical processes of PDT was investigated, which showed that the formation of singlet oxygen after treatment with PDT played a moderate important role.

Cometabolic degradation of ethyl mercaptan by phenol-utilizing Ralstonia eutropha in suspended growth and gas-recycling trickle-bed reactor.

PubMed

Sedighi, Mahsa; Zamir, Seyed Morteza; Vahabzadeh, Farzaneh

2016-01-01

The degradability of ethyl mercaptan (EM), by phenol-utilizing cells of Ralstonia eutropha, in both suspended and immobilized culture systems, was investigated in the present study. Free-cells experiments conducted at EM concentrations ranging from 1.25 to 14.42 mg/l, showed almost complete removal of EM at concentrations below 10.08 mg/l, which is much higher than the maximum biodegradable EM concentration obtained in experiments that did not utilize phenol as the primary substrate, i.e. 2.5 mg/l. The first-order kinetic rate constant (kSKS) for EM biodegradation by the phenol-utilizing cells (1.7 l/g biomass/h) was about 10 times higher than by cells without phenol utilization. Immobilized-cells experiments performed in a gas recycling trickle-bed reactor packed with kissiris particles at EM concentrations ranging from 1.6 to 36.9 mg/l, showed complete removal at all tested concentrations in a much shorter time, compared with free cells. The first-order kinetic rate constant (rmaxKs) for EM utilization was 0.04 l/h for the immobilized system compared to 0.06 for the suspended-growth culture, due to external mass transfer diffusion. Diffusion limitation was decreased by increasing the recycling-liquid flow rate from 25 to 65 ml/min. The removed EM was almost completely mineralized according to TOC and sulfate measurements. Shut down and starvation experiments revealed that the reactor could effectively handle the starving conditions and was reliable for full-scale application. Copyright © 2015 Elsevier Ltd. All rights reserved.

DNA Repair and the Evolution of Transformation in Bacillus Subtilis. II. Role of Inducible Repair

PubMed Central

Wojciechowski, M. F.; Hoelzer, M. A.; Michod, R. E.

1989-01-01

In Bacillus subtilis, DNA repair and recombination are intimately associated with competence, the physiological state in which the bacterium can bind, take up and recombine exogenous DNA. Previously, we have shown that the homologous DNA transformation rate (ratio of transformants to total cells) increases with increasing UV dosage if cells are transformed after exposure to UV radiation (UV-DNA), whereas the transformation rate decreases if cells are transformed before exposure to UV (DNA-UV). In this report, by using different DNA repair-deficient mutants, we show that the greater increase in transformation rate in UV-DNA experiments than in DNA-UV experiments does not depend upon excision repair or inducible SOS-like repair, although certain quantitative aspects of the response do depend upon these repair systems. We also show that there is no increase in the transformation rate in a UV-DNA experiment when repair and recombination proficient cells are transformed with nonhomologous plasmid DNA, although the results in a DNA-UV experiment are essentially unchanged by using plasmid DNA. We have used din operon fusions as a sensitive means of assaying for the expression of genes under the control of the SOS-like regulon in both competent and noncompetent cell subpopulations as a consequence of competence development and our subsequent experimental treatments. Results indicate that the SOS-like system is induced in both competent and noncompetent subpopulations in our treatments and so should not be a major factor in the differential response in transformation rate observed in UV-DNA and DNA-UV treatments. These results provide further support to the hypothesis that the evolutionary function of competence is to bring DNA into the cell for use as template in the repair of DNA damage. PMID:2497048

Initiation of oncogenic transformation in human mammary epithelial cells by charged particles

NASA Technical Reports Server (NTRS)

Yang, T. C.; Georgy, K. A.; Craise, L. M.; Durante, M.

1997-01-01

Experimental studies have shown that high linear-energy transfer (LET) charged particles can be more effective than x-rays and gamma-rays in inducing oncogenic transformation in cultured cells and tumors in animals. Based on these results, experiments were designed and performed with an immortal human mammary epithelial cell line (H184B5), and several clones transformed by heavy ions were obtained. Cell fusion experiments were subsequently done, and results indicate that the transforming gene(s) is recessive. Chromosome analysis with fluorescence in situ hybridization (FISH) techniques also showed additional translocations in transformed human mammary epithelial cells. In addition, studies with these cell lines indicate that heavy ions can effectively induce deletion, break, and dicentrics. Deletion of tumor suppressor gene(s) and/or formation of translocation through DNA double strand breaks is a likely mechanism for the initiation of oncogenic transformation in human mammary epithelial cells.

Electrophoresis experiments in microgravity

NASA Technical Reports Server (NTRS)

Snyder, Robert S.; Rhodes, Percy H.

1991-01-01

The use of the microgravity environment to separate and purify biological cells and proteins has been a major activity since the beginning of the NASA Microgravity Science and Applications program. Purified populations of cells are needed for research, transplantation and analysis of specific cell constituents. Protein purification is a necessary step in research areas such as genetic engineering where the new protein has to be separated from the variety of other proteins synthesized from the microorganism. Sufficient data are available from the results of past electrophoresis experiments in space to show that these experiments were designed with incomplete knowledge of the fluid dynamics of the process including electrohydrodynamics. However, electrophoresis is still an important separation tool in the laboratory and thermal convection does limit its performance. Thus, there is a justification for electrophoresis but the emphasis of future space experiments must be directed toward basic research with model experiments to understand the microgravity environment and fluid analysis to test the basic principles of the process.

Cloned Hemoglobin Genes Enhance Growth Of Cells

NASA Technical Reports Server (NTRS)

Khosla, Chaitan; Bailey, James E.

1991-01-01

Experiments show that portable deoxyribonucleic acid (DNA) sequences incorporated into host cells make them produce hemoglobins - oxygen-binding proteins essential to function of red blood cells. Method useful in several biotechnological applications. One, enhancement of growth of cells at higher densities. Another, production of hemoglobin to enhance supplies of oxygen in cells, for use in chemical reactions requiring oxygen, as additive to serum to increase transport of oxygen, and for binding and separating oxygen from mixtures of gases.

Effect of Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

PubMed

Lupatov, A Yu; Kim, Ya S; Bystrykh, O A; Vakhrushev, I V; Pavlovich, S V; Yarygin, K N; Sukhikh, G T

2017-02-01

We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.

Thymocytes may persist and differentiate without any input from bone marrow progenitors

PubMed Central

Peaudecerf, Laetitia; Lemos, Sara; Galgano, Alessia; Krenn, Gerald; Vasseur, Florence; Di Santo, James P.; Ezine, Sophie

2012-01-01

Thymus transplants can correct deficiencies of the thymus epithelium caused by the complete DiGeorge syndrome or FOXN1 mutations. However, thymus transplants were never used to correct T cell–intrinsic deficiencies because it is generally believed that thymocytes have short intrinsic lifespans. This notion is based on thymus transplantation experiments where it was shown that thymus-resident cells were rapidly replaced by progenitors originating in the bone marrow. In contrast, here we show that neonatal thymi transplanted into interleukin 7 receptor–deficient hosts harbor populations with extensive capacity to self-renew, and maintain continuous thymocyte generation and export. These thymus transplants reconstitute the full diversity of peripheral T cell repertoires one month after surgery, which is the earliest time point studied. Moreover, transplantation experiments performed across major histocompatibility barriers show that allogeneic transplanted thymi are not rejected, and allogeneic cells do not induce graft-versus-host disease; transplants induced partial or total protection to infection. These results challenge the current dogma that thymocytes cannot self-renew, and indicate a potential use of neonatal thymus transplants to correct T cell–intrinsic deficiencies. Finally, as found with mature T cells, they show that thymocyte survival is determined by the competition between incoming progenitors and resident cells. PMID:22778388

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

PubMed

Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

2015-05-01

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.

Generation Of Functional Insulin-Producing Cells In The Gut By Foxo1 Ablation

PubMed Central

Talchai, Chutima; Xuan, Shouhong; Kitamura, Tadahiro; DePinho, Ronald A.; Accili, Domenico

2012-01-01

Restoration of regulated insulin secretion is the ultimate goal of type 1 diabetes therapy. Here we show that, surprisingly, somatic ablation of Foxo1 in Neurog3+ enteroendocrine progenitor cells gives rise to gut insulin-positive cells (Ins+) that express markers of mature β-cells, and secrete bioactive insulin as well as C-peptide in response to glucose and sulfonylureas. Lineage tracing experiments show that gut Ins+ cells arise cell-autonomously from Foxo1-deficient cells. Inducible Foxo1 ablation in adult mice also results in the generation of gut Ins+ cells. Following ablation by the β-cell toxin, streptozotocin, gut Ins+ cells regenerate and produce insulin, reversing hyperglycemia in mice. The data indicate that Neurog3+ enteroendocrine progenitors require active Foxo1 to prevent differentiation into Ins+ cells. Foxo1 ablation in gut epithelium may provide an approach to restore insulin production in type 1 diabetes. PMID:22406641

Bacillus subtilis MreB paralogues have different filament architectures and lead to shape remodelling of a heterologous cell system.

PubMed

Soufo, Hervé Joël Defeu; Graumann, Peter L

2010-12-01

Like many bacteria, Bacillus subtilis cells contain three actin-like MreB proteins. We show that the three paralogues, MreB, Mbl and MreBH, have different filament architectures in a heterologous cell system, and form straight filaments, helices or ring structures, different from the regular helical arrangement in B. subtilis cells. However, when coexpressed, they colocalize into a single filamentous helical structure, showing that the paralogues influence each other's filament architecture. Ring-like MreBH structures can be converted into MreB-like helical filaments by a single point mutation affecting subunit contacts, showing that MreB paralogues feature flexible filament arrangements. Time-lapse and FRAP experiments show that filaments can extend as well as shrink at both ends, and also show internal rearrangement, suggesting that filaments consist of overlapping bundles of shorter filaments that continuously turn over. Upon induction in Escherichia coli cells, B. subtilis MreB (BsMreB) filaments push the cells into strikingly altered cell morphology, showing that MreB filaments can change cell shape. E. coli cells with a weakened cell wall were ruptured upon induction of BsMreB filaments, suggesting that the bacterial actin orthologue may exert force against the cell membrane and envelope, and thus possibly plays an additional mechanical role in bacteria. © 2010 Blackwell Publishing Ltd.

Transcriptional Regulation of Type 11 17β-Hydroxysteroid Dehydrogenase Expression in Prostate Cancer Cells

PubMed Central

Rotinen, Mirja; Villar, Joaquín; Celay, Jon; Serrano, Irantzu; Notario, Vicente; Encío, Ignacio

2011-01-01

Type 11 Hydroxysteroid (17-beta) dehydrogenase (HSD17B11) catalyzes the conversion of 5α-androstan-3α,17β-diol into androsterone suggesting that it may play an important role in androgen metabolism. We previously described that overexpression of C/EBPα or C/EBPβ induced HSD17B11 expression in HepG2 cells but this process was not mediated by the CCAAT boxes located within its proximal promoter region. Here, we study HSD17B11 transcriptional regulation in prostate cancer (PC) cells. Transfection experiments showed that the region −107/+18 is sufficient for promoter activity in PC cells. Mutagenesis analysis indicated that Sp1 and C/EBP binding sites found in this region are essential for promoter activity. Additional experiments demonstrated that ectopic expression of Sp1 and C/EBPα upregulated HSD17B11 expression only in PC cell lines. Through DAPA and ChIP assays, specific recruitment of Sp1 and C/EBPα to the HSD17B11 promoter was detected. These results show that HSD17B11 transcription in PC cells is regulated by Sp1 and C/EBPα. PMID:21549806

Mutagenic and antimutagenic effects of aqueous extract of rosemary (Rosmarinus officinalis L.) on meristematic cells of Allium cepa.

PubMed

Felicidade, I; Lima, J D; Pesarini, J R; Monreal, A C D; Mantovani, M S; Ribeiro, L R; Oliveira, R J

2014-11-28

Polyphenolic compounds present in rosemary were found to have antioxidant properties, anticarcinogenic activity, and to increase the detoxification of pro-carcinogens. The aim of the study was to determine the effect the aqueous extract of rosemary (AER) on mutagenicity induced by methylmethane sulfonate in meristematic cells of Allium cepa, as well as to describe its mode of action. Anti-mutagenicity experiments were carried out with 3 different concentrations of AER, which alone showed no mutagenic effects. In antimutagenicity experiments, AER showed chemopreventive activity in cultured meristematic cells of A. cepa against exposure to methylmethane sulfonate. Additionally, post-treatment and simultaneous treatment using pre-incubation protocols were the most effective. Evaluation of different protocols and the percent reduction in DNA indicated bioantimutagenic as well desmutagenic modes of action for AER. AER may be chemopreventive and antimutagenic.

Microbial Community Response during the Iron Fertilization Experiment LOHAFEX

PubMed Central

Thiele, Stefan; Ramaiah, Nagappa; Amann, Rudolf

2012-01-01

Iron fertilization experiments in high-nutrient, low-chlorophyll areas are known to induce phytoplankton blooms. However, little is known about the response of the microbial community upon iron fertilization. As part of the LOHAFEX experiment in the southern Atlantic Ocean, Bacteria and Archaea were monitored within and outside an induced bloom, dominated by Phaeocystis-like nanoplankton, during the 38 days of the experiment. The microbial production increased 1.6-fold (thymidine uptake) and 2.1-fold (leucine uptake), while total cell numbers increased only slightly over the course of the experiment. 454 tag pyrosequencing of partial 16S rRNA genes and catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH) showed that the composition and abundance of the bacterial and archaeal community in the iron-fertilized water body were remarkably constant without development of typical bloom-related succession patterns. Members of groups usually found in phytoplankton blooms, such as Roseobacter and Gammaproteobacteria, showed no response or only a minor response to the bloom. However, sequence numbers and total cell numbers of the SAR11 and SAR86 clades increased slightly but significantly toward the end of the experiment. It seems that although microbial productivity was enhanced within the fertilized area, a succession-like response of the microbial community upon the algal bloom was averted by highly effective grazing. Only small-celled members like the SAR11 and SAR86 clades could possibly escape the grazing pressure, explaining a net increase of those clades in numbers. PMID:23064339

Spin glass model for cell reprogramming

NASA Astrophysics Data System (ADS)

Pusuluri, Sai Teja; Castillo, Horacio E.

2014-03-01

Recent experiments show that differentiated cells can be reprogrammed to become pluripotent stem cells. The possible cell fates can be modeled as attractors in a dynamical system, the ``epigenetic landscape.'' Both cellular differentiation and reprogramming can be described in the landscape picture as motion from one attractor state to another attractor state. We use a simple model based on spin glass theory that can construct a simulated epigenetic landscape starting from the experimental genomic data. We modify the model to incorporate experimental reprogramming protocols. Our simulations successfully reproduce several reprogramming experiments. We probe the robustness of the results against random changes in the model, explore the importance of asymmetric interactions between transcription factors and study the importance of histone modification errors in reprogramming.

Removing Escherichia coli from water using zinc oxide-coated zeolite.

PubMed

Wang, Lingling; Wu, Wenlin; Xie, Xiaolan; Chen, Hongbin; Lin, Jianming; Dionysiou, Dionysios D

2018-05-11

The removal of Escherichia coli (E. coli) from water by zinc oxide-coated zeolite (ZOCZ) and ZOCZ's antibacterial properties were examined in laboratory experiments using plate counting method and tests of cell apoptosis. Batch experiments showed that ZOCZ has a maximum removal capacity for E. coli of about 4.34 × 10 6  CFU g -1  at 25 °C. Element mappings confirm that zinc ions accumulate in the E. coli cells causing cell death. Pseudo-second-order kinetics and Freundlich isotherms were found to best describe the removal of E. coli, suggesting that a multilayer of E. coli cells forms on the surface of ZOCZ particles. Copyright © 2018 Elsevier Ltd. All rights reserved.

The gastrin/cholecystokinin-B receptor on prostate cells--a novel target for bifunctional prostate cancer imaging.

PubMed

Sturzu, Alexander; Klose, Uwe; Sheikh, Sumbla; Echner, Hartmut; Kalbacher, Hubert; Deeg, Martin; Nägele, Thomas; Schwentner, Christian; Ernemann, Ulrike; Heckl, Stefan

2014-02-14

The means of identifying prostate carcinoma and its metastases are limited. The contrast agents used in magnetic resonance imaging clinical diagnostics are not taken up into the tumor cells, but only accumulate in the interstitial space of the highly vasculated tumor. We examined the gastrin/cholecystokinin-B receptor as a possible target for prostate-specific detection using the C-terminal seven amino acid sequence of the gastrin peptide hormone. The correct sequence and a scrambled control sequence were coupled to the fluorescent dye rhodamine and the magnetic resonance imaging contrast agent gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Expression analysis of the gastrin receptor mRNA was performed by reverse transcriptase polymerase chain reaction on PC3 prostate carcinoma cells, U373 glioma, U2OS osteosarcoma and Colo205 colon carcinoma cells. After having confirmed elevated expression of gastrin receptor in PC3 cells and very low expression of the receptor in Colo205 cells, these two cell lines were used to create tumor xenografts on nude mice for in vivo experiments. Confocal lasers scanning microscopy and magnetic resonance imaging showed a high specificity of the correct conjugate for the PC3 xenografts. Staining of the PC3 xenografts was much weaker with the scrambled conjugate while the Colo205 xenografts showed no marked staining with any of the conjugates. In vitro experiments comparing the correct and scrambled conjugates on PC3 cells by magnetic resonance relaxometry and fluorescence-activated cell sorting confirmed markedly higher specificity of the correct conjugate. The investigations show that the gastrin receptor is a promising tumor cell surface target for future prostate-cancer-specific imaging applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Vitamin K2 improves proliferation and migration of bovine skeletal muscle cells in vitro.

PubMed

Rønning, Sissel Beate; Pedersen, Mona Elisabeth; Berg, Ragnhild Stenberg; Kirkhus, Bente; Rødbotten, Rune

2018-01-01

Skeletal muscle function is highly dependent on the ability to regenerate, however, during ageing or disease, the proliferative capacity is reduced, leading to loss of muscle function. We have previously demonstrated the presence of vitamin K2 in bovine skeletal muscles, but whether vitamin K has a role in muscle regulation and function is unknown. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to assess a potential effect of vitamin K2 (MK-4) during myogenesis of muscle cells. Cell viability experiments demonstrate that the amount of ATP produced by the cells was unchanged when MK-4 was added, indicating viable cells. Cytotoxicity analysis show that MK-4 reduced the lactate dehydrogenase (LDH) released into the media, suggesting that MK-4 was beneficial to the muscle cells. Cell migration, proliferation and differentiation was characterised after MK-4 incubation using wound scratch analysis, immunocytochemistry and real-time PCR analysis. Adding MK-4 to the cells led to an increased muscle proliferation, increased gene expression of the myogenic transcription factor myod as well as increased cell migration. In addition, we observed a reduction in the fusion index and relative gene expression of muscle differentiation markers, with fewer complex myotubes formed in MK-4 stimulated cells compared to control cells, indicating that the MK-4 plays a significant role during the early phases of muscle proliferation. Likewise, we see the same pattern for the relative gene expression of collagen 1A, showing increased gene expression in proliferating cells, and reduced expression in differentiating cells. Our results also suggest that MK-4 incubation affect low density lipoprotein receptor-related protein 1 (LRP1) and the low-density lipoprotein receptor (LDLR) with a peak in gene expression after 45 min of MK-4 incubation. Altogether, our experiments show that MK-4 has a positive effect on muscle cell migration and proliferation, which are two important steps during early myogenesis.

Insulin receptor in mouse neuroblastoma cell line N18TG2: binding properties and visualization with colloidal gold.

PubMed

Sartori, C; Stefanini, S; Bernardo, A; Augusti-Tocco, G

1992-08-01

Insulin function in the nervous system is still poorly understood. Possible roles as a neuromodulator and as a growth factor have been proposed (Baskin et al., 1987, Ann. Rev. Physiol. 49, 335-347). Stable cell lines may provide an appropriate experimental system for the analysis of insulin action on the various cellular components of the central nervous system. We report here a study to investigate the presence and the properties of insulin specific binding sites in the murine neuroblastoma line, N18TG2, together with insulin action on cell growth and metabolism. Also, receptor internalization has been studied. Binding experiments, carried out in standard conditions at 20 degrees C, enabled us to demonstrate that these cells bind insulin in a specific manner, thus confirming previous findings on other cell lines. Saturation curves showed the presence of two binding sites with Kd 0.3 and 9.7 nM. Competition experiments with porcine and bovine insulin showed an IC50 of 1 and 10 nM, respectively. Competition did not occur in the presence of the unrelated hormones ACTH and FSH. Dissociation experiments indicated the existence of an internalization process of the ligand-receptor complex; this was confirmed by an ultrastructural study using gold conjugated insulin. As far as the insulin action in N18TG2 cells is concerned, physiological concentrations stimulate cell proliferation, whereas no stimulation of glucose uptake was observed, indicating that insulin action in these cells is not mediated by general metabolic effects. On the basis of these data, N18TG2 line appears to be a very suitable model for further studies of the neuronal type insulin receptors, and possibly insulin specific action on the nervous system.

Using simulated fluorescence cell micrographs for the evaluation of cell image segmentation algorithms.

PubMed

Wiesmann, Veit; Bergler, Matthias; Palmisano, Ralf; Prinzen, Martin; Franz, Daniela; Wittenberg, Thomas

2017-03-18

Manual assessment and evaluation of fluorescent micrograph cell experiments is time-consuming and tedious. Automated segmentation pipelines can ensure efficient and reproducible evaluation and analysis with constant high quality for all images of an experiment. Such cell segmentation approaches are usually validated and rated in comparison to manually annotated micrographs. Nevertheless, manual annotations are prone to errors and display inter- and intra-observer variability which influence the validation results of automated cell segmentation pipelines. We present a new approach to simulate fluorescent cell micrographs that provides an objective ground truth for the validation of cell segmentation methods. The cell simulation was evaluated twofold: (1) An expert observer study shows that the proposed approach generates realistic fluorescent cell micrograph simulations. (2) An automated segmentation pipeline on the simulated fluorescent cell micrographs reproduces segmentation performances of that pipeline on real fluorescent cell micrographs. The proposed simulation approach produces realistic fluorescent cell micrographs with corresponding ground truth. The simulated data is suited to evaluate image segmentation pipelines more efficiently and reproducibly than it is possible on manually annotated real micrographs.

GPER1-mediated IGFBP-1 induction modulates IGF-1-dependent signaling in tamoxifen-treated breast cancer cells.

PubMed

Vaziri-Gohar, Ali; Houston, Kevin D

2016-02-15

Tamoxifen, a selective estrogen receptor modulator, is a commonly prescribed adjuvant therapy for estrogen receptor-α (ERα)-positive breast cancer patients. To determine if extracellular factors contribute to the modulation of IGF-1 signaling after tamoxifen treatment, MCF-7 cells were treated with IGF-1 in conditioned medium (CM) obtained from 4-OHT-treated MCF-7 cells and the accumulation of phospho-Akt (S473) was measured. CM inhibited IGF-1-dependent cell signaling and suggesting the involvement of extracellular factors (ie. IGFBPs). A significant increase in IGFBP-1 mRNA and extracellular IGFBP-1 protein was observed in 4-OHT-treated MCF-7 cells. Knockdown experiments demonstrated that both GPER1 and CREB mediate IGFBP-1 induction. Furthermore, experiments showed that 4-OHT-dependent IGFBP-1 transcription is downstream of GPER1-activation in breast cancer cells. Additionally, neutralization and knockdown experiments demonstrated a role for IGFBP-1 in the observed inhibition of IGF-1 signaling. These results suggested that 4-OHT inhibits IGF-1 signaling via GPER1 and CREB mediated extracellular IGFBP-1 accumulation in breast cancer cells. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Study of Fluorescent Imaging of Se (IV) in Living Cells Using a Turn-on Fluorescent Probe Based on a Rhodamine Spirolactame Derivative.

PubMed

Guan, Mingming; Mi, Hongyu; Xu, Hui; Fei, Qiang; Shan, Hongyan; Huan, Yanfu; Lv, Shaowu; Feng, Guodong

2017-03-01

A highly selective fluorescent probe 2-(2-(2-aminoethylamino)ethyl)-3',6'-bis(ethylamino)-2',7'-dimethylspiro[isoindoline-1,9'-xanthen]-3-one (ABDO) for Se (IV) had been synthesized in our earlier report. In this study, this fluorescent sensor is applied on analysis fluorescent imaging of Se (IV) in Hela cells. The experiment conditions, such as the MTT assay, different concentration of saline, incubated time of Hela cells with ABDO and Se (IV), and intracellular action position of Se (IV), are investigated. Through a series of experiments, the fluorescent image of Se (IV) in Hela cells can be observed when the cells cultured by 2 μM ABDO and 2 μM Se (IV) for 210 min. And the intracellular action position of Se (IV) is verified after the co-localization experiments are done. It is mitochondria. These experimental results show that ABDO will be an eagerly anticipated sensor for fluorescent imaging analysis of selenium ion in living cells. Besides, we also can use the complexes of ABDO-Se to observe morphology and distribution of mitochondria in cells like JG-B.

Viscoelastic and biochemical properties of erythrocytes during storage with SAG-M at +4 degrees C.

PubMed

Farges, E; Grebe, R; Baumann, M

2002-01-01

During storage at +4 degrees C, red blood cells undergo biochemical and physicochemical modifications, which alter their rheological characteristics especially the deformability. Even so until now not precisely defined deformability is undoubtedly a function of whole cell elasticity and viscosity. In a previous study we have investigated changes of elasticity of whole RBCs during a 6 weeks storage by quasi-static experiments using our Cell-Elastometer method. Since the changes in deformability we observed with that experimental approach have not been significant we extended the hard/software capabilities of this instrument to enable dynamic measurements also. We applied this modified hard-/software set-up to examine again changes in viscoelasticity of erythrocytes from concentrates during a six weeks storage at a blood bank. The cells were resuspended in CPD-SAG-M and stored at +4 degrees C. Quasi-static and dynamic experiments were performed on stored erythrocytes and showed for both significant changes in elasticity and viscoelasticity from the fourth week on. So it can be stated that due to our experimental results decrease in deformability of RBCs during storage occurs after a four weeks period of relative stability. To get further insight in changes of underlying or related biochemical properties according experiments have been performed in parallel. Especially the decrease in ATP showed a nearly parallel time course with a significant decrease after the 4th week. All other parameters especially the 2,3 DPG level showed a nearly linear de- or increase with time which are in accordance with the results of the additionally performed elongation experiments. Our quasi-static and dynamic deformability measurements have been proven to provide a simple and reliable tool to follow up erythrocyte senescence during storage where a pronounced change in mechanical properties may be used as an indicator for a change in bioviability. This has to be verified in further experiments.

Interallelic complementation of mutations in propionic acidemia by microinjection of mutant cDNAs into fibroblasts of affected patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loyer, M.; Leclerc, D.; Gravel, R.A.

1994-09-01

Propionic acidemia is a rare autosomal recessive disorder resulting from defects of the {alpha} or {beta} subunit of biotin-dependent propionyl-CoA carboxylase (PCC). Mutations are assigned to defects of the PCCA ({alpha} subunit) or PCCB ({beta} subunit) gene through complementation studies after somatic fusion of patient cell lines. About two-thirds of patients with {beta} subunit defects (complementation group pccBC) show interallelic complementation in cell fusion experiments (subgroups pccB and pccC), monitored by the PCC-dependent metabolisms of {sup 14}C-propionate. Most patient cell lines are heteroallelic for two different mutations, leaving ambiguous the identity of the mutation participating in interallelic complementation. To identifymore » the complementing mutations, we have expressed {beta}-subunit cDNAs containing individual mutations by microinjection of the cDNAs in recipient cells from patients with {beta} subunit defects. Correction of the PCC defect was monitored by autoradiography of {sup 14}C-propionate incorporation. In some experiments, cDNAs were co-injected with a plasmid expressing the E. coli lacZ gene as a positive control for successful injection. Two mutations from the pccB subgroup showed complementation when injected into pccC cells; dupKICK140-143 and Pro228Leu. Similarly, two mutations from the pccC subgroup complemented after injection into pccB cells; {Delta}Ile408 and Arg410Trp. No mutation complemented with mutation of the pccBC group which are classified as non-complementing in cell fusion experiments. The results show that the complementing pccB mutations are found in the N-terminal half of the {beta} subunit, while the complementing pccC mutations cluxter at a site in the C-terminal half. The latter site is a candidate for the propionyl-CoA binding site based on sequence identity with a region of transcarboxylase from Propionibacterium shermanii.« less

FOXC1 Regulates Expression of Prostaglandin Receptors Leading to an Attenuated Response to Latanoprost.

PubMed

Doucette, Lance P; Footz, Tim; Walter, Michael A

2018-05-01

This study examines the effect of FOXC1 on the prostaglandin pathway in order to explore FOXC1's role in the prostaglandin-resistant glaucoma phenotype commonly seen in Axenfeld-Rieger syndrome. Binding and transcriptional activity of FOXC1 to the gene coding for the EP3 prostaglandin receptor (PTGER3) were evaluated through ChIP-qPCR and luciferase-based assays. Immortalized trabecular meshwork cells (TM1) and HeLa cells had FOXC1 mRNA reduced via siRNA interference. qPCR and Western blot experiments were conducted to examine the changes in prostaglandin receptor expression brought about by lowered FOXC1. TM1 cells were then treated with 10 μM latanoprost acid and/or an siRNA for FOXC1. The expression of fibronectin and matrix metalloproteinase 9 were evaluated via qPCR in each treatment condition. ChIP-qPCR and luciferase experiments confirmed that FOXC1 binds to and activates transcription of the EP3 gene prostaglandin receptor. qPCR and Western experiments in HeLa and TM1 cells showed that FOXC1 siRNA knockdown results in significantly lowered EP3 levels (protein and RNA). In addition, RNA levels of the other prostaglandin receptor genes EP1 (PTGER1), EP2 (PTGER2), EP4 (PTGER4), and FP (PTGFR) were altered when FOXC1 was knocked down in TM1 and HeLa cells. Analysis of fibronectin expression in TM1 cells after treatment with 10 μM latanoprost acid showed a statistically significant increase in expression; this increase was abrogated by cotreatment with a siRNA for FOXC1. We show the abrogation of latanoprost signalling when FOXC1 is knocked down via siRNA in a trabecular meshwork cell line. We propose that the lower levels of active FOXC1 in Axenfeld-Rieger syndrome patients with glaucoma account for the lack of response to prostaglandin-based medications.

Covariation of metabolic rates and cell size in coccolithophores

NASA Astrophysics Data System (ADS)

Aloisi, G.

2015-08-01

Coccolithophores are sensitive recorders of environmental change. The size of their coccosphere varies in the ocean along gradients of environmental conditions and provides a key for understanding the fate of this important phytoplankton group in the future ocean. But interpreting field changes in coccosphere size in terms of laboratory observations is hard, mainly because the marine signal reflects the response of multiple morphotypes to changes in a combination of environmental variables. In this paper I examine the large corpus of published laboratory experiments with coccolithophores looking for relations between environmental conditions, metabolic rates and cell size (a proxy for coccosphere size). I show that growth, photosynthesis and, to a lesser extent, calcification covary with cell size when pCO2, irradiance, temperature, nitrate, phosphate and iron conditions change. With the exception of phosphate and temperature, a change from limiting to non-limiting conditions always results in an increase in cell size. An increase in phosphate or temperature (below the optimum temperature for growth) produces the opposite effect. The magnitude of the coccosphere-size changes observed in the laboratory is comparable to that observed in the ocean. If the biological reasons behind the environment-metabolism-size link are understood, it will be possible to use coccosphere-size changes in the modern ocean and in marine sediments to investigate the fate of coccolithophores in the future ocean. This reasoning can be extended to the size of coccoliths if, as recent experiments are starting to show, coccolith size reacts to environmental change proportionally to coccosphere size. The coccolithophore database is strongly biased in favour of experiments with the coccolithophore Emiliania huxleyi (E. huxleyi; 82 % of database entries), and more experiments with other species are needed to understand whether these observations can be extended to coccolithophores in general. I introduce a simple model that simulates the growth rate and the size of cells forced by nitrate and phosphate concentrations. By considering a simple rule that allocates the energy flow from nutrient acquisition to cell structure (biomass) and cell maturity (biological complexity, eventually leading to cell division), the model is able to reproduce the covariation of growth rate and cell size observed in laboratory experiments with E. huxleyi when these nutrients become limiting. These results support ongoing efforts to interpret coccosphere and coccolith size measurements in the context of climate change.

Trastuzumab- and Fab' fragment-modified curcumin PEG-PLGA nanoparticles: preparation and evaluation in vitro and in vivo.

PubMed

Duan, Dongyu; Wang, Aiping; Ni, Ling; Zhang, Liping; Yan, Xiuju; Jiang, Ying; Mu, Hongjie; Wu, Zimei; Sun, Kaoxiang; Li, Youxin

2018-01-01

Nanoparticles (NPs) modified with bio-ligands represent a promising strategy for active targeted drug delivery to tumour. However, many targeted ligands, such as trastuzumab (TMAB), have high molecular weight, limiting their application for targeting. In this study, we prepared Fab' (antigen-binding fragments cut from TMAB)-modified NPs (Fab'-NPs) with curcumin (Cur) as a model drug for more effective targeting of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu), which is overexpressed on breast cancer cells. The release kinetics was conducted by dialysis bags. The ability to kill HER2-overexpressing BT-474 cells of Fab'-Cur-NPs compared with TMAB-Cur-NPs was conducted by cytotoxicity experiments. Qualitative and quantitative cell uptake studies using coumarin-6 (fluorescent probe)-loaded NPs were performed by fluorescence microscopy and flow cytometry. Pharmacokinetics and biodistribution experiments in vivo were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The release kinetics showed that both Fab'-Cur-NPs and TMAB-Cur-NPs provided continuous, slow release of curcumin for 72 h, with no significant difference. In vitro cytotoxicity experiments showed that Fab'-Cur-NPs manifested prominent ability to kill HER2-overexpressing BT-474 cells compared with TMAB-Cur-NPs. Qualitative and quantitative cell uptake studies indicated that the accumulation of Fab'-NPs was greater than that of TMAB-NPs in BT-474 (HER2+) cells; However, there was no significant difference in MDA-MB-231 (HER2-) cells. Pharmacokinetics and biodistribution experiments in vivo demonstrated that the half-life (t1/2) and area under the blood concentration-time curve (AUC0-t) of Fab'-Cur-NPs increased 5.30-fold and 1.76-fold relative to those of TMAB-Cur-NPs, respectively. Furthermore, the tumor accumulation of Fab'-Cur-NPs was higher than that of TMAB-Cur-NPs. Fab' fragment has greater capacity than the intact antibody to achieve tumor targeting through NP-based delivery.

Modulation of osteoblast attachment and growth in vitro by inertial forces

NASA Astrophysics Data System (ADS)

Kacena, Melissa Ann

1999-11-01

Spaceflight exploration and associated experiments show that human bones lose in density during inertial unloading, due principally to their demineralization. This research project examines the effect of gravity on osteoblast attachment and function in various inertial environments. Chicken calvarial osteoblasts were cultured under the following inertial conditions: spaceflight, simulated shuttle launch accelerations and vibrations, centrifugation, clino-rotation, and inversion. Cultures exposed to these conditions were compared with cultures grown in the laboratory as static 1G controls. Electron and light microscopy revealed the number of total osteoblasts attached to their substrate. Biochemical assays discerned changes in viable cell number, alkaline phosphatase levels, and mineralization. Immunohistochemical assays were used to investigate differences in cytoskeletal and extracellular matrix protein concentrations in the cultures, the percentage of proliferative cells, and cell viability. Compared to controls, spaceflight results indicated that the number of attached osteoblast cells was reduced. Launch simulation data indicated that the associated accelerations and vibrations may contribute to the reduction of attached osteoblasts in spaceflight cultures. Following centrifugation, the number of attached cells was unaltered; however, immunostaining of actin, fibronectin, and vinculin did show alterations in cultures exposed to hypergravity. Confluent cultures that were right side up, inverted, and clino-rotated contained a comparable number of attached cells and functioned similarly on the basis of measured alkaline phosphatase and bound calcium content. Sparse clino-rotated or inverted cultures showed an immediate response of diminished viable osteoblast numbers, but this effect disappeared with time and all remaining attached cells functioned similarly (APase and bound calcium). On the basis of these data osteoblast attachment and function in confluent cultures is minimally, if at all, affected by alterations in inertial environments. However, in sparse cultures about half as many cells are found attached initially. The remaining attached cells appear to multiply and function normally. These results suggest that the effects of spaceflight on bone are thus not likely to be caused by direct intrinsic effects of gravity on single osteoblasts that can be simulated in laboratory experiments in vitro experiments.

Moissanite anvil cell design for Giga-Pascal nuclear magnetic resonance.

PubMed

Meier, Thomas; Herzig, Tobias; Haase, Jürgen

2014-04-01

A new design of a non-magnetic high-pressure anvil cell for nuclear magnetic resonance (NMR) experiments at Giga-Pascal pressures is presented, which uses a micro-coil inside the pressurized region for high-sensitivity NMR. The comparably small cell has a length of 22 mm and a diameter of 18 mm, so it can be used with most NMR magnets. The performance of the cell is demonstrated with external-force vs. internal-pressure experiments, and the cell is shown to perform well at pressures up to 23.5 GPa using 800 μm 6H-SiC large cone Boehler-type anvils. (1)H, (23)Na, (27)Al, (69)Ga, and (71)Ga NMR test measurements are presented, which show a resolution of better than 4.5 ppm, and an almost maximum possible signal-to-noise ratio.

SILAC-Based Quantitative Proteomic Analysis of Human Lung Cell Response to Copper Oxide Nanoparticles

PubMed Central

Edelmann, Mariola J.; Shack, Leslie A.; Naske, Caitlin D.; Walters, Keisha B.; Nanduri, Bindu

2014-01-01

Copper (II) oxide (CuO) nanoparticles (NP) are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level. PMID:25470785

Inflammatory sensitization of nociceptors depends on activation of NMDA receptors in DRG satellite cells.

PubMed

Ferrari, Luiz Fernando; Lotufo, Celina Monteiro; Araldi, Dionéia; Rodrigues, Marcos A; Macedo, Larissa P; Ferreira, Sérgio H; Parada, Carlos Amilcar

2014-12-23

The present study evaluated the role of N-methyl-D-aspartate receptors (NMDARs) expressed in the dorsal root ganglia (DRG) in the inflammatory sensitization of peripheral nociceptor terminals to mechanical stimulation. Injection of NMDA into the fifth lumbar (L5)-DRG induced hyperalgesia in the rat hind paw with a profile similar to that of intraplantar injection of prostaglandin E2 (PGE2), which was significantly attenuated by injection of the NMDAR antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-AP-5) in the L5-DRG. Moreover, blockade of DRG AMPA receptors by the antagonist 6,7-dinitroquinoxaline-2,3-dione had no effect in the PGE2-induced hyperalgesia in the paw, showing specific involvement of NMDARs in this modulatory effect and suggesting that activation of NMDAR in the DRG plays an important role in the peripheral inflammatory hyperalgesia. In following experiments we observed attenuation of PGE2-induced hyperalgesia in the paw by the knockdown of NMDAR subunits NR1, NR2B, NR2D, and NR3A with antisense-oligodeoxynucleotide treatment in the DRG. Also, in vitro experiments showed that the NMDA-induced sensitization of cultured DRG neurons depends on satellite cell activation and on those same NMDAR subunits, suggesting their importance for the PGE2-induced hyperalgesia. In addition, fluorescent calcium imaging experiments in cultures of DRG cells showed induction of calcium transients by glutamate or NMDA only in satellite cells, but not in neurons. Together, the present results suggest that the mechanical inflammatory nociceptor sensitization is dependent on glutamate release at the DRG and subsequent NMDAR activation in satellite glial cells, supporting the idea that the peripheral hyperalgesia is an event modulated by a glutamatergic system in the DRG.

Why large cells dominate estuarine phytoplankton

USGS Publications Warehouse

Cloern, James E.

2018-01-01

Surveys across the world oceans have shown that phytoplankton biomass and production are dominated by small cells (picoplankton) where nutrient concentrations are low, but large cells (microplankton) dominate when nutrient-rich deep water is mixed to the surface. I analyzed phytoplankton size structure in samples collected over 25 yr in San Francisco Bay, a nutrient-rich estuary. Biomass was dominated by large cells because their biomass selectively grew during blooms. Large-cell dominance appears to be a characteristic of ecosystems at the land–sea interface, and these places may therefore function as analogs to oceanic upwelling systems. Simulations with a size-structured NPZ model showed that runs of positive net growth rate persisted long enough for biomass of large, but not small, cells to accumulate. Model experiments showed that small cells would dominate in the absence of grazing, at lower nutrient concentrations, and at elevated (+5°C) temperatures. Underlying these results are two fundamental scaling laws: (1) large cells are grazed more slowly than small cells, and (2) grazing rate increases with temperature faster than growth rate. The model experiments suggest testable hypotheses about phytoplankton size structure at the land–sea interface: (1) anthropogenic nutrient enrichment increases cell size; (2) this response varies with temperature and only occurs at mid-high latitudes; (3) large-cell blooms can only develop when temperature is below a critical value, around 15°C; (4) cell size diminishes along temperature gradients from high to low latitudes; and (5) large-cell blooms will diminish or disappear where planetary warming increases temperature beyond their critical threshold.

Aggregation via the Red, Dry, and Rough Morphotype Is Not a Virulence Adaptation in Salmonella enterica Serovar Typhimurium▿

PubMed Central

White, A. P.; Gibson, D. L.; Grassl, G. A.; Kay, W. W.; Finlay, B. B.; Vallance, B. A.; Surette, M. G.

2008-01-01

The Salmonella rdar (red, dry, and rough) morphotype is an aggregative and resistant physiology that has been linked to survival in nutrient-limited environments. Growth of Salmonella enterica serovar Typhimurium was analyzed in a variety of nutrient-limiting conditions to determine whether aggregation would occur at low cell densities and whether the rdar morphotype was involved in this process. The resulting cultures consisted of two populations of cells, aggregated and nonaggregated, with the aggregated cells preferentially displaying rdar morphotype gene expression. The two groups of cells could be separated based on the principle that aggregated cells were producing greater amounts of thin aggregative fimbriae (Tafi or curli). In addition, the aggregated cells retained some physiological characteristics of the rdar morphotype, such as increased resistance to sodium hypochlorite. Competitive infection experiments in mice showed that nonaggregative ΔagfA cells outcompeted rdar-positive wild-type cells in all tissues analyzed, indicating that aggregation via the rdar morphotype was not a virulence adaptation in Salmonella enterica serovar Typhimurium. Furthermore, in vivo imaging experiments showed that Tafi genes were not expressed during infection but were expressed once Salmonella was passed out of the mice into the feces. We hypothesize that the primary role of the rdar morphotype is to enhance Salmonella survival outside the host, thereby aiding in transmission. PMID:18195033

An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro.

PubMed

Zhang, Li; Wu, Yenan; Li, Xiang; Wei, Shao; Xing, Yiming; Lian, Zhengxing; Han, Hongbing

2018-01-01

Chicken embryonic stem cells (cESCs) obtained from stage X embryos provide a novel model for the study of avian embryonic development. A new way to maintain cESCs for a long period in vitro still remains unexplored. We found that the cESCs showed stem cell-like properties in vitro for a long term with the support of DF-1 feeder and basic culture medium supplemented with human basic fibroblast growth factor (hbFGF), mouse stem cell factor (mSCF), and human leukemia inhibitory factor (hLIF). During the long culture period, the cESCs showed typical ES cell morphology and expressed primitive stem cell markers with a relatively stable proliferation rate and high telomerase activity. These cells also exhibited the capability to differentiate into cardiac myocytes, smooth muscle cells, neural cells, osteoblast, and adipocyte in vitro . Chimera chickens were produced by cESCs cultured for 25 passages with this new culture system. The experiments showed that DF-1 was the optimal feeder and hbFGF was an important factor for maintaining the pluripotency of cESCs in vitro .

Drosophila Glypicans Regulate Follicle Stem Cell Maintenance and Niche Competition.

PubMed

Su, Tsu-Yi; Nakato, Eriko; Choi, Pui Yee; Nakato, Hiroshi

2018-04-09

Adult stem cells reside in specialized microenvironments, called niches, which provide signals for stem cells to maintain their undifferentiated and self-renewing state. To maintain stem cell quality, several types of stem cells are known to be regularly replaced by progenitor cells through niche competition. However, the cellular and molecular bases for stem cell competition for niche occupancy are largely unknown. Here, we show that two Drosophila members of the glypican family of heparan sulfate proteoglycans (HSPGs), Dally and Dally-like (Dlp), differentially regulate follicle stem cell (FSC) maintenance and FSC competitiveness for niche occupancy. Lineage analyses of glypican mutant FSC clones showed that dally is essential for normal FSC maintenance. In contrast, dlp is a hyper-competitive mutation: dlp mutant FSC progenitors often eventually occupy the entire epithelial sheet. RNAi knockdown experiments showed that Dally and Dlp play both partially redundant and distinct roles in regulating Jak/Stat, Wg and Hh signaling in FSCs. The Drosophila FSC system offers a powerful genetic model to study the mechanisms by which HSPGs exert specific functions in stem cell replacement and competition. Copyright © 2018, Genetics.

MicroRNA-1 overexpression increases chemosensitivity of non-small cell lung cancer cells by inhibiting autophagy related 3-mediated autophagy.

PubMed

Hua, Li; Zhu, Guirong; Wei, Jianguo

2018-05-30

Non-small cell lung cancer (NSCLC) is a major type of lung cancer. Drug resistance is a enormous obstacle for cancer treatment. Copious microRNAs (miRNAs) have been demonstrated to be implicated in drug resistance in NSCLC. In the present study, RT-qPCR assay revealed that microRNA-1 (miR-1) expression was downregulated in DDP resistant NSCLC tissues and cells. Western blot assay presented a remarkable increase of LC3B-II/LC3B-I ratio and a notable decline of p62 level in DDP resistant NSCLC cells, while these effects were weakened by miR-1. GFP-LC3 puncta experiment showed that ectopic expression of miR-1 induced a noticeable downregulation of GFP-LC3 positive cell percentage in DDP resistant NSCLC cells. Bioinformatical analysis and luciferase assay revealed that autophagy related 3 (ATG3) was a target of miR-1. Also, western blot and RT-qPCR assays manifested that ATG3 was highly expressed in DDP resistant NSCLC tissues and cells. Additionally, miR-1 inhibited ATG3 expression and ATG3 upregulation abolished miR-1-meidated autophagy inhibition in DDP resistant NSCLC cells. Cell Counting Kit-8 (CCK-8) assay showed that the half maximal inhibitory concentration (IC 50 ) of cisplatin (DDP) was reduced in miR-1-enforced DDP resistant NSCLC cells, but was restored following the overexpression of ATG3. Flow cytometry experiments further showed that miR-1 overexpression induced a significant upregulation of apoptotic rate and ATG3 restoration weakened miR-1-induced apoptosis in DDP resistant NSCLC cells. Collectively, our study validated that miR-1 overexpression improved DDP sensitivity of NSCLC cells by inhibiting ATG3-mediated autophagy, providing a potential therapeutic target for easing chemoresistance of anti-tumor drugs. This article is protected by copyright. All rights reserved.

Isolation and characterization of chromosome-gain and increase-in-ploidy mutants in yeast.

PubMed

Chan, C S; Botstein, D

1993-11-01

We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37 degrees. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37 degrees. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth.

Assembly of live micro-organisms on microstructured PDMS stamps by convective/capillary deposition for AFM bio-experiments

NASA Astrophysics Data System (ADS)

Dague, E.; Jauvert, E.; Laplatine, L.; Viallet, B.; Thibault, C.; Ressier, L.

2011-09-01

Immobilization of live micro-organisms on solid substrates is an important prerequisite for atomic force microscopy (AFM) bio-experiments. The method employed must immobilize the cells firmly enough to enable them to withstand the lateral friction forces exerted by the tip during scanning but without denaturing the cell interface. In this work, a generic method for the assembly of living cells on specific areas of substrates is proposed. It consists in assembling the living cells within the patterns of microstructured, functionalized poly-dimethylsiloxane (PDMS) stamps using convective/capillary deposition. This versatile approach is validated by applying it to two systems of foremost importance in biotechnology and medicine: Saccharomyces cerevisiae yeasts and Aspergillus fumigatus fungal spores. We show that this method allows multiplexing AFM nanomechanical measurements by force spectroscopy on S. cerevisiae yeasts and high-resolution AFM imaging of germinated Aspergillus conidia in buffer medium. These two examples clearly demonstrate the immense potential of micro-organism assembly on functionalized, microstructured PDMS stamps by convective/capillary deposition for performing rigorous AFM bio-experiments on living cells.

Cellular pressure and volume regulation and implications for cell mechanics

NASA Astrophysics Data System (ADS)

Jiang, Hongyuan; Sun, Sean

2013-03-01

In eukaryotic cells, small changes in cell volume can serve as important signals for cell proliferation, death and migration. Volume and shape regulation also directly impacts the mechanics of the cell and multi-cellular tissues. Recent experiments found that during mitosis, eukaryotic cells establish a preferred steady volume and pressure, and the steady volume and pressure can robustly adapt to large osmotic shocks. Here we develop a mathematical model of cellular pressure and volume regulation, incorporating essential elements such as water permeation, mechano-sensitive channels, active ion pumps and active stresses in the actomyosin cortex. The model can fully explain the available experimental data, and predicts the cellular volume and pressure for several models of cell cortical mechanics. Furthermore, we show that when cells are subjected to an externally applied load, such as in an AFM indentation experiment, active regulation of volume and pressure leads to complex cellular response. We found the cell stiffness highly depends on the loading rate, which indicates the transport of water and ions might contribute to the observed viscoelasticity of cells.

Drosophila heart cell movement to the midline occurs through both cell autonomous migration and dorsal closure.

PubMed

Haack, Timm; Schneider, Matthias; Schwendele, Bernd; Renault, Andrew D

2014-12-15

The Drosophila heart is a linear organ formed by the movement of bilaterally specified progenitor cells to the midline and adherence of contralateral heart cells. This movement occurs through the attachment of heart cells to the overlying ectoderm which is undergoing dorsal closure. Therefore heart cells are thought to move to the midline passively. Through live imaging experiments and analysis of mutants that affect the speed of dorsal closure we show that heart cells in Drosophila are autonomously migratory and part of their movement to the midline is independent of the ectoderm. This means that heart formation in flies is more similar to that in vertebrates than previously thought. We also show that defects in dorsal closure can result in failure of the amnioserosa to properly degenerate, which can physically hinder joining of contralateral heart cells leading to a broken heart phenotype. Copyright © 2014 Elsevier Inc. All rights reserved.

Biotechnology

NASA Image and Video Library

2001-08-04

In August 2001, principal investigator Jeanne Becker sent human ovarian tumor cells to the International Space Station (ISS) aboard the STS-105 mission. The tumor cells were cultured in microgravity for a 14 day growth period and were analyzed for changes in the rate of cell growth and synthesis of associated proteins. In addition, they were evaluated for the expression of several proteins that are the products of oncogenes, which cause the transformation of normal cells into cancer cells. This photo, which was taken by astronaut Frank Culbertson who conducted the experiment for Dr. Becker, shows two cell culture bags containing LN1 ovarian carcinoma cell cultures.

Investigating Genomic Mechanisms of Treatment Resistance in Castration Resistant Prostate Cancer

DTIC Science & Technology

2015-05-01

and genomically profiled. Figure 3 shows data from a series of cell- line experiments showing that PC3 prostate cancer cells are recoverable and...coursework until the second-half of the grant period. I am enrolled in the UCSF Biomedical Sciences Graduate Program class BMS 255: Genetics : Basic... Genetics and Genomics. This class is set to start in January 2016. Given a large number of clinical, teaching, and research duties I will plan to enroll

Dispersal, density dependence, and population dynamics of a fungal microbe on leaf surfaces.

PubMed

Woody, Scott T; Ives, Anthony R; Nordheim, Erik V; Andrews, John H

2007-06-01

Despite the ubiquity and importance of microbes in nature, little is known about their natural population dynamics, especially for those that occupy terrestrial habitats. Here we investigate the dynamics of the yeast-like fungus Aureobasidium pullulans (Ap) on apple leaves in an orchard. We asked three questions. (1) Is variation in fungal population density among leaves caused by variation in leaf carrying capacities and strong density-dependent population growth that maintains densities near carrying capacity? (2) Do resident populations have competitive advantages over immigrant cells? (3) Do Ap dynamics differ at different times during the growing season? To address these questions, we performed two experiments at different times in the growing season. Both experiments used a 2 x 2 factorial design: treatment 1 removed fungal cells from leaves to reveal density-dependent population growth, and treatment 2 inoculated leaves with an Ap strain engineered to express green fluorescent protein (GFP), which made it possible to track the fate of immigrant cells. The experiments showed that natural populations of Ap vary greatly in density due to sustained differences in carrying capacities among leaves. The maintenance of populations close to carrying capacities indicates strong density-dependent processes. Furthermore, resident populations are strongly competitive against immigrants, while immigrants have little impact on residents. Finally, statistical models showed high population growth rates of resident cells in one experiment but not in the other, suggesting that Ap experiences relatively "good" and "bad" periods for population growth. This picture of Ap dynamics conforms to commonly held, but rarely demonstrated, expectations of microbe dynamics in nature. It also highlights the importance of local processes, as opposed to immigration, in determining the abundance and dynamics of microbes on surfaces in terrestrial systems.

Consciousness can reduce the voltage of the output signal of solar cell

NASA Astrophysics Data System (ADS)

Cao, Dayong

2011-03-01

When the sun's light radiate on the solar cell, it can produce the output signal as the pho- tocurrent. We use the Data Acquisition Modules to record the voltage of the output signals. The v1 is voltage of the photocurrent of solar cell1; The v2 is the one of solar cell2. And these two solar cells stay side by side. When we record the voltages from the morning to the noon, the voltages will go up, and the v1 is bigger than the v2 during this time. But in other experi- menter, not only sun's light ratiade on two solar cells, but also consciousness act on two solar cells. Not only I can use consciousness to reduce the growth voltage of the output signals, but also can change the v1 to be littler than the v2. The experiment was conducted on Sep. 2010. When light of lamp radiate on two solar cells, I can reduce v1, at the same time, can augment v2. These experiments had been finished in Los Angeles, Oct. 26th. And the experiment show that the consciousness active function differ from the passive function of conditioned reflex (of Pavlov). There is the physical system of the mass, energy, space and time-MEST; There is the spirited system of the mind, consciousness, emotion and desire-MECD; the information system is the code system. We can use the consciousness change the electron-structure of solar cell by the interaction of the information.

[Recombinant adeno-associated virus mediated RNA interference of angiogenin expression inhibits cell growth of human lung adenocarcinoma].

PubMed

Li, Bai-Ling; Zhang, Guan-Xin; Hou, Xiao-Lei; Tan, Meng-Wei; Yuan, Yang; Liu, Xiao-Hong; Gong, De-Jun; Huang, Sheng-Dong

2009-03-01

To study the inhibition of angiogenin (ANG) expression in human lung squamous cancer cell strain-A549 through adeno-associated virus (AAV)-mediated RNA-interference, and therefore to observe its effect on the growth of cancer cells and tumor formation. Recombinant AAV expressing H1-promoter-induced small-interference- RNA (siRNA) targeting ANG (AAV-shANG) was constructed, and then transfected into A549 cells. A549 cells and cells transfected with AAV-Null were used as the control groups. The effects of the reduced expression of ANG by RNAi from AAV-shANG on the growth, formation, reproduction, apoptosis, and microvessel-density of the carcinoma were observed. In vitro experiment showed that AAV-shANG was constructed successfully, There was an significant decrease in the expression of ANG protein 72 h after transfection, compared with the normal A459 cells and AAV-Null cells (P < 0.01). Cell cycle analysis showed that the proliferation index (PI) of normal A549 cells, AAV-Null cells and AAVshANG cells were 0.32 +/- 0.29, 0.35 +/- 0.38 and 0.31 +/- 0.43, respectively. There was no statistic difference in the PIs among the 3 groups (P > 0.05). In vivo experiment using thymus-defect mice showed that, there was an remarkable reduction in the mass and volume of tumors in AAV-shANG transfected group, compared to the control groups. Microvessel-density was 9.4 +/- 1.5, 9.8 +/- 2.1 and 5.7 +/- 1.9, respectively in the 3 groups, a statistic difference among the AAV-shANG-transfected group, the normal A549 group and the AAV-Null transfected group. The percentages of apoptotic cells in each group were (7.7 +/- 3.1)%, (8.5 +/- 5.4)%, (17.1 +/- 8.6)%, respectively, the experimental group being higher than those of the control groups. Positive rates of PCNA were (84.8 +/- 9.7)%, (85.8 +/- 9.8)%, and (70.4 +/- 10.1)%, respectively, the AAV-shANG transfected cancer cells showing a lower PCNA index than the control groups. AAV-mediated expression of siRNA could reduce the expression of ANG in cancer cells, significantly enough to inhibit cell proliferation, promote cell apoptosis and inhibit tumor growth.

Colloidal Au and Au-alloy catalysts for direct borohydride fuel cells: Electrocatalysis and fuel cell performance

NASA Astrophysics Data System (ADS)

Atwan, Mohammed H.; Macdonald, Charles L. B.; Northwood, Derek O.; Gyenge, Elod L.

Supported colloidal Au and Au-alloys (Au-Pt and Au-Pd, 1:1 atomic ratio) on Vulcan XC-72 (with 20 wt% metal load) were prepared by the Bönneman method. The electrocatalytic activity of the colloidal metals with respect to borohydride electro-oxidation for fuel cell applications was investigated by voltammetry on static and rotating electrodes, chronoamperometry, chronopotentiometry and fuel cell experiments. The fundamental electrochemical techniques showed that alloying Au, a metal that leads to the maximum eight-electron oxidation of BH 4 -, with Pd or Pt, well-known catalysts of dehydrogenation reactions, improved the electrode kinetics of BH 4 - oxidation. Fuel cell experiments corroborated the kinetic studies. Using 5 mg cm -2 colloidal metal load on the anode, it was found that Au-Pt was the most active catalyst giving a cell voltage of 0.47 V at 100 mA cm -2 and 333 K, while under identical conditions the cell voltage using colloidal Au was 0.17 V.

Transcriptome analysis illuminates the nature of the intracellular interaction in a vertebrate-algal symbiosis

PubMed Central

Burns, John A; Zhang, Huanjia; Hill, Elizabeth; Kim, Eunsoo; Kerney, Ryan

2017-01-01

During embryonic development, cells of the green alga Oophila amblystomatis enter cells of the salamander Ambystoma maculatum forming an endosymbiosis. Here, using de novo dual-RNA seq, we compared the host salamander cells that harbored intracellular algae to those without algae and the algae inside the animal cells to those in the egg capsule. This two-by-two-way analysis revealed that intracellular algae exhibit hallmarks of cellular stress and undergo a striking metabolic shift from oxidative metabolism to fermentation. Culturing experiments with the alga showed that host glutamine may be utilized by the algal endosymbiont as a primary nitrogen source. Transcriptional changes in salamander cells suggest an innate immune response to the alga, with potential attenuation of NF-κB, and metabolic alterations indicative of modulation of insulin sensitivity. In stark contrast to its algal endosymbiont, the salamander cells did not exhibit major stress responses, suggesting that the host cell experience is neutral or beneficial. DOI: http://dx.doi.org/10.7554/eLife.22054.001 PMID:28462779

Ultrasound Stimulation of Insulin Release from Pancreatic Beta Cells

NASA Astrophysics Data System (ADS)

Suarez Castellanos, Ivan M.

Type 2 diabetes (T2D) mellitus is a complex metabolic disease that has reached epidemic proportions in the United States and around the world. Controlling T2D is often difficult as pharmacological management routinely requires complex therapy with multiple medications, and loses its effectiveness over time. The objective of this dissertation was to explore a novel, non-pharmacological approach that utilizes the application of ultrasound energy to stimulate insulin release. Our experiments have focused on determination of effectiveness and safety of ultrasound application in stimulation of insulin release from the pancreatic beta cells. Our results showed that ultrasound treatment, applied at frequencies of 800 kHz and 1 MHz and intensities of 0.5 W/cm2 and 1 W/cm2, did not produce any significant effects on cell viability compared to sham group as assessed with trypan blue dye exclusion test and MTT cytotoxicity assay. ELISA quantification of insulin release from beta cells resulting from ultrasound treatment showed clinically-significant amounts of released insulin as compared to sham-treated beta cells. Carbon fiber amperometry detection of secretory events from dopamine-loaded beta cells treated with ultrasound showed that release of secretory content could be temporally controlled by careful selection of ultrasound parameters. Both ELISA and amperometry experiments demonstrated that ultrasound-stimulated insulin release is a calcium-dependent process, potentially mediated by the mechanical effects of ultrasound. This study demonstrated that therapeutic ultrasound is a technique capable of stimulating the release of insulin from pancreatic beta cells in a safe, effective and controlled manner.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Romey, G.; Quast, U.; Pauron, D.

This paper shows the interaction of the cardiotonic agent 4-(3-(4-diphenylmethyl-1-piperazinyl)-2-hydroxypropoxy)-1H-indole-2-carbonitrile (DPI 201-106) and its optic enantiomers R-DPI (205-429) and S-DPI (205-430) with the Na/sup +/ channel of a variety of excitable cells. Voltage-clamp experiments show that DPI 201-106 acts on neuroblastoma cells and rat cardiac cells. S-DPI (205-430) increases the peak Na/sup +/ current, slows down the kinetics of Na/sup +/ channel inactivation, and is cardiotonic on heart cells. Conversely, R-DPI (205-429) reduces the peak Na/sup +/ current and blocks Na/sup +/ channel activity and cardiac contractions. Binding experiments using radioactively labeled toxins indicate that DPI 201-106 and its enantiomersmore » do not interact with sites already identified for tetrodotoxin or sea anemone and scorpion toxins. DPI 201-106 and its enantiomers inhibit binding of a /sup 3/H-labeled batrachotoxin derivative, (/sup 3/H)batrachotoxinin A 20-..cap alpha..-benzoate, to brain membranes. The dissociation constant of the complex formed between the Na/sup +/ channel and both R-DPI and S-DPI is K/sub d/ approx. 100 nM. /sup 22/Na/sup +/ uptake experiments using different cell types have shown that R and S enantiomers of DPI 201-106 are active on the different Na/sup +/ channel subtypes with similar IC/sub 50/ values. These results are discussed in relation with the cardiotonic properties of DPI 201-106 that are not accompanied by cardiotoxic effects.« less

Interaction of Synuclein and Inflammation in Dopaminergic Neurodegeneration

DTIC Science & Technology

2010-07-01

synuclein in cell culture. Specific Aim II In vivo studies: we are now optimizing immunostaining for human -synuclein in order to distinguish...responsible for this response. Recent cell culture experiments seem to point us in the right direction for the answer as it has been shown that human BE-M17...synuclein. Furthermore, Zhou et al (2002) showed that, in primary cell cultures derived from the embryonic human mesencephalon overexpressing either

Censoring of self-reactive B cells by follicular dendritic cell-displayed self-antigen

PubMed Central

Yau, Irene W.; Cato, Matthew H.; Jellusova, Julia; Hurtado de Mendoza, Tatiana; Brink, Robert; Rickert, Robert C.

2013-01-01

In the secondary lymphoid organs, intimate contact with follicular dendritic cells (FDCs) is required for B cell retention and antigen-driven selection during the germinal center response. However, selection of self-reactive B cells by antigen on FDCs has not been addressed. To this end, we generated a mouse model to conditionally express a membrane-bound self-antigen on FDCs, and monitor the fate of developing self-reactive B cells. Here, we show that self-antigen displayed on FDCs mediates effective elimination of self-reactive B cells at the transitional stage. Notwithstanding, some self-reactive B cells persist beyond this checkpoint, showing evidence of antigen experience and intact proximal BCR signaling, but they are short-lived and unable to elicit T cell help. These results implicate FDCs as an important component of peripheral B cell tolerance that prevent the emergence of naïve B cells capable of responding to sequestered self-antigens. PMID:23817432

Functional convergence of Akt protein with VEGFR-1 in human endothelial progenitor cells exposed to sera from patient with type 2 diabetes mellitus.

PubMed

Hassanpour, Mehdi; Rezabakhsh, Aysa; Rahbarghazi, Reza; Nourazarian, Alireza; Nouri, Mohammad; Avci, Çığır Biray; Ghaderi, Shahrooz; Alidadyani, Neda; Bagca, Bakiye Goker; Bagheri, Hesam Saghaei

2017-11-01

Diabetes mellitus type 2 predisposes patients to various microvascular complications. In the current experiment, the potent role of diabetes mellitus was investigated on the content of VEGFR-1, -2, Tie-1 and -2, and Akt in human endothelial progenitor cells. The gene expression profile of mTOR and Hedgehog signaling pathways were measured by PCR array. The possible crosstalk between RTKs, mTOR and Hedgehog signaling was also studied by bioinformatic analysis. Endothelial progenitor cells were incubated with serum from normal and diabetic for 7days. Compared to non-treated cells, diabetic serum-induced cell apoptosis (~2-fold) and prohibited cell migration toward bFGF (p<0.001). ELISA analysis showed that diabetes exposed cells had increased abundance of Tie-1, -2 and VEGFR-2 and reduced amount of VEGFR-1 (p<0.0001) in diabetic cells. Western blotting showed a marked reduction in the protein level of Akt after cells exposure to serum from diabetic subjects (p<0.0001). PCR array revealed a significant stimulation of both mTOR and Hedgehog signaling pathways in diabetic cells (p<0.05). According to data from bioinformatic datasets, we showed VEGFR-1, -2 and Tie-2, but not Tie-1, are master regulators of angiogenesis. There is a crosstalk between RTKs and mTOR signaling by involving P62, GABARAPL1, and HTT genes. It seems that physical interaction and co-expression of Akt decreased the level of VEGFR-1 in diabetic cells. Regarding data from the present experiment, diabetic serum contributed to uncontrolled induction of both mTOR and Hedgehog signaling in endothelial progenitor cells. Diabetes mellitus induces mTOR pathway by involving receptor tyrosine kinases while Hedgehog stimulation is independent of these receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

Minichromosome maintenance protein 2 and 3 promote osteosarcoma progression via DHX9 and predict poor patient prognosis

PubMed Central

Cheng, Dong-dong; Zhang, Hui-zhen; Yuan, Jun-qing; Li, Shi-jie; Yang, Qing-cheng; Fan, Cun-yi

2017-01-01

A label free quantitative proteomic approach (SWATH™ experiment) was performed to identify tumor-associated nuclear proteins that are differentially expressed between osteosarcoma cells and osteoblast cells. By functional screening, minichromosome maintenance protein 2 (MCM2) and minichromosome maintenance protein 3 (MCM3) were found to be related to osteosarcoma cell growth. Here, we show that knockdown of MCM2 or MCM3 inhibits osteosarcoma growth in vitro and in vivo. In co-immunoprecipitation and co-localization experiments, MCM2 and MCM3 were found to interact with DExH-box helicase 9 (DHX9) in osteosarcoma cells. A rescue study showed that the decreased growth of osteosarcoma cells by MCM2 or MCM3 knockdown was reversed by DHX9 overexpression, indicating that MCM2 and MCM3 activity was DHX9-dependent. In addition, the depletion of DHX9 hindered osteosarcoma cell proliferation. Notably, MCM2 and MCM3 expression levels were positively correlated with the DHX9 expression level in tumor samples and were associated with a poor prognosis in patients with osteosarcoma. Taken together, these results suggest that the MCM2/MCM3–DHX9 axis has an important role in osteosarcoma progression. PMID:28460433

Kir 4.1 inward rectifier potassium channel is upregulated in astrocytes in a murine multiple sclerosis model.

PubMed

Mercado, Francisco; Almanza, Angélica; Rubio, Nazario; Soto, Enrique

2018-06-11

Multiple sclerosis (MS) is a high prevalence degenerative disease characterized at the cellular level by glial and neuronal cell death. The causes of cell death during the disease course are not fully understood. In this work we demonstrate that in a MS model induced by Theiler's murine encephalomyelitis virus (TMEV) infection, the inward rectifier (Kir) 4.1 potassium channel subunit is overexpressed in astrocytes. In voltage clamp experiments the inward current density from TMEV-infected astrocytes was significantly larger than in mock-infected ones. The cRNA hybridization analysis from mock- and TMEV-infected cells showed an upregulation of a potassium transport channel coding sequence. We validated this mRNA increase by RT-PCR and quantitative PCR using Kir 4.1 specific primers. Western blotting experiments confirmed the upregulation of Kir 4.1, and alignment between sequences provided the demonstration that the over-expressed gene encodes for a Kir family member. Flow cytometry showed that the Kir 4.1 protein is located mainly in the cell membrane in mock and TMEV-infected astrocytes. Our results demonstrate an increase in K + inward current in TMEV-infected glial cells, this increment may reduce the neuronal depolarization, contributing to cell resilience mechanisms. Copyright © 2018 Elsevier B.V. All rights reserved.

Compressible Convection Experiment using Xenon Gas in a Centrifuge

NASA Astrophysics Data System (ADS)

Menaut, R.; Alboussiere, T.; Corre, Y.; Huguet, L.; Labrosse, S.; Deguen, R.; Moulin, M.

2017-12-01

We present here an experiment especially designed to study compressible convection in the lab. For significant compressible convection effects, the parameters of the experiment have to be optimized: we use xenon gaz in a cubic cell. This cell is placed in a centrifuge to artificially increase the apparent gravity and heated from below. With these choices, we are able to reach a dissipation number close to Earth's outer core value. We will present our results for different heating fluxes and rotation rates. We success to observe an adiabatic gradient of 3K/cm in the cell. Studies of pressure and temperature fluctuations lead us to think that the convection takes place under the form of a single roll in the cell for high heating flux. Moreover, these fluctuations show that the flow is geostrophic due to the high rotation speed. This important role of rotation, via Coriolis force effects, in our experimental setup leads us to develop a 2D quasigeostrophic compressible model in the anelastic liquid approximation. We test numerically this model with the finite element solver FreeFem++ and compare its results with our experimental data. In conclusion, we will present our project for the next experiment in which the cubic cell will be replace by a annulus cell. We will discuss the new expected effects due to this geometry as Rossby waves and zonal flows.

Abscisic Acid Regulation of Root Hydraulic Conductivity and Aquaporin Gene Expression Is Crucial to the Plant Shoot Growth Enhancement Caused by Rhizosphere Humic Acids.

PubMed

Olaetxea, Maite; Mora, Verónica; Bacaicoa, Eva; Garnica, María; Fuentes, Marta; Casanova, Esther; Zamarreño, Angel M; Iriarte, Juan C; Etayo, David; Ederra, Iñigo; Gonzalo, Ramón; Baigorri, Roberto; García-Mina, Jose M

2015-12-01

The physiological and metabolic mechanisms behind the humic acid-mediated plant growth enhancement are discussed in detail. Experiments using cucumber (Cucumis sativus) plants show that the shoot growth enhancement caused by a structurally well-characterized humic acid with sedimentary origin is functionally associated with significant increases in abscisic acid (ABA) root concentration and root hydraulic conductivity. Complementary experiments involving a blocking agent of cell wall pores and water root transport (polyethylenglycol) show that increases in root hydraulic conductivity are essential in the shoot growth-promoting action of the model humic acid. Further experiments involving an inhibitor of ABA biosynthesis in root and shoot (fluridone) show that the humic acid-mediated enhancement of both root hydraulic conductivity and shoot growth depended on ABA signaling pathways. These experiments also show that a significant increase in the gene expression of the main root plasma membrane aquaporins is associated with the increase of root hydraulic conductivity caused by the model humic acid. Finally, experimental data suggest that all of these actions of model humic acid on root functionality, which are linked to its beneficial action on plant shoot growth, are likely related to the conformational structure of humic acid in solution and its interaction with the cell wall at the root surface. © 2015 American Society of Plant Biologists. All Rights Reserved.

Abscisic Acid Regulation of Root Hydraulic Conductivity and Aquaporin Gene Expression Is Crucial to the Plant Shoot Growth Enhancement Caused by Rhizosphere Humic Acids1

PubMed Central

Bacaicoa, Eva; Garnica, María; Fuentes, Marta; Casanova, Esther; Etayo, David; Ederra, Iñigo; Gonzalo, Ramón

2015-01-01

The physiological and metabolic mechanisms behind the humic acid-mediated plant growth enhancement are discussed in detail. Experiments using cucumber (Cucumis sativus) plants show that the shoot growth enhancement caused by a structurally well-characterized humic acid with sedimentary origin is functionally associated with significant increases in abscisic acid (ABA) root concentration and root hydraulic conductivity. Complementary experiments involving a blocking agent of cell wall pores and water root transport (polyethylenglycol) show that increases in root hydraulic conductivity are essential in the shoot growth-promoting action of the model humic acid. Further experiments involving an inhibitor of ABA biosynthesis in root and shoot (fluridone) show that the humic acid-mediated enhancement of both root hydraulic conductivity and shoot growth depended on ABA signaling pathways. These experiments also show that a significant increase in the gene expression of the main root plasma membrane aquaporins is associated with the increase of root hydraulic conductivity caused by the model humic acid. Finally, experimental data suggest that all of these actions of model humic acid on root functionality, which are linked to its beneficial action on plant shoot growth, are likely related to the conformational structure of humic acid in solution and its interaction with the cell wall at the root surface. PMID:26450705

Inhibition of Regulatory Volume Decrease Enhances the Cytocidal Effect of Hypotonic Shock in Hepatocellular Carcinoma.

PubMed

Kudou, Michihiro; Shiozaki, Atsushi; Kosuga, Toshiyuki; Ichikawa, Daisuke; Konishi, Hirotaka; Morimura, Ryo; Komatsu, Shuhei; Ikoma, Hisashi; Fujiwara, Hitoshi; Okamoto, Kazuma; Hosogi, Shigekuni; Nakahari, Takashi; Marunaka, Yoshinori; Otsuji, Eigo

2016-01-01

Background : Hypotonic shock induces cytocidal effects through cell rupture, and cancer therapy based on this mechanism has been clinically administered to hepatocellular carcinoma patients. We herein investigated the effectiveness of hypotonic shock combined with the inhibition of regulatory volume decrease as cancer therapy for hepatocellular carcinoma. Methods : Morphological changes in human hepatocellular carcinoma cell lines were observed under a differential interference contrast microscope connected to a high-speed digital video camera. Cell volume changes under hypotonic shock with or without chloride, potassium, or water channel blockers were observed using a high-resolution flow cytometer. In order to investigate cytocidal effects, the number of surviving cells was compared after exposure to hypotonic solution with and without each channel blocker (re-incubation experiment). Results : Video recordings showed that cells exposed to distilled water rapidly swelled and then ruptured. Cell volume measurements revealed regulatory volume decrease under mild hypotonic shock, whereas severe hypotonic shock increased the number of broken fragments as a result of cell rupture. Moreover, regulatory volume decrease was inhibited in cells treated with each channel blocker. Re-incubation experiments showed the cytocidal effects of hypotonic shock in cells exposed to hypotonic solution, and additional treatments with each channel blocker enhanced these effects. Conclusion : The inhibition of regulatory volume decrease with chloride, potassium, or water channel blockers may enhance the cytocidal effects of hypotonic shock in hepatocellular carcinoma. Hypotonic shock combined with the inhibition of regulatory volume decrease was a more effective therapy than hypotonic shock alone.

Inhibition of Regulatory Volume Decrease Enhances the Cytocidal Effect of Hypotonic Shock in Hepatocellular Carcinoma

PubMed Central

Kudou, Michihiro; Shiozaki, Atsushi; Kosuga, Toshiyuki; Ichikawa, Daisuke; Konishi, Hirotaka; Morimura, Ryo; Komatsu, Shuhei; Ikoma, Hisashi; Fujiwara, Hitoshi; Okamoto, Kazuma; Hosogi, Shigekuni; Nakahari, Takashi; Marunaka, Yoshinori; Otsuji, Eigo

2016-01-01

Background: Hypotonic shock induces cytocidal effects through cell rupture, and cancer therapy based on this mechanism has been clinically administered to hepatocellular carcinoma patients. We herein investigated the effectiveness of hypotonic shock combined with the inhibition of regulatory volume decrease as cancer therapy for hepatocellular carcinoma. Methods: Morphological changes in human hepatocellular carcinoma cell lines were observed under a differential interference contrast microscope connected to a high-speed digital video camera. Cell volume changes under hypotonic shock with or without chloride, potassium, or water channel blockers were observed using a high-resolution flow cytometer. In order to investigate cytocidal effects, the number of surviving cells was compared after exposure to hypotonic solution with and without each channel blocker (re-incubation experiment). Results: Video recordings showed that cells exposed to distilled water rapidly swelled and then ruptured. Cell volume measurements revealed regulatory volume decrease under mild hypotonic shock, whereas severe hypotonic shock increased the number of broken fragments as a result of cell rupture. Moreover, regulatory volume decrease was inhibited in cells treated with each channel blocker. Re-incubation experiments showed the cytocidal effects of hypotonic shock in cells exposed to hypotonic solution, and additional treatments with each channel blocker enhanced these effects. Conclusion: The inhibition of regulatory volume decrease with chloride, potassium, or water channel blockers may enhance the cytocidal effects of hypotonic shock in hepatocellular carcinoma. Hypotonic shock combined with the inhibition of regulatory volume decrease was a more effective therapy than hypotonic shock alone. PMID:27471568

Cyclin-dependent kinase inhibitor flavopiridol promotes remyelination in a cuprizone induced demyelination model

PubMed Central

Mi, Guiyun; Gao, Yunyun; Liu, Shuai; Ye, Enmao; Li, Yanyan; Jin, Xiao; Yang, Hongju; Yang, Zheng

2016-01-01

ABSTRACT The cuprizone (CPZ) model has been widely used for the studies of de-and remyelination. The CPZ-exposed mice show oligodendrocyte precursor cells (OPCs) increase and mature oligodendrocytes decrease, suggesting an imbalance between proliferation and differentiation of OPCs. In the first experiment of this study, we examined the expression of cell cycle related genes in brains of mice following CPZ administration for 5 weeks by means of microarray assay. In addition, we performed a double labeling of BrdU and Ki-67 to calculate cell cycle exit index in the mice. Our results showed that CPZ administration up-regulated the expression of 16 cell cycle related genes, but down-regulated the expression of only one in the prefrontal cortex (PFC) of mice compared to control group. The treatment inhibited potential precursor cells exit from cell cycle. In the second experiment, we evaluated effects of a CDK inhibitor flavopiridol (FLA) on CPZ-induced neuropathological changes and spatial working memory impairment in mice.FLA treatment for one week effectively attenuated the CPZ-induced increases in NG2 positive cells, microglia and astrocytes, alleviated the concurrent mature oligodendrocyte loss and myelin breakdown, and improved spatial working memory deficit in the CPZ-exposed mice. These results suggest that CPZ-induced neuropathological changes involve in dysregulation of cell cycle related genes. The therapeutic effects of FLA on CPZ-exposed mice may be related to its ability of cell cycle inhibition. PMID:27580304

Experimental study on rat NK cell activity improvement by laser acupoint irradiation

NASA Astrophysics Data System (ADS)

Yang, Dongxiao; Chen, Xiufeng; Ruan, Buqing; Yang, Feng

1998-08-01

To study the improvement of the natural killer (NK) cell activity by semiconductor laser acupoint irradiation, rats were used in this experiment and were injected immunosuppressant in their abdomen. The immunoassay was made after the surface irradiation and inner irradiation at Baihui point by semiconductor laser. The NK cell activity is an important index of immunologic function. The results showed that the NK cell activity after laser acupoint irradiation was enhanced. This enhancement is relatively important in the clinical therapy of tumor.

Moissanite anvil cell design for giga-pascal nuclear magnetic resonance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meier, Thomas; Herzig, Tobias; Haase, Jürgen

2014-04-15

A new design of a non-magnetic high-pressure anvil cell for nuclear magnetic resonance (NMR) experiments at Giga-Pascal pressures is presented, which uses a micro-coil inside the pressurized region for high-sensitivity NMR. The comparably small cell has a length of 22 mm and a diameter of 18 mm, so it can be used with most NMR magnets. The performance of the cell is demonstrated with external-force vs. internal-pressure experiments, and the cell is shown to perform well at pressures up to 23.5 GPa using 800 μm 6H-SiC large cone Boehler-type anvils. {sup 1}H, {sup 23}Na, {sup 27}Al, {sup 69}Ga, and {supmore » 71}Ga NMR test measurements are presented, which show a resolution of better than 4.5 ppm, and an almost maximum possible signal-to-noise ratio.« less

Estrogens and human papilloma virus oncogenes regulate human ether-à-go-go-1 potassium channel expression.

PubMed

Díaz, Lorenza; Ceja-Ochoa, Irais; Restrepo-Angulo, Iván; Larrea, Fernando; Avila-Chávez, Euclides; García-Becerra, Rocío; Borja-Cacho, Elizabeth; Barrera, David; Ahumada, Elías; Gariglio, Patricio; Alvarez-Rios, Elizabeth; Ocadiz-Delgado, Rodolfo; Garcia-Villa, Enrique; Hernández-Gallegos, Elizabeth; Camacho-Arroyo, Ignacio; Morales, Angélica; Ordaz-Rosado, David; García-Latorre, Ethel; Escamilla, Juan; Sánchez-Peña, Luz Carmen; Saqui-Salces, Milena; Gamboa-Dominguez, Armando; Vera, Eunice; Uribe-Ramírez, Marisela; Murbartián, Janet; Ortiz, Cindy Sharon; Rivera-Guevara, Claudia; De Vizcaya-Ruiz, Andrea; Camacho, Javier

2009-04-15

Ether-à-go-go-1 (Eag1) potassium channels are potential tools for detection and therapy of numerous cancers. Here, we show human Eag1 (hEag1) regulation by cancer-associated factors. We studied hEag1 gene expression and its regulation by estradiol, antiestrogens, and human papillomavirus (HPV) oncogenes (E6/E7). Primary cultures from normal placentas and cervical cancer tissues; tumor cell lines from cervix, choriocarcinoma, keratinocytes, and lung; and normal cell lines from vascular endothelium, keratinocytes, and lung were used. Reverse transcription-PCR (RT-PCR) experiments and Southern blot analysis showed Eag1 expression in all of the cancer cell types, normal trophoblasts, and vascular endothelium, in contrast to normal keratinocytes and lung cells. Estradiol and antiestrogens regulated Eag1 in a cell type-dependent manner. Real-time RT-PCR experiments in HeLa cells showed that Eag1 estrogenic regulation was strongly associated with the expression of estrogen receptor-alpha. Eag1 protein was detected by monoclonal antibodies in normal placenta and placental blood vessels. Patch-clamp recordings in normal trophoblasts treated with estradiol exhibited potassium currents resembling Eag1 channel activity. Eag1 gene expression in keratinocytes depended either on cellular immortalization or the presence of HPV oncogenes. Eag1 protein was found in keratinocytes transfected with E6/E7 HPV oncogenes. Cell proliferation of E6/E7 keratinocytes was decreased by Eag1 antibodies inhibiting channel activity and by the nonspecific Eag1 inhibitors imipramine and astemizole; the latter also increased apoptosis. Our results propose novel oncogenic mechanisms of estrogen/antiestrogen use and HPV infection. We also suggest Eag1 as an early indicator of cell proliferation leading to malignancies and a therapeutic target at early stages of cellular hyperproliferation.

Proteolytic processing of endogenous and recombinant beta 4 integrin subunit

PubMed Central

1992-01-01

The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin. PMID:1500432

Microbial Mineral Weathering for Nutrient Acquisition Releases Arsenic

NASA Astrophysics Data System (ADS)

Mailloux, B. J.; Alexandrova, E.; Keimowitz, A.; Wovkulich, K.; Freyer, G.; Stolz, J.; Kenna, T.; Pichler, T.; Polizzotto, M.; Dong, H.; Radloff, K. A.; van Geen, A.

2008-12-01

Tens of millions of people in Southeast Asia drink groundwater contaminated with naturally occurring arsenic. The process of arsenic release from the sediment to the groundwater remains poorly understood. Experiments were performed to determine if microbial mineral weathering for nutrient acquisition can serve as a potential mechanism for arsenic mobilization. We performed microcosm experiments with Burkholderia fungorum, phosphate free artificial groundwater, and natural apatite. Controls included incubations with no cells and with killed cells. Additionally, samples were treated with two spikes - an arsenic spike, to show that arsenic release is independent of the initial arsenic concentration, and a phosphate spike to determine whether release occurs at field relevant phosphate conditions. We show in laboratory experiments that phosphate-limited cells of Burkholderia fungorum mobilize ancillary arsenic from apatite as a by-product of mineral weathering for nutrient acquisition. The released arsenic does not undergo a redox transformation but appears to be solubilized from the apatite mineral lattice as arsenate during weathering. Apatite has been shown to be commonly present in sediment samples from Bangladesh aquifers. Analysis of apatite purified from the Ganges, Brahamputra, Meghna drainage basin shows 210 mg/kg of arsenic, which is higher than the average crustal level. Finally, we demonstrate the presence of the microbial phenotype that releases arsenic from apatite in Bangladesh sediments. These results suggest that microbial weathering for nutrient acquisition could be an important mechanism for arsenic mobilization.

Effect of minerals on accumulation of Cs by fungus Saccaromyces cerevisiae.

PubMed

Ohnuki, Toshihiko; Sakamoto, Fuminori; Yamasaki, Shinya; Kozai, Naofumi; Shiotsu, Hiroyuki; Utsunomiya, Satoshi; Watanabe, Naoko; Kozaki, Tamotsu

2015-06-01

The accumulation of Cs by unicellular fungus of Saccharomyces cerevisiae in the presence of minerals has been studied to elucidate the role of microorganisms in the migration of radioactive Cs in the environment. Two different types of experiments were employed: experiments using stable Cs to examine the effect of a carbon source on the accumulation of Cs, and accumulation experiments of radioactive Cs from agar medium containing (137)Cs and zeolite, vermiculite, phlogopite, smectite, mica, or illite as mineral supplements. In the former type of experiments, the Cs-accumulated cells were analyzed by scanning electron microscopy equipped with energy dispersive X-ray analysis (SEM-EDS). In the latter type, the radioactivity in the yeast cells was measured by an autoradiography technique. When a carbon source was present, higher amounts of Cs accumulated in the cells than in the resting condition without a carbon source. Analyses with SEM-EDS showed that no mineral formed on the cell surface. These results indicate that the yeast cells accumulate Cs by adsorption on the cell surface and intracellular accumulation. In the presence of minerals in the agar medium, the radioactivity in the yeast cells was in the order of mica > smectite, illite > vermiculite, phlogopite, zeolite. This order is inversely correlated to the ratio of the concentration of radioactive Cs between the minerals and the medium solution. These results strongly suggest that the yeast accumulates radioactive Cs competitively with minerals. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ovarian Tumor Cells Studied Aboard the International Space Station (ISS)

NASA Technical Reports Server (NTRS)

2001-01-01

In August 2001, principal investigator Jeanne Becker sent human ovarian tumor cells to the International Space Station (ISS) aboard the STS-105 mission. The tumor cells were cultured in microgravity for a 14 day growth period and were analyzed for changes in the rate of cell growth and synthesis of associated proteins. In addition, they were evaluated for the expression of several proteins that are the products of oncogenes, which cause the transformation of normal cells into cancer cells. This photo, which was taken by astronaut Frank Culbertson who conducted the experiment for Dr. Becker, shows two cell culture bags containing LN1 ovarian carcinoma cell cultures.

E-cigarette vapour is not inert and exposure can lead to cell damage.

PubMed

Holliday, Richard; Kist, Ralf; Bauld, Linda

2016-03-01

In vitro experiments were performed on normal epithelial cells as well as head and neck squamous cell carcinoma (HNSCC) cell lines. The widely available cell line HaCat, a spontaneously transformed immortal keratinocyte and the HNSCC cell lines HN30 and UMSCC10B were used. Cells were exposed to nicotine-containing and nicotine-free vapour extract from two popular e-cigarette brands for periods ranging from 48 hours to eight weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapour nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. In conclusion, our study strongly suggests that electronic cigarettes are not as safe as their marketing makes them appear to the public. Our in vitro experiments employing two brands of e-cigs show that at biologically relevant doses, vapourised e-cig liquids induce increased DNA strand breaks and cell death, and decreased clono- genic survival in both normal epithelial and HNSCC cell lines independently of nicotine content. Further research is needed to definitively determine the long-term effects of e-cig usage, as well as whether the DNA damage shown in our study as a result of e-cig exposure will lead to mutations that ultimately result in cancer.

Regulation of monocyte cell fate by blood vessels mediated by Notch signalling.

PubMed

Gamrekelashvili, Jaba; Giagnorio, Roberto; Jussofie, Jasmin; Soehnlein, Oliver; Duchene, Johan; Briseño, Carlos G; Ramasamy, Saravana K; Krishnasamy, Kashyap; Limbourg, Anne; Kapanadze, Tamar; Ishifune, Chieko; Hinkel, Rabea; Radtke, Freddy; Strobl, Lothar J; Zimber-Strobl, Ursula; Napp, L Christian; Bauersachs, Johann; Haller, Hermann; Yasutomo, Koji; Kupatt, Christian; Murphy, Kenneth M; Adams, Ralf H; Weber, Christian; Limbourg, Florian P

2016-08-31

A population of monocytes, known as Ly6C(lo) monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6C(hi) monocytes into Ly6C(lo) monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation.

Regulation of monocyte cell fate by blood vessels mediated by Notch signalling

PubMed Central

Gamrekelashvili, Jaba; Giagnorio, Roberto; Jussofie, Jasmin; Soehnlein, Oliver; Duchene, Johan; Briseño, Carlos G.; Ramasamy, Saravana K.; Krishnasamy, Kashyap; Limbourg, Anne; Häger, Christine; Kapanadze, Tamar; Ishifune, Chieko; Hinkel, Rabea; Radtke, Freddy; Strobl, Lothar J.; Zimber-Strobl, Ursula; Napp, L. Christian; Bauersachs, Johann; Haller, Hermann; Yasutomo, Koji; Kupatt, Christian; Murphy, Kenneth M.; Adams, Ralf H.; Weber, Christian; Limbourg, Florian P.

2016-01-01

A population of monocytes, known as Ly6Clo monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6Chi monocytes into Ly6Clo monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation. PMID:27576369

Attachment and spreadout study of 3T3 cells onto PP track etched films

NASA Astrophysics Data System (ADS)

Smolko, Eduardo; Mazzei, Ruben; Tadey, Daniel; Lombardo, Daniel

2001-12-01

Polymer surface modifications are obtained by the application of radiation treatments and other physico-chemical methods: fission fragment (ff) irradiation and etching. The biocompatibility of the surface is then observed by cell seeding and cell adhesion experiments. Approaches to improvement of the cell adhesion are obtained by different methods: for example, in PS, cell adhesion is improved after ion implantation; in PMMA, after bombarding the polymer, the surface is reconditioned with surfactants and proteins and in PVDF, cell adhesion is assayed on nuclear tracks membranes. In this work, we obtained important cell adhesion improvements in PP films by irradiation with swift heavy ions and subsequent etching of the nuclear tracks. We use BOPP (isotactic -25 μm thickness). Irrradiations were performed with a Cf-252 californium ff source. The source has a heavy ff and a light one, with 160-200 MeV energy divided among them corresponding to ff energies between 1 and 2 MeV/amu. A chemical etching procedure consisting of a solution of sulphuric acid and chromium three oxide at 85 °C was used. The 3T3 NIH fibroblast cell line was used for the cell adhesion experiment. Here we report for the first time, the results of a series of experiments by varying the ff fluence and the etching time showing that attachment and spreadout of cells are very much improved in this cell line according to the number of pores and the pore size.

Analysis of surface properties of fixed and live cells using derivatized agarose beads.

PubMed

Navarro, Vanessa M; Walker, Sherri L; Badali, Oliver; Abundis, Maria I; Ngo, Lylla L; Weerasinghe, Gayani; Barajas, Marcela; Zem, Gregory; Oppenheimer, Steven B

2002-01-01

A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes

NASA Astrophysics Data System (ADS)

Weinberger, Andreas; Walter, Vivien; MacEwan, Sarah R.; Schmatko, Tatiana; Muller, Pierre; Schroder, André P.; Chilkoti, Ashutosh; Marques, Carlos M.

2017-03-01

Although cationic cell-penetrating peptides (CPPs) are able to bind to cell membranes, thus promoting cell internalization by active pathways, attachment of cargo molecules to CPPs invariably reduces their cellular uptake. We show here that CPP binding to lipid bilayers, a simple model of the cell membrane, can be recovered by designing cargo molecules that self-assemble into spherical micelles and increase the local interfacial density of CPP on the surface of the cargo. Experiments performed on model giant unilamellar vesicles under a confocal laser scanning microscope show that a family of thermally responsive elastin-like polypeptides that exhibit temperature-triggered micellization can promote temperature triggered attachment of the micelles to membranes, thus rescuing by self-assembly the cargo-induced loss of the CPP affinity to bio-membranes.

Slimeware: engineering devices with slime mold.

PubMed

Adamatzky, Andrew

2013-01-01

The plasmodium of the acellular slime mold Physarum polycephalum is a gigantic single cell visible to the unaided eye. The cell shows a rich spectrum of behavioral patterns in response to environmental conditions. In a series of simple experiments we demonstrate how to make computing, sensing, and actuating devices from the slime mold. We show how to program living slime mold machines by configurations of repelling and attracting gradients and demonstrate the workability of the living machines on tasks of computational geometry, logic, and arithmetic.

Release and fate of fluorocarbons in a shredder residue landfill cell: 1. Laboratory experiments.

PubMed

Scheutz, Charlotte; Fredenslund, Anders M; Nedenskov, Jonas; Kjeldsen, Peter

2010-11-01

The shredder residues from automobiles, home appliances and other metal-containing products are often disposed in landfills, as recycling technologies for these materials are not common in many countries. Shredder waste contains rigid and soft foams from cushions and insulation panels blown with fluorocarbons. The objective of this study was to use laboratory experiments to estimate fluorocarbon release and attenuation processes in a monofill shredder residue (SR) landfill cell. Waste from the open SR landfill cell at the AV Miljø landfill in Denmark was sampled at three locations. The waste contained 1-3% metal and a relatively low fraction of rigid polyurethane (PUR) foam particles. The PUR waste contained less blowing agent (CFC-11) than predicted from a release model. However, CFC-11 was steadily released in an aerobic bench scale experiment. Anaerobic waste incubation bench tests showed that SRSR produced significant methane (CH(4)), but at rates that were in the low end of the range observed for municipal solid waste. Aerobic and anaerobic batch experiments showed that processes in SRSR potentially can attenuate the fluorocarbons released from the SRSR itself: CFC-11 is degraded under anaerobic conditions with the formation of degradation products, which are being degraded under CH(4) oxidation conditions prevailing in the upper layers of the SR. Copyright © 2010 Elsevier Ltd. All rights reserved.

Transient gene and microRNA expression profile changes of confluent human fibroblast cells in space

NASA Astrophysics Data System (ADS)

Wu, Honglu; Story, Michael; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Lu, Tao

2016-07-01

Microgravity, or an altered gravity environment from the Earth1g, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of these cells. Whether non-proliferating cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted onboard the International Space Station (ISS), confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days, respectively, for investigations of gene and miRNA expression profile changes in these cells. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67 positive cells. Gene and miRNA expression data indicated activation of NFkB and other growth related pathways involving HGF and Vegf along with down regulation of the Let-7 miRNA family. On Day 14 when the cells were mostly non-proliferating, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples with respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for αa-tubulin and fibronectin showed no difference between flown and ground samples. Taken together, our study suggests that in true non-dividing human fibroblast cells in culture, microgravity experienced in space has little effect on the gene and miRNA expression profiles.

Human umbilical cord blood stem cells show PDGF-D–dependent glioma cell tropism in vitro and in vivo

PubMed Central

Gondi, Christopher S.; Veeravalli, Krishna Kumar; Gorantla, Bharathi; Dinh, Dzung H.; Fassett, Dan; Klopfenstein, Jeffrey D.; Gujrati, Meena; Rao, Jasti S.

2010-01-01

Despite advances in clinical therapies and technologies, the prognosis for patients with malignant glioma is poor. Neural stem cells (NSCs) have a chemotactic tropism toward glioma cells. The use of NSCs as carriers of therapeutic agents for gliomas is currently being explored. Here, we demonstrate that cells isolated from the umbilical cord blood show mesenchymal characteristics and can differentiate to adipocytes, osteocytes, and neural cells and show tropism toward cancer cells. We also show that these stem cells derived from the human umbilical cord blood (hUCB) induce apoptosis-like cell death in the glioma cell line SNB19 via Fas-mediated caspase-8 activation. From our glioma tropism studies, we have observed that hUCB cells show tropism toward glioma cells in vitro, in vivo, and ex vivo. We determined that this migration is partially dependent on the expression levels of platelet-derived growth factor (PDGF)-D from glioma cells and have observed that local concentration gradient of PDGF-D is sufficient to cause migration of hUCB cells toward the gradient as seen from our brain slice cultures. In our animal experiment studies, we observed that intracranially implanted SNB19 green fluorescent protein cells induced tropism of the hUCB cells toward themselves. In addition, the ability of these hUCBs to inhibit established intracranial tumors was also observed. We also determined that the migration of stem cells toward glioma cells was partially dependent on PDGF secreted by glioma cells and that the presence of PDGF-receptor (PDGFR) on hUCB is required for migration. Our results demonstrate that hUCB are capable of inducing apoptosis in human glioma cells and also show that glioma tropism and hUCB tropism toward glioma cells are partially dependent on the PDGF/PGGFR system. PMID:20406896

Acanthamoeba castellanii is not be an adequate model to study human adenovirus interactions with macrophagic cells

PubMed Central

Cateau, Estelle; Leveque, Nicolas; Kaaki, Sihem; Beby-Defaux, Agnès; Rodier, Marie-Hélène

2017-01-01

Free living amoebae (FLA) including Acanthamoeba castellanii, are protozoa that feed on different microorganisms including viruses. These microorganisms show remarkable similarities with macrophages in cellular structures, physiology or ability to phagocyte preys, and some authors have therefore wondered whether Acanthamoeba and macrophages are evolutionary related. It has been considered that this amoeba may be an in vitro model to investigate relationships between pathogens and macrophagic cells. So, we intended in this study to compare the interactions between a human adenovirus strain and A. castellanii or THP-1 macrophagic cells. The results of molecular and microscopy techniques following co-cultures experiments have shown that the presence of the adenovirus decreased the viability of macrophages, while it has no effect on amoebic viability. On another hand, the viral replication occurred only in macrophages. These results showed that this amoebal model is not relevant to explore the relationships between adenoviruses and macrophages in in vitro experiments. PMID:28591183

Perspectives of boron-neutron capture therapy of malignant brain tumors

NASA Astrophysics Data System (ADS)

Kanygin, V. V.; Kichigin, A. I.; Krivoshapkin, A. L.; Taskaev, S. Yu.

2017-09-01

Boron neutron capture therapy (BNCT) is characterized by a selective effect directly on the cells of malignant tumors. The carried out research showed the perspective of the given kind of therapy concerning malignant tumors of the brain. However, the introduction of BNCT into clinical practice is hampered by the lack of a single protocol for the treatment of patients and the difficulty in using nuclear reactors to produce a neutron beam. This problem can be solved by using a compact accelerator as a source of neutrons, with the possibility of installation in a medical institution. Such a neutron accelerator for BNCT was developed at Budker Institute of Nuclear Physics, Novosibirsk. A neutron beam was obtained on this accelerator, which fully complies with the requirements of BNCT, as confirmed by studies on cell cultures and experiments with laboratory animals. The conducted experiments showed the relative safety of the method with the absence of negative effects on cell cultures and living organisms, and also confirmed the effectiveness of BNCT for malignant brain tumors.

Enhancing osseointegration of orthopedic implants with titania nanotube surfaces

NASA Astrophysics Data System (ADS)

Baker, Erin A.

Introduction: As joint arthroplasty surgical procedures increase annually, the development of new strategies, including novel materials and surface modifications, to attain solid bone-implant fixation are needed to increase implant terms of service. In this study, we evaluate two morphologies of titania nanotubes in both in vitro and in vivo experiments to quantify osseointegrative potential and material-level biocompatibility. Materials and Methods: Samples were prepared via an electrochemical etching process. Two different titania nanotube (TiNT) morphologies were produced, Aligned and Trabecular. For the in vitro experiment, Sprague Dawley (SD) rat marrow-derived bone marrow cells (BMC) were seeded on samples. Alkaline phosphatase (ALP) activity, osteocalcin (OC) expression, expression of relevant genes as well as cell attachment and morphology were assessed. In the first in vivo experiment, Kirschner wires were implanted unilaterally into SD rat femora with a TiNT-etched or unmodified (Control) implant. General health assessments and weekly body weights were recorded. At a 12-week endpoint, hematologic, systemic metal ion, and histologic analyses were performed. For the second in vivo experiment, Kirschner wires were implanted bilaterally into SD rat femora, with a TiNT-etched implant in one femora and unmodified (Control) implant as an internal control. At 4- and 12-week endpoints, femora were assessed via biomechanics, undecalcified histology, micro-computed tomography (muCT), and backscattered electron imaging (BEI) to characterize de novo bone formation. Results: In vitro experiments demonstrated BMC attachment and differentiation into osteoblasts as well as greater ALP activity, OC expression, total cell counts, and gene expression (of Col1a1, IGF-1, and osteonectin) on TiNT surfaces versus Controls. Cells on TiNT-etched substrates were smaller in diameter and more eccentric than Controls. In the first in vivo experiment, there were significant differences in body weight between groups at Weeks 9 and 11. There were no significant differences in red or white blood cell function between TiNT groups and Control. Aluminum levels in the lungs were significantly greater in the Trabecular TiNT group compared to Control. Histologic analysis showed significantly fewer granulocytes and neutrophils in the distal region of Trabecular TiNT-implanted femora as well as significantly fewer foreign body giant/multinucleated cells and neutrophils in the midshaft region of Aligned TiNT-implanted femora versus Controls. In the second in vivo experiment, at 12 weeks, microCT analysis showed TiNT implants generated greater bone formation than Controls. Histologic analysis demonstrated 1.5 times greater bone-implant contact in TiNT groups than Controls at 12 weeks. TiNT groups exhibited 1.3 to 3.7 times greater strength of fixation than Controls during pull-out testing. Discussion and Conclusions: In vitro data confirmed BMC attachment and differentiation into osteoblasts as well as osteoblastic phenotypic behavior. A clinically-relevant in vivo model of femoral intramedullary fixation, showed increased bone formation and quality in femora implanted with TiNT-etched implants versus Controls. A second in vivo study showed that TiNT surfaces do not generate systemic effects and may beneficially modulate the periprosthetic inflammatory environment.

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments.

PubMed

Van Valen, David A; Kudo, Takamasa; Lane, Keara M; Macklin, Derek N; Quach, Nicolas T; DeFelice, Mialy M; Maayan, Inbal; Tanouchi, Yu; Ashley, Euan A; Covert, Markus W

2016-11-01

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments

DOE PAGES

Van Valen, David A.; Kudo, Takamasa; Lane, Keara M.; ...

2016-11-04

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domainsmore » of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.« less

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Valen, David A.; Kudo, Takamasa; Lane, Keara M.

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domainsmore » of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.« less

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments

PubMed Central

Van Valen, David A.; Lane, Keara M.; Quach, Nicolas T.; Maayan, Inbal

2016-01-01

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems. PMID:27814364

The dual effects of polar methanolic extract of Hypericum perforatum L. in bladder cancer cells

NASA Astrophysics Data System (ADS)

Nseyo, U. O.; Nseyo, O. U.; Shiverick, K. T.; Medrano, T.; Mejia, M.; Stavropoulos, N.; Tsimaris, I.; Skalkos, D.

2007-02-01

Introduction and background: We have reported on the polar methanolic fraction (PMF) of Hypericum Perforatum L as a novel photosensitizing agent for photodynamic therapy (PDT) and photodynamic diagnosis (PDD). PMF has been tested in human leukemic cells, HL-60 cells, cord blood hemopoietic progenitor cells, bladder cancers derived from metastatic lymph node (T-24) and primary papillary bladder lesion (RT-4). However, the mechanisms of the effects of PMF on these human cell lines have not been elucidated. We have investigated mechanisms of PMF + light versus PMF-alone (dark experiment) in T-24 human bladder cancer cells. Methods: PMF was prepared from an aerial herb of HPL which was brewed in methanol and extracted with ether and methanol. Stock solutions of PMF were made in DSMO and stored in dark conditions. PMF contains 0.57% hypericin and 2.52% hyperforin. The T24 cell line was obtained from American Type Culture Collection (ATCC). In PDT treatment, PMF (60μg/ml) was incubated with cells, which were excited with laser light (630nm) 24 hours later. Apoptosis was determined by DNA fragmentation/laddering assay. DNA isolation was performed according to the manufacture's instructions with the Kit (Oncogene Kit#AM41). Isolated DNA samples were separated by electrophoresis in 1.5% in agarose gels and bands were visualized by ethidium bromide labeling. The initial cell cycle analysis and phase distribution was by flow cytometry. DNA synthesis was measured by [3H] thymidine incorporation, and cell cycle regulatory proteins were assayed by Western immunoblot. Results: The results of the flow cytometry showed PMF +light induced significant (40%) apoptosis in T24 cells, whereas Light or PMF alone produced little apoptosis. The percentage of cells in G 0/G I phase was decreased by 25% and in G2/M phase by 38%. The main impact was observed on the S phase which was blocked by 78% from the specific photocytotoxic process. DNA laddering analysis showed that PMF (60μg/ml) + light at 630nm induced DNA fragmentation in a light dose-dependent manner; in contrast, PMF or light alone did not induce DNA fragmentation. In separate experiments, PMF alone treatment produced a dose-dependent DNA synthesis with a 90% inhibition at a concentration of 25μg/ml (IC90 = 25μg/ml). Expression of p53 and p27 cell cycle regulatory proteins was not altered by PMF alone, however, a dose-dependent increase in p21 expression was observed that correlates with PMF concentrations. Cyclin A and cyclin B protein levels showed a clear decrease inverse to the concentration of PMF. In the absence of light treatment, flow cytometry analysis showed that PMF alone results in G 0/G I cell cycle arrest, with a 2-fold increase in G 0/G I cells concomitant with 50% decrease in cells in both S and G II/M phases. However, flow cytometry on PMF alone-treated cells did not show sub G 0/G I peak, further evidence of the lack of apoptosis as a mechanism of effect of PMF in the dark. Conclusions: With respect to light treatment, apoptosis appears to play a vital role in PDT-induced cytotoxicity. The flow cytometry and DNA laddering results revealed that T24 cells demonstrated apoptotic responses in PMF-mediated PDT. Experiments conducted with PMF alone showed a dose-dependent inhibition of DNA synthesis associated with G 0/G I cell cycle arrest and the extract is able to coordinate changes in key cell cycle regulatory proteins in human bladder cancer cells. Both experimental conditions suggest PMF as a potent and effect anti-proliferative agent in cancer chemoprevention and therapy of human urothelial carcinoma cells.

Experiments on solar photovoltaic power generation using concentrator and liquid cooling

NASA Technical Reports Server (NTRS)

Beam, B. H.; Hansen, C. F.

1975-01-01

Calculations and experimental data are presented leading to the development of a practical, economical solar photovoltaic power supply. The concept involves concentration of sunlight up to about 100 times normal solar intensity in a solar tracking collector and directing this to an array of solar cells. The cells are immersed in water circulated from a thermal reservoir which limits cell temperature rise to about 20 C above ambient during the day and which cools to ambient temperature during the night. Experiments were conducted on solar cells using a Fresnel lens for magnification, a telescope equatorial mount with clock drive, and tap water circulated through the solar cell holder cavity. Test results show that cells operate satisfactorily under these conditions. Power outputs achieved experimentally with cell optimized for 25 suns were linear with concentration to about 15 suns. Cells optimized for 100 suns were not available, but a corresponding linear relation of power output with concentration is anticipated. Test results have been used in a design analysis of the cost of systems utilizing this technique.

Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates.

PubMed Central

Forus, A; Høifødt, H K; Overli, G E; Myklebost, O; Fodstad, O

1999-01-01

AIM: The presence of malignant cells in the blood and bone marrow of patients with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells might be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and characterisation of malignant cells present in bone marrow. METHODS: In spiking experiments, SKBR3 cells were mixed with mononuclear cells in known proportions to mimic bone marrow samples with micrometastatic cells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the MOC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and int2 copy numbers were also determined in 15 primary breast cancers, and bone marrow samples from patients with amplification were analysed for micrometastatic cells by immunomagnetic enrichment and FISH. RESULTS: In model experiments, cells with amplification could be detected in bead selected fractions when ratios of tumour cells (SKBR3) to mononuclear cells were as low as 10:10(7). Among the tumour samples, eight showed increased copy numbers of erbB2 and/or int2, and three of these patients had detectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tumour cells/10(7) mononuclear cells, respectively. The patient with 540 tumour cells/10(7) mononuclear cells showed high level amplification of erbB2 and suffered from a particularly aggressive disease, whereas the patient with 4000 tumour cells/10(7) mononuclear cells had favourable disease progression. CONCLUSION: These results demonstrate the feasibility and advantage of combining immunomagnetic selection and FISH characterisation of cancer cells in bone marrow samples. It is possible that molecular characterisation of such cells could provide prognostically valuable information. PMID:10474684

An Integrin-Targeting RGDK-Tagged Nanocarrier: Anticancer Efficacy of Loaded Curcumin.

PubMed

Das, Krishnendu; Nimushakavi, Sahithi; Chaudhuri, Arabinda; Das, Prasanta Kumar

2017-05-22

Herein we report the design and development of α 5 β 1 integrin-specific noncovalent RGDK-lipopeptide-functionalized single-walled carbon nanotubes (SWNTs) that selectively deliver the anticancer drug curcumin to tumor cells. RGDK tetrapeptide-tagged amphiphiles were synthesized that efficiently disperse SWNTs with a suspension stability index of >80 % in cell culture media. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)- and lactate dehydrogenase (LDH)-based cell viability assays in tumor (B16F10 melanoma) and noncancerous (NIH3T3 mouse fibroblast) cells revealed the non-cytotoxic nature of these RGDK-lipopeptide-SWNT conjugates. Cellular uptake experiments with monoclonal antibodies against α v β 3 , α v β 5 , and α 5 β 1 integrins showed that these SWNT nanovectors deliver their cargo (Cy3-labeled oligonucleotides, Cy3-oligo) to B16F10 cells selectively via α 5 β 1 integrin. Notably, the nanovectors failed to deliver the Cy3-oligo to NIH3T3 cells. The RGDK-SWNT is capable of delivering the anticancer drug curcumin to B16F10 cells more efficiently than NIH3T3 cells, leading to selective killing of B16F10 cells. Results of Annexin V binding based flow cytometry experiments are consistent with selective killing of tumor cells through the late apoptotic pathway. Biodistribution studies in melanoma (B16F10)-bearing C57BL/6J mice showed tumor-selective accumulation of curcumin intravenously administered via RGDK-lipopeptide-SWNT nanovectors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Aptamer-facilitated mass cytometry.

PubMed

Mironov, Gleb G; Bouzekri, Alexandre; Watson, Jessica; Loboda, Olga; Ornatsky, Olga; Berezovski, Maxim V

2018-05-01

Mass cytometry is a novel cell-by-cell analysis technique, which uses elemental tags instead of fluorophores. Sample cells undergo rapid ionization in inductively coupled plasma and the ionized elemental tags are then analyzed by means of time-of-flight mass spectrometry. Benefits of the mass cytometry approach are in no need for compensation, the high number of detection channels (up to 100) and low background noise. In this work, we applied a biotinylated aptamer against human PTK7 receptor for characterization of positive (human acute lymphoblastic leukemia) and negative (human Burkitt's lymphoma) cells by a mass cytometry instrument. Our proof of principal experiments showed that biotinylated aptamers in conjunction with metal-labeled neutravidin can be successfully utilized for mass cytometry experiments at par with commercially available antibodies. Graphical abstract Biotinylated aptamers in conjunction with metal-labeled neutravidin bind to cell biomarkers, and then injected into the inductively coupled plasma (ICP) source, where cells are vaporized, atomized, and ionized in the plasma for subsequent mass spectrometry (MS) analysis of lanthanide metals.

Antiproliferative and proapoptotic effects of bisphenol A on human trophoblastic JEG-3 cells.

PubMed

Morice, Lucie; Benaîtreau, Delphine; Dieudonné, Marie-Noëlle; Morvan, Corinne; Serazin, Valérie; de Mazancourt, Philippe; Pecquery, René; Dos Santos, Esther

2011-07-01

Different studies performed in rodents revealed that bisphenol-A (BPA), an environmental compound, altered early embryonic development. However, little is known concerning the direct effects of BPA on human implantation process. Thus, we decided to study in vitro BPA's effects on proliferative capacities of the human trophoblastic cell line, JEG-3. For this purpose, we first have shown that JEG-3 cells express the specific BPA receptor, namely estrogen-related receptor γ1 (ERRγ1). Secondly, we demonstrated that BPA did not exert any cytotoxic action in JEG-3 cells up to 10(-6)M. Moreover [(3)H]-thymidine incorporation experiments revealed that BPA significantly reduced cell proliferation. The results also showed that BPA induced JEG-3 apoptosis capacity as reflected by DNA fragmentation experiments. In conclusion, we describe here the direct impact of BPA on trophoblastic cell number mediated through both anti-proliferative and pro-apoptotic effects. Copyright © 2011 Elsevier Inc. All rights reserved.

Elastic light scattering from single cells: orientational dynamics in optical trap.

PubMed

Watson, Dakota; Hagen, Norbert; Diver, Jonathan; Marchand, Philippe; Chachisvilis, Mirianas

2004-08-01

Light-scattering diagrams (phase functions) from single living cells and beads suspended in an optical trap were recorded with 30-ms time resolution. The intensity of the scattered light was recorded over an angular range of 0.5-179.5 degrees using an optical setup based on an elliptical mirror and rotating aperture. Experiments revealed that light-scattering diagrams from biological cells exhibit significant and complex time dependence. We have attributed this dependence to the cell's orientational dynamics within the trap. We have also used experimentally measured phase function information to calculate the time dependence of the optical radiation pressure force on the trapped particle and show how it changes depending on the orientation of the particle. Relevance of these experiments to potential improvement in the sensitivity of label-free flow cytometry is discussed.

Gallium Arsenide solar cell radiation damage experiment

NASA Technical Reports Server (NTRS)

Maurer, R. H.; Kinnison, J. D.; Herbert, G. A.; Meulenberg, A.

1991-01-01

Gallium arsenide (GaAs) solar cells for space applications from three different manufactures were irradiated with 10 MeV protons or 1 MeV electrons. The electrical performance of the cells was measured at several fluence levels and compared. Silicon cells were included for reference and comparison. All the GaAs cell types performed similarly throughout the testing and showed a 36 to 56 percent power areal density advantage over the silicon cells. Thinner (8-mil versus 12-mil) GaAs cells provide a significant weight reduction. The use of germanium (Ge) substrates to improve mechanical integrity can be implemented with little impact on end of life performance in a radiation environment.

Role of a cumulus parameterization scheme in simulating atmospheric circulation and rainfall in the nine-layer Goddard Laboratory for Atmospheres General Circulation Model

NASA Technical Reports Server (NTRS)

Sud, Y. C.; Chao, Winston C.; Walker, G. K.

1992-01-01

The influence of a cumulus convection scheme on the simulated atmospheric circulation and hydrologic cycle is investigated by means of a coarse version of the GCM. Two sets of integrations, each containing an ensemble of three summer simulations, were produced. The ensemble sets of control and experiment simulations are compared and differentially analyzed to determine the influence of a cumulus convection scheme on the simulated circulation and hydrologic cycle. The results show that cumulus parameterization has a very significant influence on the simulation circulation and precipitation. The upper-level condensation heating over the ITCZ is much smaller for the experiment simulations as compared to the control simulations; correspondingly, the Hadley and Walker cells for the control simulations are also weaker and are accompanied by a weaker Ferrel cell in the Southern Hemisphere. Overall, the difference fields show that experiment simulations (without cumulus convection) produce a cooler and less energetic atmosphere.

Global architecture of the F-actin cytoskeleton regulates cell shape-dependent endothelial mechanotransduction.

PubMed

Shao, Yue; Mann, Jennifer M; Chen, Weiqiang; Fu, Jianping

2014-03-01

Uniaxial stretch is an important biophysical regulator of cell morphology (or shape) and functions of vascular endothelial cells (ECs). However, it is unclear whether and how cell shape can independently regulate EC mechanotransductive properties under uniaxial stretch. Herein, utilizing a novel uniaxial cell-stretching device integrated with micropost force sensors, we reported the first experimental evidence showing cell shape-dependent EC mechanotransduction via cytoskeleton (CSK) contractile forces in response to uniaxial stretch. Combining experiments and theoretical modeling from first principles, we showed that it was the global architecture of the F-actin CSK that instructed the cell shape-dependent EC mechanotransductive process. Furthermore, a cell shape-dependent nature was relayed in EC mechanotransduction via dynamic focal adhesion (FA) assembly. Our results suggested a novel mechanotransductive process in ECs wherein the global architecture of the F-actin CSK, governed by cell shape, controls mechanotransduction via CSK contractile forces and force-dependent FA assembly under uniaxial stretch.

The dynamic behavior of chemically "stiffened" red blood cells in microchannel flows.

PubMed

Forsyth, Alison M; Wan, Jiandi; Ristenpart, William D; Stone, Howard A

2010-07-01

The rigidity of red blood cells (RBCs) plays an important role in whole blood viscosity and is correlated with several cardiovascular diseases. Two chemical agents that are commonly used to study cell deformation are diamide and glutaraldehyde. Despite diamide's common usage, there are discrepancies in the literature surrounding diamide's effect on the deformation of RBCs in shear and pressure-driven flows; in particular, shear flow experiments have shown that diamide stiffens cells, while pressure-driven flow in capillaries did not give this result. We performed pressure-driven flow experiments with RBCs in a microfluidic constriction and quantified the cell dynamics using high-speed imaging. Diamide, which affects RBCs by cross-linking spectrin skeletal membrane proteins, did not reduce deformation and showed an unchanged effective strain rate when compared to healthy cells. In contrast, glutaraldehyde, which is a non-specific fixative that acts on all components of the cell, did reduce deformation and showed increased instances of tumbling, both of which are characteristic features of stiffened, or rigidified, cells. Because glutaraldehyde increases the effective viscosity of the cytoplasm and lipid membrane while diamide does not, one possible explanation for our results is that viscous effects in the cytoplasm and/or lipid membrane are a dominant factor in dictating dynamic responses of RBCs in pressure-driven flows. Finally, literature on the use of diamide as a stiffening agent is summarized, and provides supporting evidence for our conclusions. Copyright 2010 Elsevier Inc. All rights reserved.

CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

PubMed

Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

2016-11-16

For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

Specific Accumulation of Tumor-Derived Adhesion Factor in Tumor Blood Vessels and in Capillary Tube-Like Structures of Cultured Vascular Endothelial Cells

NASA Astrophysics Data System (ADS)

Akaogi, Kotaro; Okabe, Yukie; Sato, Junji; Nagashima, Yoji; Yasumitsu, Hidetaro; Sugahara, Kazuyuki; Miyazaki, Kaoru

1996-08-01

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

Screening and Identification of Cryopreservative Agents for Human Cellular Biotechnology Experiments in Microgravity

NASA Technical Reports Server (NTRS)

Love,J.; Elliott, T.; Das, G. C.; Hammond, D. K.; Schwarzkopf, R. J.; Jones, L. B.; Baker, T. L.

2006-01-01

Dimethyl sulfoxide (DMSO) has been used as a standard cryopreservative agent for mammalian cell culture; however, prolonged exposure of thawed cells to DMSO can alter cell growth. While DMSO is easily eliminated in ground-based experiments, removal of DMSO in flight-based experiments is more difficult due to various on-orbit constraints. Failure of cryopreservation is due to a number of factors, including intracellular ice formation, solute effect, and apoptotic cell death following thawing. One objective of this study is to identify and characterize an alternative cryopreservative that could be used on the International Space Station (ISS). We systematically screened for potential permeating and non-permeating agents using a human colorectal carcinoma cell line, MIP-101. Cells were suspended in cryopreservation solution and frozen either following a two-step procedure involving initial cooling at -1 C/min overnight followed by storage in liquid nitrogen (LN2) vapor, or by freezing cells directly in the LN2 vapor phase at -10 C/min. Ability to preserve cellular function after one cycle of freeze-thawing was assessed by the recovery of viable cells in short and long-term cell culture experiments. Results showed that permeating preservatives glycerol (G) and ethylene glycol (EG) had an efficacy (80-110%) comparable to, if not better than, 7.5% DMSO; but, propylene glycol (PG) had a somewhat lesser efficacy. Among the non-permeating preservatives, trehalose, raffinose, and dextran exhibited significant protective effect (50-80%) relative to that offered by 7.5% DMSO, but at -10 C and not at -1 C/min cooling rate. Preliminary data thus suggest that a combination of permeating and non-permeating agents may have improved efficacy as a cryoprotectant and serve as an alternate to DMSO for experimentation on ISS.

Flow dynamics and salt transport in a coastal aquifer driven by a stratified saltwater body: Lab experiment and numerical modeling

NASA Astrophysics Data System (ADS)

Oz, Imri; Shalev, Eyal; Yechieli, Yoseph; Gavrieli, Ittai; Gvirtzman, Haim

2014-04-01

This paper examines the transient development and the steady-state configuration of groundwater within a coastal aquifer adjacent to a stratified saltwater body. Such systems consist of three different water types: the regional fresh groundwater, and low and high salinity brines forming the upper and lower water layers of the stratified water body, respectively. The dynamics, location and the geometry of the interfaces and the density-driven circulation flows that develop in the aquifer are examined using laboratory experiments and numerical modeling at the same scale. The results show that the transient intrusion of the different water bodies into the aquifer takes place at different rates, and that the locations of the interfaces between them change with time, before reaching steady-state. Under steady-state conditions both the model and the experiments show the existence of three interfaces between the three water types. The numerical model, which is calibrated against the salinity distribution and groundwater discharge rate in the laboratory experiments, allows the quantification of the flow rates and flow patterns within the aquifer. These flow patterns, which cannot be derived from laboratory experiments, show the transient development of three circulation cells which are confined between the three interfaces. These results confirm the hypothesis that has been previously suggested based solely on a steady-state numerical modeling defined by a conceptual understanding. Parametric analysis shows that the creation of three circulation cells and three interfaces is limited to certain conditions and defines the ranges for the creation of this unique system.

Turbulent unmixing: how marine turbulence drives patchy distributions of motile phytoplankton

NASA Astrophysics Data System (ADS)

Durham, William; Climent, Eric; Barry, Michael; de Lillo, Filippo; Boffetta, Guido; Cencini, Massimo; Stocker, Roman

2013-11-01

Centimeter-scale patchiness in the distribution of phytoplankton increases the efficacy of many important ecological interactions in the marine food web. We show that turbulent fluid motion, usually synonymous with mixing, instead triggers intense small-scale patchiness in the distribution of motile phytoplankton. We use a suite of experiments, direct numerical simulations of turbulence, and analytical tools to show that turbulent shear and acceleration directs the motility of cells towards well-defined regions of flow, increasing local cell concentrations more than ten fold. This motility-driven `unmixing' offers an explanation for why motile cells are often more patchily distributed than non-motile cells and provides a mechanistic framework to understand how turbulence, whose strength varies profoundly in marine environments, impacts ocean productivity.

The Influence of Neuronal Density and Maturation on Network Activity of Hippocampal Cell Cultures: A Methodological Study

PubMed Central

Menegon, Andrea; Ferrigno, Giancarlo; Pedrocchi, Alessandra

2013-01-01

It is known that cell density influences the maturation process of in vitro neuronal networks. Neuronal cultures plated with different cell densities differ in number of synapses per neuron and thus in single neuron synaptic transmission, which results in a density-dependent neuronal network activity. Although many authors provided detailed information about the effects of cell density on neuronal culture activity, a dedicated report of density and age influence on neuronal hippocampal culture activity has not yet been reported. Therefore, this work aims at providing reference data to researchers that set up an experimental study on hippocampal neuronal cultures, helping in planning and decoding the experiments. In this work, we analysed the effects of both neuronal density and culture age on functional attributes of maturing hippocampal cultures. We characterized the electrophysiological activity of neuronal cultures seeded at three different cell densities, recording their spontaneous electrical activity over maturation by means of MicroElectrode Arrays (MEAs). We had gather data from 86 independent hippocampal cultures to achieve solid statistic results, considering the high culture-to-culture variability. Network activity was evaluated in terms of simple spiking, burst and network burst features. We observed that electrical descriptors were characterized by a functional peak during maturation, followed by a stable phase (for sparse and medium density cultures) or by a decrease phase (for high dense neuronal cultures). Moreover, 900 cells/mm2 cultures showed characteristics suitable for long lasting experiments (e.g. chronic effect of drug treatments) while 1800 cells/mm2 cultures should be preferred for experiments that require intense electrical activity (e.g. to evaluate the effect of inhibitory molecules). Finally, cell cultures at 3600 cells/mm2 are more appropriate for experiments in which time saving is relevant (e.g. drug screenings). These results are intended to be a reference for the planning of in vitro neurophysiological and neuropharmacological experiments with MEAs. PMID:24386305

The influence of neuronal density and maturation on network activity of hippocampal cell cultures: a methodological study.

PubMed

Biffi, Emilia; Regalia, Giulia; Menegon, Andrea; Ferrigno, Giancarlo; Pedrocchi, Alessandra

2013-01-01

It is known that cell density influences the maturation process of in vitro neuronal networks. Neuronal cultures plated with different cell densities differ in number of synapses per neuron and thus in single neuron synaptic transmission, which results in a density-dependent neuronal network activity. Although many authors provided detailed information about the effects of cell density on neuronal culture activity, a dedicated report of density and age influence on neuronal hippocampal culture activity has not yet been reported. Therefore, this work aims at providing reference data to researchers that set up an experimental study on hippocampal neuronal cultures, helping in planning and decoding the experiments. In this work, we analysed the effects of both neuronal density and culture age on functional attributes of maturing hippocampal cultures. We characterized the electrophysiological activity of neuronal cultures seeded at three different cell densities, recording their spontaneous electrical activity over maturation by means of MicroElectrode Arrays (MEAs). We had gather data from 86 independent hippocampal cultures to achieve solid statistic results, considering the high culture-to-culture variability. Network activity was evaluated in terms of simple spiking, burst and network burst features. We observed that electrical descriptors were characterized by a functional peak during maturation, followed by a stable phase (for sparse and medium density cultures) or by a decrease phase (for high dense neuronal cultures). Moreover, 900 cells/mm(2) cultures showed characteristics suitable for long lasting experiments (e.g. chronic effect of drug treatments) while 1800 cells/mm(2) cultures should be preferred for experiments that require intense electrical activity (e.g. to evaluate the effect of inhibitory molecules). Finally, cell cultures at 3600 cells/mm(2) are more appropriate for experiments in which time saving is relevant (e.g. drug screenings). These results are intended to be a reference for the planning of in vitro neurophysiological and neuropharmacological experiments with MEAs.

RNA editing is induced by type I interferon in esophageal squamous cell carcinoma.

PubMed

Zhang, Jinyao; Chen, Zhaoli; Tang, Zefang; Huang, Jianbing; Hu, Xueda; He, Jie

2017-07-01

In recent years, abnormal RNA editing has been shown to play an important role in the development of esophageal squamous cell carcinoma, as such abnormal editing is catalyzed by ADAR (adenosine deaminases acting on RNA). However, the regulatory mechanism of ADAR1 in esophageal squamous cell carcinomas remains largely unknown. In this study, we investigated ADAR1 expression and its association with RNA editing in esophageal squamous cell carcinomas. RNA sequencing applied to esophageal squamous cell carcinoma clinical samples showed that ADAR1 expression was correlated with the expression of STAT1, STAT2, and IRF9. In vitro experiments showed that the abundance of ADAR1 protein was associated with the induced activation of the JAK/STAT pathway by type I interferon. RNA sequencing results showed that treatment with type I interferon caused an increase in the number and degree of RNA editing in esophageal squamous cell carcinoma cell lines. In conclusion, the activation of the JAK/STAT pathway is a regulatory mechanism of ADAR1 expression and causes abnormal RNA editing profile in esophageal squamous cell carcinoma. This mechanism may serve as a new target for esophageal squamous cell carcinoma therapy.

Influence of temperature on the aging behavior of 18650-type lithium ion cells: A comprehensive approach combining electrochemical characterization and post-mortem analysis

NASA Astrophysics Data System (ADS)

Friesen, Alex; Mönnighoff, Xaver; Börner, Markus; Haetge, Jan; Schappacher, Falko M.; Winter, Martin

2017-02-01

The understanding of the aging behavior of lithium ion batteries in automotive and energy storage applications is essential for the acceptance of the technology. Therefore, aging experiments were conducted on commercial 18650-type state-of-the-art cells to determine the influence of the temperature during electrochemical cycling on the aging behavior of the different cell components. The cells, based on Li(Ni0.5Co0.2Mn0.3)O2 (NCM532)/graphite, were aged at 20 °C and 45 °C to different states of health. The electrochemical performance of the investigated cells shows remarkable differences depending on the cycling temperature. At contrast to the expected behavior, the cells cycled at 45 °C show a better electrochemical performance over lifetime than the cells cycled at 20 °C. Comprehensive post-mortem analyses revealed the main aging mechanisms, showing a complex interaction between electrodes and electrolyte. The main aging mechanisms of the cells cycled at 45 °C differ strongly at contrast to cells cycled at 20 °C. A strong correlation between the formed SEI, the electrolyte composition and the electrochemical performance over lifetime was observed.

[Study on garlic oil combined with 5-FU induced apoptosis of adenoid cystic carcinoma cell line ACC-M].

PubMed

Wu, Fayin; Zhou, Hefeng; Fan, Zhiying; Zhu, Yawen; Li, Yongye; Yao, Yukun; Ran, Dan

2014-02-01

To observe the effect of garlic oil combined with 5-FU induced apoptosis of adenoid cystic carcinoma cell line ACC-M. Human salivary in adenoid cystic carcinoma cell line AC-M was cultured, divided into the experimental group (5-FU group, garlic oil group, garlic oil + 5-FU group) and the control group, to observe the growth activity of tumor cells by MTT methods; to analyse the changes of cell cycle and apoptosis rate by flow cytometry. MTT experiments showed that 5-FU, garlic oil, garlic oil and 5-FU on ACC-M cells have inhibition in different concentration, with the increase of concentration and action time of the rise; Cell cycle analysis showed significant changes in flow cytometry. With the increase of concentration and the acting time, the G0/G1, phase of the cell ratio increased, S had no significant change, but G2/M phase cells decreased. Apoptosis rate display showed garlic oil combined with 5-FU induced apoptosis of ACC-M cells was significantly stronger than single group. Garlic oil can effectively induce the apoptosis of adenoid cystic carcinoma cell line ACC-M. The effect of garlic oil combined with 5-FU on ACC-M cells was stronger than the garlic oil, 5-FU used alone.

A CD13-targeting peptide integrated protein inhibits human liver cancer growth by killing cancer stem cells and suppressing angiogenesis.

PubMed

Zheng, Yan-Bo; Gong, Jian-Hua; Liu, Xiu-Jun; Li, Yi; Zhen, Yong-Su

2017-05-01

CD13 is a marker of angiogenic endothelial cells, and recently it is proved to be a biomarker of human liver cancer stem cells (CSCs). Herein, the therapeutic effects of NGR-LDP-AE, a fusion protein composed of CD13-targeting peptide NGR and antitumor antibiotic lidamycin, on human liver cancer and its mechanism were studied. Western blot and immunofluorescence assay demonstrated that CD13 (WM15 epitope) was expressed in both human liver cancer cell lines and vascular endothelial cells, while absent in normal liver cells. MTT assay showed that NGR-LDP-AE displayed potent cytotoxicity to cultured tumor cell lines with IC 50 values at low nanomolar level. NGR-LDP-AE inhibited tumorsphere formation of liver cancer cells, and the IC 50 values were much lower than that in MTT assay, indicating selectively killing of CSCs. In endothelial tube formation assay, NGR-LDP-AE at low cytotoxic dose significantly inhibited the formation of intact tube networks. Animal experiment demonstrated that NGR-LDP-AE inhibited the growth of human liver cancer xenograft. Immunohistochemical analysis showed that NGR-LDP-AE induced the down-regulation of CD13. In vitro experiment using cultured tumor cells also confirmed this result. NGR-LDP-AE activated both apoptotic and autophagic pathways in cultured tumor cells, while the induced autophagy protected cells from death. Conclusively, NGR-LDP-AE exerts its antitumor activity via killing liver CSCs and inhibiting angiogenesis. With one targeting motif, NGR-LDP-AE acts on both liver CSCs and angiogenic endothelial cells. It is a promising dual targeting fusion protein for liver cancer therapy, especially for advanced or relapsed cancers. © 2017 Wiley Periodicals, Inc.

Human CIK Cells Loaded with Au Nanorods as a Theranostic Platform for Targeted Photoacoustic Imaging and Enhanced Immunotherapy and Photothermal Therapy

NASA Astrophysics Data System (ADS)

Yang, Yao; Zhang, Jingjing; Xia, Fangfang; Zhang, Chunlei; Qian, Qirong; Zhi, Xiao; Yue, Caixia; Sun, Rongjin; Cheng, Shangli; Fang, Shan; Jin, Weilin; Yang, Yuming; Cui, Daxiang

2016-06-01

How to realize targeted photoacoustic imaging, enhanced immunotherapy, and photothermal therapy of gastric cancer has become a great challenge. Herein, we reported for the first time that human cytokine-induced killer cells (CIK) loaded with gold nanorods were used for targeted photoacoustic imaging, enhanced immunotherapy, and photothermal therapy of gastric cancer. Silica-modified gold nanorods were prepared; then incubated with human cytokine-induced killer cells (CIK), resultant human CIK cells loaded with Au nanorods were evaluated for their cytotoxicity, targeted ability of gastric cancer in vitro and in vivo, immunotherapy, and photothermal therapy efficacy. In vitro cell experiment shows that human CIK cells labeled with gold nanorods actively target gastric cancer MGC803 cells, inhibit growth of MGC803 cells by inducing cell apoptosis, and kill MGC803 cells under low power density near-infrared (NIR) laser treatment (808-nm continuous wave laser, 1.5 W/cm2, 3 min). In vivo experiment results showed that human CIK cells labeled with gold nanorods could target actively and image subcutaneous gastric cancer vessels via photoacoustic imaging at 4 h post-injection, could enhance immunotherapy efficacy by up-regulating cytokines such as IL-1, IL-12, IL-2, IL-4, IL-17, and IFN-γ, and kill gastric cancer tissues by photothermal therapy via direct injection into tumor site under near-infrared (NIR) laser irradiation. High-performance human CIK cells labeled with Au nanorods are a good novel theranostic platform to exhibit great potential in applications such as tumor-targeted photoacoustic imaging, enhanced immunotherapy, and photothermal therapy in the near future.

Bortezomib in Kidney Transplantation

PubMed Central

Raghavan, Rajeev; Jeroudi, Abdallah; Achkar, Katafan; Gaber, A. Osama; Patel, Samir J.; Abdellatif, Abdul

2010-01-01

Although current therapies for pretransplant desensitization and treatment of antibody-mediated rejection (AMR) have had some success, they do not specifically deplete plasma cells that produce antihuman leukocyte antigen (HLA) antibodies. Bortezomib, a proteasome inhibitor approved for the treatment of multiple myeloma (a plasma cell neoplasm), induces plasma cell apoptosis. In this paper we review the current body of literature regarding the use of this biological agent in the field of transplantation. Although limited experience with bortezomib may seem to show promise in the realm of transplant recipients desensitization and treatment of AMR, there is also experience that may suggest otherwise. Bortezomib's role in desensitization protocols and treatment of AMR will be defined better as more clinical data and trials become available. PMID:20953363

Cell micro-irradiation with MeV protons counted by an ultra-thin diamond membrane

NASA Astrophysics Data System (ADS)

Barberet, Philippe; Pomorski, Michal; Muggiolu, Giovanna; Torfeh, Eva; Claverie, Gérard; Huss, Cédric; Saada, Samuel; Devès, Guillaume; Simon, Marina; Seznec, Hervé

2017-12-01

We report the development of thin single crystal diamond membranes suitable for dose control in targeted cell irradiation experiments with a proton microbeam. A specific design was achieved to deliver single protons with a hit detection efficiency approaching 100%. The membranes have thicknesses between 1.8 and 3 μm and are used as vacuum windows on the microbeam line. The impact of these transmission detectors on the microbeam spot size is estimated by Monte-Carlo simulations, indicating that a beam lateral resolution below 2 μm is achieved. This is confirmed by experiments showing the accumulation online of X-ray Repair Cross-Complementing protein 1 (XRCC1)-Green Fluorescent Protein (GFP) at DNA damaged sites in living cells.

Rationale for two phase polymer system microgravity separation experiments

NASA Technical Reports Server (NTRS)

Brooks, D. E.; Bamberger, S. B.; Harris, J. M.; Vanalstine, J.

1984-01-01

The two-phase systems that result when aqueous solutions of dextran and poly(ethylene glycol) are mixed at concentrations above a few percent are discussed. They provide useful media for the partition and isolation of macromolecules and cell subpopulations. By manipulating their composition, separations based on a variety of molecular and surface properties are achieved, including membrane hydrophobic properties, cell surface charge, and membrane antigenicity. Work on the mechanism of cell partition shows there is a randomizing, nonthermal energy present which reduces separation resolution. This stochastic energy is probably associated with hydrodynamic interactions present during separation. Because such factors should be markedly reduced in microgravity, a series of shuttle experiments to indicate approaches to increasing the resolution of the procedure are planned.

In Situ Optical Observation of High-Temperature Geological Processes With the Moissanite Cell

NASA Astrophysics Data System (ADS)

Walte, N.; Keppler, H.

2005-12-01

A major drawback of existing techniques in experimental earth and material sciences is the inability to observe ongoing high-temperature processes in situ during an experiment. Examples for important time-dependent processes include the textural development of rocks and oxide systems during melting and crystallization, solid-state and melt-present recrystallization and Ostwald ripening, and bubble nucleation and growth during degassing of glasses and melts. The investigation of these processes by post-mortem analysis of a quenched microstructure is time consuming and often unsatisfactory. Here, we introduce the moissanite cell that allows optical in situ observation of long-term experiments at high temperatures. Moissanite is a transparent gem-quality type of SiC that is characterized by its hardness and superior chemical and thermal resistance. Two moissanite windows with a thickness and diameter of several millimeters are placed into sockets of fired pyrophyllite and fixed onto two opposite metal plates. The sockets are wrapped with heating wire and each window is connected to a thermocouple for temperature control. The sample is placed directly between the moissanite windows and the cell is assembled similarly to a large diamond anvil cell. In situ observation of the sample is done with a microscope through observation windows and movies are recorded with an attached digital camera. Our experiments with the new cell show that temperatures above 1200°C can be maintained and observed in a sample for several days without damaging the cell nor the windows. Time-lapse movies of melting and crystallizing natural and synthetic rocks and of degassing glasses and melts will be presented to show the potential of the new technique for experimental earth and material science.

A Novel Microfluidic Cell Co-culture Platform for the Study of the Molecular Mechanisms of Parkinson's Disease and Other Synucleinopathies.

PubMed

Fernandes, João T S; Chutna, Oldriska; Chu, Virginia; Conde, João P; Outeiro, Tiago F

2016-01-01

Although, the precise molecular mechanisms underlying Parkinson's disease (PD) are still elusive, it is now known that spreading of alpha-synuclein (aSyn) pathology and neuroinflammation are important players in disease progression. Here, we developed a novel microfluidic cell-culture platform for studying the communication between two different cell populations, a process of critical importance not only in PD but also in many biological processes. The integration of micro-valves in the device enabled us to control fluid routing, cellular microenvironments, and to simulate paracrine signaling. As proof of concept, two sets of experiments were designed to show how this platform can be used to investigate specific molecular mechanisms associated with PD. In one experiment, naïve H4 neuroglioma cells were co-cultured with cells expressing aSyn tagged with GFP (aSyn-GFP), to study the release and spreading of the protein. In our experimental set up, we induced the release of the contents of aSyn-GFP producing cells to the medium and monitored the protein's diffusion. In another experiment, H4 cells were co-cultured with N9 microglial cells to assess the interplay between two cell lines in response to environmental stimuli. Here, we observed an increase in the levels of reactive oxygen species in H4 cells cultured in the presence of activated N9 cells, confirming the cross talk between different cell populations. In summary, the platform developed in this study affords novel opportunities for the study of the molecular mechanisms involved in PD and other neurodegenerative diseases.

Mechanoreceptor Cells on the Tertiary Pulvini of Mimosa pudica L.

PubMed Central

Világi, Ildikó; Varró, Petra; Kristóf, Zoltán

2007-01-01

Special red cells were found on the adaxial surface of tertiary pulvini of Mimosa pudica and experiments performed to determine the origin and function of these cells. Using anatomical (light, scanning electron and transmission electron microscopy) and electrophysiological techniques, we have demonstrated that these red cells are real mechanoreceptor cells. They can generate receptor potential following mechanical stimuli and they are in connection with excitable motor cells (through plasmodesmata). We also provide evidence that these red cells are derived from stomatal subsidiary cells and not guard cells. As histochemical studies show red cells contain tannin, which is important in development of action potentials and movements of plants. These cells could be one of unidentified mechanoreceptors of mimosa. PMID:19517007

Changing pattern of the subcellular distribution of erythroblast macrophage protein (Emp) during macrophage differentiation.

PubMed

Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

2007-01-01

Erythroblast macrophage protein (Emp) mediates the attachment of erythroid cells to macrophages and is required for normal differentiation of both cell lineages. In erythroid cells, Emp is believed to be involved in nuclear extrusion, however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data show that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture shows that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data show that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation and suggest that Emp may be involved in multiple cellular functions.

*CHANGING PATTERN OF THE SUBCELLULAR DISTRIBUTION OF ERYTHROBLAST MACROPHAGE PROTEIN (EMP) DURING MACROPHAGE DIFFERENTIATION

PubMed Central

Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

2007-01-01

Erythroblast macrophage protein (Emp), mediates the attachment of erythroid cells to macrophages, and is required for normal differentiation of both cell lineages. In erythroid cells Emp is believed to be involved in nuclear extrusion however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data shows that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture show that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data shows that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation, and suggests that Emp may be involved in multiple cellular functions. PMID:17071116

Enhanced expression of SRPK2 contributes to aggressive progression and metastasis in prostate cancer.

PubMed

Zhuo, Yang Jia; Liu, Ze Zhen; Wan, Song; Cai, Zhi Duan; Xie, Jian Jiang; Cai, Zhou da; Song, Sheng da; Wan, Yue Ping; Hua, Wei; Zhong, Wei de; Wu, Chin Lee

2018-06-01

Serine/Arginine-Rich Protein-Specific Kinase-2 (SRSF protein kinase-2, SRPK2) is up-regulated in multiple human tumors. However, the expression, function and clinical significance of SRPK2 in prostate cancer (PCa) has not yet been understood. We therefore aimed to determine the association of SRPK2 with tumor progression and metastasis in PCa patients in our present study. The expression of SRPK2 was detected by some public datasets and validated using a clinical tissue microarray (TMA) by immunohistochemistry. The association of SRPK2 expression with various clinicopathological characteristics of PCa patients was subsequently statistically analyzed based on the The Cancer Genome Atlas (TCGA) dataset and clinical TMA. The effects of SRPK2 on cancer cell proliferation, migration, invasion, cell cycle progression, apoptosis and tumor growth were then respectively investigated using in vitro and in vivo experiments. First, public datasets showed that SRPK2 expression was greater in PCa tissues when compared with non-cancerous tissues. Statistical analysis demonstrated that high expression of SRPK2 was significantly correlated with a higher Gleason Score, advanced pathological stage and the presence of tumor metastasis in the TCGA Dataset (all P < 0.01). Similar correlations between SRPK2 and a higher Gleason Score or advanced pathological stage were also identified in the TMA (P < 0.05). Kaplan-Meier curve analyses showed that the biochemical recurrence (BCR)-free time of PCa patients with SRPK2 high expression was shorter than for those with SRPK2 low expression (P < 0.05). Second, cell function experiments in PCa cell lines revealed that enhanced SRPK2 expression could promote cell proliferation, migration, invasion and cell cycle progression but suppress tumor cell apoptosis in vitro. Xenograft experiments showed that SRPK2 promoted tumor growth in vivo. In conclusion, our data demonstrated that SRPK2 may play an important role in the progression and metastasis of PCa, which suggests that it might be a potential therapeutic target for PCa clinical therapy. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Simultaneous recording of electrical activity and the underlying ionic currents in NG108-15 cells cultured on gold substrate.

PubMed

Acosta-García, Ma Cristina; Morales-Reyes, Israel; Jiménez-Anguiano, Anabel; Batina, Nikola; Castellanos, N P; Godínez-Fernández, R

2018-02-01

This paper shows the simultaneous recording of electrical activity and the underlying ionic currents by using a gold substrate to culture NG108-15 cells. Cells grown on two different substrates (plastic Petri dishes and gold substrates) were characterized quantitatively through scanning electron microscopy (SEM) as well as qualitatively by optical and atomic force microscopy (AFM). No significant differences were observed between the surface area of cells cultured on gold substrates and Petri dishes, as indicated by measurements performed on SEM images. We also evaluated the electrophysiological compatibility of the cells through standard patch-clamp experiments by analyzing features such as the resting potential, membrane resistance, ionic currents, etc. Cells grown on both substrates showed no significant differences in their dependency on voltage, as well as in the magnitude of the Na+ and K+ current density; however, cells cultured on the gold substrate showed a lower membrane capacitance when compared to those grown on Petri dishes. By using two separate patch-clamp amplifiers, we were able to record the membrane current with the conventional patch-clamp technique and through the gold substrate simultaneously. Furthermore, the proposed technique allowed us to obtain simultaneous recordings of the electrical activity (such as action potentials firing) and the underlying membrane ionic currents. The excellent conductivity of gold makes it possible to overcome important difficulties found in conventional electrophysiological experiments such as those presented by the resistance of the electrolytic bath solution. We conclude that the technique here presented constitutes a solution to the problem of the simultaneous recording of electrical activity and the underlying ionic currents, which for decades, had been solved only partially.

Feeding Frequency Affects Cultured Rat Pituitary Cells in Low Gravity

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Grindeland, R. E.; Salada, T.; Cenci, R.; Krishnan, K.; Mukai, C.; Nagaoka, S.

1996-01-01

In this report, we describe the results of a rat pituitary cell culture experiment done on STS-65 in which the effect of cell feeding on the release of the six anterior pituitary hormones was studied. We found complex microgravity related interactions between the frequency of cell feeding and the quantity and quality (i.e. biological activity) of some of the six hormones released in flight. Analyses of growth hormone (GH) released from cells into culture media on different mission days using gel filtration and ion exchange chromatography yielded qualitatively similar results between ground and flight samples. Lack of cell feeding resulted in extensive cell clumping in flight (but not ground) cultures. Vigorous fibroblast growth occurred in both ground and flight cultures fed 4 times. These results are interpreted within the context of autocrine and or paracrine feedback interactions. Finally the payload specialist successfully prepared a fresh trypsin solution in microgravity, detached the cells from their surface and reinserted them back into the culture chamber. These cells reattached and continued to release hormone in microgravity. In summary, this experiment shows that pituitary cells are microgravity sensitive and that coupled operations routinely associated with laboratory cel1 culture can also be accomplished in low gravity.

Noninvasive and label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Wei, Xunbin

2015-03-01

Melanoma is a malignant tumor of melanocytes. Circulating melanoma cell has high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC). PAFC is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. In our research, we developed in vitro experiments to prove the ability of PAFC system of detecting PA signals from melanoma cells. For in vivo experiments, we constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells B16F10 with subcutaneous injection. PA signals were detected in the blood vessels of mouse ears in vivo. By counting circulating melanoma cells termly, we obtained the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation. Our PAFC system is an efficient tool to monitor melanoma metastases, cancer recurrence and therapeutic efficacy.

Gold and Iron-Gold Nanoparticles for Intracellular Tracking and in Vivo Medical Applicatons

NASA Astrophysics Data System (ADS)

Fu, Wei

2005-03-01

We have fabricated Au and Fe-Au nanoparticles for potential use in ex vivo experiments such as intracellular tracking, as well as a variety of in vivo medical applications. In order to improve their targeting potential, circulation time and flexibility, gold NPs were surface modified using a hetero-bifunctional poly(ethylene glycol) (PEG, MW 1,500) spacers. A coumarin-PEG-gold NP complex was formed and cell viability studies and optical fluorescence experiments were carried out demonstrating the use of these surface-modified gold NPs for drug delivery, gene therapy and cell trafficking experiments. Fe-Au nanoparticles were also fabricated and show significant contrast enhancement in MRI studies through a substantial reduction of the T2 relaxation time.

Involvement of activated leukocytes in the regulation of plasma levels of acute phase proteins in microgravity simulation experiments

NASA Astrophysics Data System (ADS)

Larina, Olga; Bekker, Anna; Turin-Kuzmin, Alexey

2016-07-01

Earth-based studies of microgravity effects showed the induction of the mechanisms of acute phase reaction (APR). APR comprises the transition of stress-sensitive protein kinases of macrophages and other responsive cells into the active state and the phosphorylation of transcription factors which in turn stimulate the production of acute-phase reaction cytokines. Leukocyte activation is accompanied by the acceleration of the formation of oxygen radicals which can serve a functional indice of leukocyte cell state. The series of events at acute phase response result in selective changes in the synthesis of a number of secretory blood proteins (acute phase proteins, APPs) in liver cells thus contributing the recovery of homeostasis state in the organism. Earlier experiment with head-down tilt showed the increase in plasma concentrations of two cytokine mediators of acute phase response, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) being the outcome of the activation of producer cells, foremost, leukocytes. In experiment with 4-day dry immersion chemiluminescent (ChL) reply of the whole blood samples to a test stimulus were studied along with the measurements of plasma levels of APPs, namely, alpha1-antitrypsin (alpha1-AT), alpha1-acid glycoprotein (alpha1-AGP), alpha2-macroglobulin (alpha2-M), ceruloplasmin (Cer), haptoglobin (Hp), C3-complement component (C3), C-reactive protein (CRP). Eight individuals aged 21.2 ± 3.2 years were the test subjects in the investigation. Protein studies showed a noticeable increase in the mean plasma levels of all APPs measured in experiment thus producing the evidence of the activation of acute phase response mechanisms while individual patterns revealed variability during the immersion period. The overall trends were similar to these in the previous immersion series. The augment in the strength of signal in stimulated light emission tests was higher after 1- and 2-day of immersion exposure than before the experiment. The effects obtained in this survey suggest the enhancement of the synthesis of active oxygen species by blood phagocytes at the initial stages of adaptation to immersion conditions. The gain of chemiluminescence signal correlated with maximal augment in APP concentrations registered in the course of 4-day immersion. Moreover, in the only case with zero effects in chemiluminescent reply stable APP levels were obtained. The data from functional studies performed with phagocytic cells in the experiment with dry immersion corroborate their implication in acute phase mechanisms participating in the adaptation to simulated microgravity conditions.

On-chip gradient generation in 256 microfluidic cell cultures: simulation and experimental validation.

PubMed

Somaweera, Himali; Haputhanthri, Shehan O; Ibraguimov, Akif; Pappas, Dimitri

2015-08-07

A microfluidic diffusion diluter was used to create a stable concentration gradient for dose response studies. The microfluidic diffusion diluter used in this study consisted of 128 culture chambers on each side of the main fluidic channel. A calibration method was used to find unknown concentrations with 12% error. Flow rate dependent studies showed that changing the flow rates generated different gradient patterns. Mathematical simulations using COMSOL Multi-physics were performed to validate the experimental data. The experimental data obtained for the flow rate studies agreed with the simulation results. Cells could be loaded into culture chambers using vacuum actuation and cultured for long times under low shear stress. Decreasing the size of the culture chambers resulted in faster gradient formation (20 min). Mass transport into the side channels of the microfluidic diffusion diluter used in this study is an important factor in creating the gradient using diffusional mixing as a function of the distance. To demonstrate the device's utility, an H2O2 gradient was generated while culturing Ramos cells. Cell viability was assayed in the 256 culture chambers, each at a discrete H2O2 concentration. As expected, the cell viability for the high concentration side channels increased (by injecting H2O2) whereas the cell viability in the low concentration side channels decreased along the chip due to diffusional mixing as a function of distance. COMSOL simulations were used to identify the effective concentration of H2O2 for cell viability in each side chamber at 45 min. The gradient effects were confirmed using traditional H2O2 culture experiments. Viability of cells in the microfluidic device under gradient conditions showed a linear relationship with the viability of the traditional culture experiment. Development of the microfluidic device used in this study could be used to study hundreds of concentrations of a compound in a single experiment.

Radioprotective activity of Gentiana lutea extract and mangiferin.

PubMed

Menkovic, Nebojsa; Juranic, Zorica; Stanojkovic, Tatjana; Raonic-Stevanovic, Tatjana; Savikin, Katarina; Zdunić, Gordana; Borojevic, Nenad

2010-11-01

Radioprotective/sensitizing actions of Gentiana lutea aqueous-ethanol extract and mangiferin on radiation-induced effects on different types of cells were investigated. The study focused on the decreasing survival of normal human immunocompetent cells, the survival of the malignant cells in vitro, and the survival of ex vivo irradiated cells before and after consumption of the extract by healthy volunteers. The in vitro experiments showed that mangiferin could inhibit cytotoxic action of ionizing irradiation (doses of 6 and 8 Gy) only on normal resting human PBMC, not stimulated for proliferation. Orally consumed G. lutea extract showed the potential to reduce the cytotoxic effect of x-ray irradiation on normal human immunocompetent cells PBMC of some healthy people, without changing the susceptibility of malignant cells to be destroyed by irradiation. Since the radioprotective effect was individually dependent, further clinical studies are needed. Copyright © 2010 John Wiley & Sons, Ltd.

Laser annealing of ion implanted CZ silicon for solar cell junction formation

NASA Technical Reports Server (NTRS)

Katzeff, J. S.

1981-01-01

The merits of large spot size pulsed laser annealing of phosphorus implanted, Czochralski grown silicon for function formation of solar cells are evaluated. The feasibility and requirements are also determined to scale-up a laser system to anneal 7.62 cm diameter wafers at a rate of one wafer/second. Results show that laser annealing yields active, defect-free, shallow junction devices. Functional cells with AM 1 conversion efficiencies up to 15.4% for 2 x 2 cm and 2 x 4 cm sizes were attained. For larger cells, 7.62 cm dia., conversion efficiencies ranged up to 14.5%. Experiments showed that texture etched surfaces are not compatible with pulsed laser annealing due to the surface melting caused by the laser energy. When compared with furnace annealed cells, the laser annealed cells generally exhibited conversion efficiencies which were equal to or better than those furnace annealed. In addition, laser annealing has greater throughput potential.

RNA-programmed genome editing in human cells

PubMed Central

Jinek, Martin; East, Alexandra; Cheng, Aaron; Lin, Steven; Ma, Enbo; Doudna, Jennifer

2013-01-01

Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3′ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells. DOI: http://dx.doi.org/10.7554/eLife.00471.001 PMID:23386978

Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes.

PubMed

Rakers, Lena; Grill, David; Matos, Anna L L; Wulff, Stephanie; Wang, Da; Börgel, Jonas; Körsgen, Martin; Arlinghaus, Heinrich F; Galla, Hans-Joachim; Gerke, Volker; Glorius, Frank

2018-05-01

Cholesterol is an essential component of most biological membranes and serves important functions in controlling membrane integrity, organization, and signaling. However, probes to follow the dynamic distribution of cholesterol in live cells are scarce and so far show only limited applicability. Herein, we addressed this problem by synthesizing and characterizing a class of versatile and clickable cholesterol-based imidazolium salts. We show that these cholesterol analogs faithfully mimic the biophysical properties of natural cholesterol in phospholipid mono- and bilayers, and that they integrate into the plasma membrane of cultured and primary human cells. The membrane-incorporated cholesterol analogs can be specifically labeled by click chemistry and visualized in live-cell imaging experiments that show a distribution and behavior comparable with that of endogenous membrane cholesterol. These results indicate that the cholesterol analogs can be used to reveal the dynamic distribution of cholesterol in live cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Single Cell Force Spectroscopy for Quantification of Cellular Adhesion on Surfaces

NASA Astrophysics Data System (ADS)

Christenson, Wayne B.

Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is alpha Mbeta2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering quantitative insights.

Isolation and Characterization of Chromosome-Gain and Increase-in-Ploidy Mutants in Yeast

PubMed Central

Chan, CSM.; Botstein, D.

1993-01-01

We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37°. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37°. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth. PMID:8293973

Exposure of magnetic bacteria to simulated mobile phone-type RF radiation has no impact on mortality.

PubMed

Cranfield, Charles G; Wieser, Heinz Gregor; Dobson, Jon

2003-09-01

The interaction of mobile phone RF emissions with biogenic magnetite in the human brain has been proposed as a potential mechanism for mobile phone bioeffects. This is of particular interest in light of the discovery of magnetite in human brain tissue. Previous experiments using magnetite-containing bacteria exposed directly to emissions from a mobile phone have indicated that these emissions might be causing greater levels of cell death in these bacterial populations when compared to sham exposures. A repeat of these experiments examining only the radio frequency (RF) global system for mobile communication (GSM) component of the mobile phone signal in a well-defined waveguide system (REFLEX), shows no significant change in cell mortality compared to sham exposures. A nonmagnetite containing bacterial cell strain (CC-26) with similar genotype and phenotype to the magnetotactic bacteria was used as a control. These also showed no significant change in cell mortality between RF and sham exposed samples. Results indicate that the RF components of mobile phone exposure do not appear to be responsible for previous findings indicating cell mortality as a result of direct mobile phone exposure. A further mobile phone emission component that should be investigated is the 2-Hz magnetic field pulse generated by battery currents during periods of discontinuous transmission.

Trastuzumab- and Fab′ fragment-modified curcumin PEG-PLGA nanoparticles: preparation and evaluation in vitro and in vivo

PubMed Central

Ni, Ling; Zhang, Liping; Yan, Xiuju; Jiang, Ying; Mu, Hongjie; Wu, Zimei; Sun, Kaoxiang; Li, Youxin

2018-01-01

Introduction Nanoparticles (NPs) modified with bio-ligands represent a promising strategy for active targeted drug delivery to tumour. However, many targeted ligands, such as trastuzumab (TMAB), have high molecular weight, limiting their application for targeting. In this study, we prepared Fab’ (antigen-binding fragments cut from TMAB)-modified NPs (Fab′-NPs) with curcumin (Cur) as a model drug for more effective targeting of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu), which is overexpressed on breast cancer cells. Material and methods The release kinetics was conducted by dialysis bags. The ability to kill HER2-overexpressing BT-474 cells of Fab′-Cur-NPs compared with TMAB-Cur-NPs was conducted by cytotoxicity experiments. Qualitative and quantitative cell uptake studies using coumarin-6 (fluorescent probe)-loaded NPs were performed by fluorescence microscopy and flow cytometry. Pharmacokinetics and biodistribution experiments in vivo were assessed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Results The release kinetics showed that both Fab′-Cur-NPs and TMAB-Cur-NPs provided continuous, slow release of curcumin for 72 h, with no significant difference. In vitro cytotoxicity experiments showed that Fab′-Cur-NPs manifested prominent ability to kill HER2-overexpressing BT-474 cells compared with TMAB-Cur-NPs. Qualitative and quantitative cell uptake studies indicated that the accumulation of Fab′-NPs was greater than that of TMAB-NPs in BT-474 (HER2+) cells; However, there was no significant difference in MDA-MB-231 (HER2−) cells. Pharmacokinetics and biodistribution experiments in vivo demonstrated that the half-life (t1/2) and area under the blood concentration-time curve (AUC0-t) of Fab′-Cur-NPs increased 5.30-fold and 1.76-fold relative to those of TMAB-Cur-NPs, respectively. Furthermore, the tumor accumulation of Fab′-Cur-NPs was higher than that of TMAB-Cur-NPs. Conclusion Fab′ fragment has greater capacity than the intact antibody to achieve tumor targeting through NP-based delivery. PMID:29606874

Design and optimization of reverse-transcription quantitative PCR experiments.

PubMed

Tichopad, Ales; Kitchen, Rob; Riedmaier, Irmgard; Becker, Christiane; Ståhlberg, Anders; Kubista, Mikael

2009-10-01

Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells. A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We found that intersubject differences became easily confounded by sample heterogeneity for single cells and solid tissue. In cell cultures and blood, the noise from the RT and qPCR steps contributed substantially to the overall error because the sampling noise was less pronounced. We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells, and we recommend the use of RT replicates when working with blood. We show how an optimal sampling plan can be calculated for a limited budget. .

In vitro cytotoxicity of galvanically coupled magnesium-titanium particles on human osteosarcoma SAOS2 cells: A potential cancer therapy.

PubMed

Kim, Jua; Gilbert, Jeremy L

2018-04-10

Osteosarcoma is a malignant bone cancer that occurs mostly in children and young adults. This study investigated the cytotoxicity of Mg and Mg-Ti microparticles to human osteosarcoma cells. Osteosarcoma cells were killed in a dosage-dependent manner when cells, with a cell seeding density of 30,000 cells/cm 2 , were cultured with 0 to 2500 µg/mL of Mg or Mg-Ti in cell culture media for 24-72 h. Mg-Ti killed cells more effectively, where 1250 µg/mL of Mg-Ti killed cells completely by 24 h, while 2500 µg/mL of Mg killed nearly all cells, but not all. Killing due to particle corrosion occurred mostly during the first 24 h, and so the percent cell viability between 24 and 72 h showed not much variability. However, the measurement of live and dead cell numbers, over the timeframe of 24-72 h, showed more insight, such as cell recovery. If particle concentrations were low, the number of live cells increased after 24 h, indicating cell proliferation. If particle concentrations were high, the number of live cells either remained steady or decreased, indicating cell quiescence or continued killing, respectively. Increase in the number of dead cells also indicated killing, while plateau meant discontinued killing. In addition, repeated killing of recovered cells exhibited the same dose-dependent killing profile as the initial experiment, implying little development of cell resistance to treatment. These results, together, show that osteosarcoma cells are susceptible to killing by way of exposure to corroding particles, showing highly effective killing using the galvanic couple of Mg-Ti. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc.

Cell growth, division, and death in cohesive tissues: A thermodynamic approach

NASA Astrophysics Data System (ADS)

Yabunaka, Shunsuke; Marcq, Philippe

2017-08-01

Cell growth, division, and death are defining features of biological tissues that contribute to morphogenesis. In hydrodynamic descriptions of cohesive tissues, their occurrence implies a nonzero rate of variation of cell density. We show how linear nonequilibrium thermodynamics allows us to express this rate as a combination of relevant thermodynamic forces: chemical potential, velocity divergence, and activity. We illustrate the resulting effects of the nonconservation of cell density on simple examples inspired by recent experiments on cell monolayers, considering first the velocity of a spreading front, and second an instability leading to mechanical waves.

Safety Performance of Small Lithium-Ion Cells in High Voltage Batteries

NASA Technical Reports Server (NTRS)

Cowles, Philip R.; Darcy, Eric C.; Davies, Frank J.; Jeevarajan, Judith A.; Spurrett, Robert P.

2003-01-01

Topics covered include: Small-cell EAPU work done by NASA-JSC & COM DEV; Looking at safety features (short circuit protection - PTCs); Early tests showed that long strings do not withstand short circuit; a) Some PTCs experience large negative voltages; b) Destructive results. Solution: group cells into shorter substrings, with bypass diodes Work included: a) Tests with single cells shorted; b) Tests with single cells with imposed-negative voltages; c) 6s, 7s and 8s string shorts; and d) Tests with protection scheme in place, on 12s and 41s x 5p.

ARC-2006-ACD06-0091-020

NASA Image and Video Library

2006-06-05

Space shuttle STS-121 FIT (Fly Immunity and Tumors) payload. Using Drosophila (fruit fly) to complete the experiments. Computer screen showing green fluorescent protein used to visualize blood cells in Drosophila (fruit fly).

Immunological unresponsiveness in mice. II. Cellular basis of immunological unresponsiveness induced in foetal and neonatal mice by transfer of human gamma-globulin by the maternal route.

PubMed Central

Shinka, S; Komatsu, T; Dohi, Y; Amano, T

1979-01-01

The cellular basis of the mechanism of immunological tolerance to human gamma-globulin (H gamma G) induced in foetal and neonatal mice by materno-foetal or materno-neonatal transfer after a single injection of tolerogen (deaggregated H gamma G) into the mothers was investigated using a cell transfer system and assays of passive haemagglutinating antibodies and plaque-forming cells to H gamma G. The results demonstrated that B cells are mainly involved in the tolerance induced on the fourteenth day of gestation, whereas inactivation of T cells may account for the tolerance induced on the eighteenth day of gestation and in the neonatal stage. Treatment of the mothers with tolerogen and then anti-H gamma G serum reduced the tolerance induced on the fourteenth day of gestation, but did not affect that induced on the eighteenth day of gestation and in the neonatal stage. Cell transfer experiments showed that B-cell tolerance induced on the fourteenth day of gestation was prevented by passive antibody, while T-cell tolerance induced on the eighteenth day of gestation and in the neonatal stage was not affected by passive antibody. Assay of the anti-DNP antibody response after immunization with DNP10-H gamma G showed that treatment of mice with the tolerogen on the eighteenth day of gestation, but not the fourteenth day of gestation, inactivated H gamma G-reactive helper cells. The significance of these results is discussed in relation to the results of the cell transfer experiments described as above. PMID:89080

Different in vitro cellular responses to tamoxifen treatment in polydimethylsiloxane-based devices compared to normal cell culture.

PubMed

Wang, Lingyu; Yu, Linfen; Grist, Samantha; Cheung, Karen C; Chen, David D Y

2017-11-15

Cell culture systems based on polydimethylsiloxane (PDMS) microfluidic devices offer great flexibility because of their simple fabrication and adaptability. PDMS devices also make it straightforward to set up parallel experiments and can facilitate process automation, potentially speeding up the drug discovery process. However, cells grown in PDMS-based systems can develop in different ways to those grown with conventional culturing systems because of the differences in the containers' surfaces. Despite the growing number of studies on microfluidic cell culture devices, the differences in cellular behavior in PDMS-based devices and normal cell culture systems are poorly characterized. In this work, we investigated the proliferation and autophagy of MCF7 cells cultured in uncoated and Parylene-C coated PDMS wells. Using a quantitative method combining solid phase extraction and liquid chromatography mass spectrometry we developed, we showed that Tamoxifen uptake into the surfaces of uncoated PDMS wells can change the drug's effective concentration in the culture medium, affecting the results of Tamoxifen-induced autophagy and cytotoxicity assays. Such changes must be carefully analyzed before transferring in vitro experiments from a traditional culture environment to a PDMS-based microfluidic system. We also found that cells cultured in Parylene-C coated PDMS wells showed similar proliferation and drug response characteristics to cells cultured in standard polystyrene (PS) plates, indicating that Parylene-C deposition offers an easy way of limiting the uptake of small molecules into porous PDMS materials and significantly improves the performance of PDMS-based device for cell related research. Copyright © 2017 Elsevier B.V. All rights reserved.

Pathological proof of cellular death in radiofrequency ablation therapy and correlation with flash echo imaging--an experiment study.

PubMed

Fujiki, Kei

2004-01-01

The aims of this study were to clarify the geographic distribution of complete cell death in the radiofrequency ablated area in a porcine liver experiment, and to evaluate the efficacy of ultrasonography using contrast media in detecting the area of Radiofrequency-induced cell death. Radiofrequency ablation was performed at 3 sites in each liver in seven swine with a RF2000TM radiofrequency generator using an expandable type needle electrode. The ablation area was investigated histologically by Hematoxylin-Eosin staining and NADH staining. The area of radiofrequency-induced cell death was correlated to the ultrasonographic findings using contrast media, by means of contrast harmonic imaging, flash echo imaging-subtraction and flash echo imaging-power Doppler. The ablation area showed three distinct regions. Although the HE staining did not indicate necrosis, the NADH staining showed a complete loss of cellular activity in the inner and middle layers of the ablation area. However, in the outer layer cells displaying cellular integrity were intermingled with the necrotic cells, indicating that some of the cells in this layer had a chance to survive. Further, in some cases the outer layer of the ablated area had irregular margins. The flash-echo power-doppler images were accurately correlated in size and shape to the pathologically proved region of complete cell death in the radiofrequency-induced lesions. In the marginal part of the radiofrequency ablation area, cell death was incomplete. Flash echo imaging-power doppler was a useful and sensitive real time imaging technique for accurate evaluation of the region of complete cell death.

Synthesis and characterization of 2-substituted benzimidazoles and their evaluation as anticancer agent

NASA Astrophysics Data System (ADS)

Azam, Mohammad; Khan, Azmat Ali; Al-Resayes, Saud I.; Islam, Mohammad Shahidul; Saxena, Ajit Kumar; Dwivedi, Sourabh; Musarrat, Javed; Trzesowska-Kruszynska, Agata; Kruszynski, Rafal

2015-05-01

In this work, we report a series of benzimidazole derivatives synthesized from benzene-1,2-diamine and aryl-aldehydes at room temperature. The synthesized compounds have been characterized on the basis of elemental analysis and various spectroscopic studies viz., IR, 1H- and 13C-NMR, ESI-MS as well by X-ray single X-ray crystallographic study. Interaction of these compounds with CT-DNA has been examined with fluorescence experiments and showed significant binding ability. All the synthesized compounds have been screened for their antitumor activities against various human cancer cell lines viz., Human breast adenocarcinoma cell line (MCF-7), Human leukemia cell line (THP-1), Human prostate cancer cell lines (PC-3) and adenocarcinomic human alveolar basal epithelial cell lines (A-549). Interestingly, all the compounds showed significant anticancer activity.

Transient transfection of mammalian cells using a violet diode laser

NASA Astrophysics Data System (ADS)

Torres-Mapa, Maria Leilani; Angus, Liselotte; Ploschner, Martin; Dholakia, Kishan; Gunn-Moore, Frank J.

2010-07-01

We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to ~40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.

Development of drug-loaded chitosan hollow nanoparticles for delivery of paclitaxel to human lung cancer A549 cells.

PubMed

Jiang, Jie; Liu, Ying; Wu, Chao; Qiu, Yang; Xu, Xiaoyan; Lv, Huiling; Bai, Andi; Liu, Xuan

2017-08-01

In this study, biodegradable chitosan hollow nanospheres (CHN) were fabricated using polystyrene nanospheres (PS) as templates. CHN were applied to increase the solubility of poorly water-soluble drugs. The lung cancer drug paclitaxel (PTX), which is used as a model drug, was loaded into CHN by the adsorption equilibrium method. The drug-loaded sample (PTX-CHN) offered sustained PTX release and good bioavailability. The state characterization of PTX by differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) showed that the PTX absorbed into CHN existed in an amorphous state. An in vitro toxicity experiment indicated that CHN were nontoxic as carriers of poorly water-soluble drugs. The PTX-CHN produced a marked inhibition of lung cancer A549 cells proliferation and encouraged apoptosis. A cell uptake experiment indicated that PTX-CHN was successfully taken up by lung cancer A549 cells. Furthermore, a degradation experiment revealed that CHN were readily biodegradable. These findings state clearly that CHN can be regarded as promising biomaterials for lung cancer treatment.

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology

PubMed Central

Dong, Jia; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K.N.; Knobeloch, Daniel; Gerlach, Jörg C.; Zeilinger, Katrin

2008-01-01

Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes. PMID:19003182

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology.

PubMed

Dong, Jia; Mandenius, Carl-Fredrik; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K N; Knobeloch, Daniel; Gerlach, Jörg C; Zeilinger, Katrin

2008-07-01

Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes.

Partially converted stereoscopic images and the effects on visual attention and memory

NASA Astrophysics Data System (ADS)

Kim, Sanghyun; Morikawa, Hiroyuki; Mitsuya, Reiko; Kawai, Takashi; Watanabe, Katsumi

2015-03-01

This study contained two experimental examinations of the cognitive activities such as visual attention and memory in viewing stereoscopic (3D) images. For this study, partially converted 3D images were used with binocular parallax added to a specific region of the image. In Experiment 1, change blindness was used as a presented stimulus. The visual attention and impact on memory were investigated by measuring the response time to accomplish the given task. In the change blindness task, an 80 ms blank was intersected between the original and altered images, and the two images were presented alternatingly for 240 ms each. Subjects were asked to temporarily memorize the two switching images and to compare them, visually recognizing the difference between the two. The stimuli for four conditions (2D, 3D, Partially converted 3D, distracted partially converted 3D) were randomly displayed for 20 subjects. The results of Experiment 1 showed that partially converted 3D images tend to attract visual attention and are prone to remain in viewer's memory in the area where moderate negative parallax has been added. In order to examine the impact of a dynamic binocular disparity on partially converted 3D images, an evaluation experiment was conducted that applied learning, distraction, and recognition tasks for 33 subjects. The learning task involved memorizing the location of cells in a 5 × 5 matrix pattern using two different colors. Two cells were positioned with alternating colors, and one of the gray cells was moved up, down, left, or right by one cell width. Experimental conditions was set as a partially converted 3D condition in which a gray cell moved diagonally for a certain period of time with a dynamic binocular disparity added, a 3D condition in which binocular disparity was added to all gray cells, and a 2D condition. The correct response rates for recognition of each task after the distraction task were compared. The results of Experiment 2 showed that the correct response rate in the partial 3D condition was significantly higher with the recognition task than in the other conditions. These results showed that partially converted 3D images tended to have a visual attraction and affect viewer's memory.

Long term storage in liquid nitrogen leads to only minor phenotypic and gene expression changes in the mammary carcinoma model cell line BT474.

PubMed

Fazekas, Judit; Grunt, Thomas W; Jensen-Jarolim, Erika; Singer, Josef

2017-05-23

Cancer cell lines are indispensible surrogate models in cancer research, as they can be used off-the-shelf, expanded to the desired extent, easily modified and exchanged between research groups for affirmation, reproduction or follow-up experiments.As malignant cells are prone to genomic instability, phenotypical changes may occur after certain passages in culture. Thus, cell lines have to be regularly authenticated to ensure data quality. In between experiments these cell lines are often stored in liquid nitrogen for extended time periods.Although freezing of cells is a necessary evil, little research is performed on how long-term storage affects cancer cell lines. Therefore, this study investigated the effects of a 28-year long liquid nitrogen storage period on BT474 cells with regard to phenotypical changes, differences in cell-surface receptor expression as well as cytokine and gene expressional variations. Two batches of BT474 cells, one frozen in 1986, the other directly purchased from ATCC were investigated by light microscopy, cell growth analysis, flow cytometry and cytokine as well as whole-transcriptome expression profiling. The cell lines were morphologically indifferent and showed similar growth rates and similar cell-surface receptor expression. Transcriptome analysis revealed significant differences in only 26 of 40,716 investigated RefSeq transcripts with 4 of them being up-regulated and 22 down-regulated. This study demonstrates that even after very long periods of storage in liquid nitrogen, cancer cell lines display only minimal changes in their gene expression profiles. However, also such minor changes should be carefully assessed before continuation of experiments, especially if phenotypic alterations can be additionally observed.

[The morphofunctional state of the bone marrow in lead and zinc intoxication].

PubMed

Vladimtseva, T M; Pashkevich, I A; Salmina, A B

2006-01-01

The nucleolus is a compulsory nuclear structure of all cells of eukaryotes. The quantitative and qualitative characteristics of nuclei show the functional activity of a cell, the rate of its synthesis of RNA and portents, and its metabolic state. Heavy metals (zinc chloride and lead acetate) were comparatively investigated for their effects on the nucleolar apparatus of bone marrow cells in in vivo experiments. Zinc chloride and lead acetate were ascertained to damage the nucleolar apparatus of cells, thus decreasing their transcriptional activity or irreversibly damaging them.

Collective gradient sensing and chemotaxis: modeling and recent developments

NASA Astrophysics Data System (ADS)

Camley, Brian A.

2018-06-01

Cells measure a vast variety of signals, from their environment’s stiffness to chemical concentrations and gradients; physical principles strongly limit how accurately they can do this. However, when many cells work together, they can cooperate to exceed the accuracy of any single cell. In this topical review, I will discuss the experimental evidence showing that cells collectively sense gradients of many signal types, and the models and physical principles involved. I also propose new routes by which experiments and theory can expand our understanding of these problems.

[Research progress of Lgr5-positive stem cells in the formation of organoid in 3D culture].

PubMed

He, Q Q; Li, A; Wang, M H; Gao, X

2018-06-07

Stem cell is critical to regeneration of tissue or organ of human. How to promote repair or regeneration in the tissues/organ using its pluripotency is always an important issue. Lgr5-possitive cell is one type of the stem cell-like cells capable of pluripotent differentiation in various tissues/organs of both humans and mice. Current study showed that single or small amount Lgr5-possitive stem cells can grow and form a plurality of organs in 3D culture system, and some organs can present similar biological and physiological properties with the progenitor they were derived. These studies provided new insight into future orientation, for example, Lgr5-possitive inner ear cells were confirmed as inner ear pluripotent cells population, the experiences obtained from organoid studies of Lgr5-possitive cells have certainly showed potential in the future study of inner ear stem cells. This review will focus on the recent progress associated with Lgr 5-positive stem cells forming organoids in the 3D culture.

Origin of temperature plateaus in laser-heated diamond anvil cell experiments

NASA Astrophysics Data System (ADS)

Geballe, Zachary M.; Jeanloz, Raymond

2012-06-01

Many high-pressure high-temperature studies using laser-heated diamond cells have documented plateaus in the increase of temperature with increasing laser power or with time. By modeling heat transfer in typical laser-heated diamond anvil cell experiments, we demonstrate that latent heat due to melting or other phase transformation is unlikely to be the source of observed plateaus in any previously published studies, regardless of whether pulsed or continuous lasers were used. Rather, large increases (˜10-fold) in thermal conductivity can explain some of the plateaus, and modest increases in reflectivity (tens of percent) can explain any or all of them. Modeling also shows that the sub-microsecond timescale of heating employed in recent pulsed heating experiments is fast enough compared to heat transport into and through typical insulations, but too slow compared to heat transport into metallic laser absorbers themselves to allow the detection of a large plateau due to latent heat of fusion. Four new designs are suggested for future experiments that could use the simple observation of a latent heat-induced plateau to provide reliable high-pressure melting data.

Patient-derived glioblastoma cells show significant heterogeneity in treatment responses to the inhibitor-of-apoptosis-protein antagonist birinapant

PubMed Central

Zakaria, Z; Tivnan, A; Flanagan, L; Murray, D W; Salvucci, M; Stringer, B W; Day, B W; Boyd, A W; Kögel, D; Rehm, M; O'Brien, D F; Byrne, A T; Prehn, J H M

2016-01-01

Background: Resistance to temozolomide (TMZ) greatly limits chemotherapeutic effectiveness in glioblastoma (GBM). Here we analysed the ability of the Inhibitor-of-apoptosis-protein (IAP) antagonist birinapant to enhance treatment responses to TMZ in both commercially available and patient-derived GBM cells. Methods: Responses to TMZ and birinapant were analysed in a panel of commercial and patient-derived GBM cell lines using colorimetric viability assays, flow cytometry, morphological analysis and protein expression profiling of pro- and antiapoptotic proteins. Responses in vivo were analysed in an orthotopic xenograft GBM model. Results: Single-agent treatment experiments categorised GBM cells into TMZ-sensitive cells, birinapant-sensitive cells, and cells that were insensitive to either treatment. Combination treatment allowed sensitisation to therapy in only a subset of resistant GBM cells. Cell death analysis identified three principal response patterns: Type A cells that readily activated caspase-8 and cell death in response to TMZ while addition of birinapant further sensitised the cells to TMZ-induced cell death; Type B cells that readily activated caspase-8 and cell death in response to birinapant but did not show further sensitisation with TMZ; and Type C cells that showed no significant cell death or moderately enhanced cell death in the combined treatment paradigm. Furthermore, in vivo, a Type C patient-derived cell line that was TMZ-insensitive in vitro and showed a strong sensitivity to TMZ and TMZ plus birinapant treatments. Conclusions: Our results demonstrate remarkable differences in responses of patient-derived GBM cells to birinapant single and combination treatments, and suggest that therapeutic responses in vivo may be greatly affected by the tumour microenvironment. PMID:26657652

Cuticular Waxes of Arabidopsis thaliana Shoots: Cell-Type-Specific Composition and Biosynthesis

PubMed Central

Hegebarth, Daniela; Jetter, Reinhard

2017-01-01

It is generally assumed that all plant epidermis cells are covered with cuticles, and the distinct surface geometries of pavement cells, guard cells, and trichomes imply functional differences and possibly different wax compositions. However, experiments probing cell-type-specific wax compositions and biosynthesis have been lacking until recently. This review summarizes new evidence showing that Arabidopsis trichomes have fewer wax compound classes than pavement cells, and higher amounts of especially long-chain hydrocarbons. The biosynthesis machinery generating this characteristic surface coating is discussed. Interestingly, wax compounds with similar, long hydrocarbon chains had been identified previously in some unrelated species, not all of them bearing trichomes. PMID:28686187

Increased concentration of an apparently identical cellular protein in cells transformed by either Abelson murine leukemia virus or other transforming agents.

PubMed

Rotter, V; Boss, M A; Baltimore, D

1981-04-01

Abelson murine leukemia virus (A-MuLV)-transformed cells, simian virus 40 (SV40)-transformed cells, and chemically transformed cells all have increased levels of a 50,000-molecular-weight host cell protein. The protein was detected with sera raised to the A-MuLV-transformed and chemically transformed cells and was tightly bound to T-antigen in extracts of SV40-transformed cells. Partial protease digests showed that the proteins from all three sources were indistinguishable. The three proteins were phosphorylated in cells, and the linkage of phosphate to the A-MuLV-associated P50 was to a serine residue. By immunofluorescence methods, P50-related protein was found on the surface of both normal lymphoid cells and A-MuLV-transformed lymphoid cells, but cell fractionation showed that the majority of P50 was free in the cytoplasm of the transformed cells. Immunofluorescence also showed that P50 was found in granules in the cytoplasm of both untransformed and SV40-transformed fibroblasts. Other cells gave indistinct patterns. Cocapping experiments showed that the A-MuLV-specified P120 protein is weakly associated with the surface P50-related protein of lymphoid cells, but no association of P120 and P50 could be demonstrated by immunoprecipitation methods. Although a monoclonal antiserum to P50 was used in many of these studies, the identity of the bulk P50 protein with the molecules that are reactive at the cell surface requires further study.

Flow of essential elements in subcellular fractions during oxidative stress.

PubMed

Lago, Larissa; Nunes, Emilene A; Vigato, Aryane A; Souza, Vanessa C O; Barbosa, Fernando; Sato, João R; Batista, Bruno L; Cerchiaro, Giselle

2017-02-01

Essential trace elements are commonly found in altered concentrations in the brains of patients with neurodegenerative diseases. Many studies in trace metal determination and quantification are conducted in tissue, cell culture or whole brain. In the present investigation, we determined by ICP-MS Fe, Cu, Zn, Ca, Se, Co, Cr, Mg, and Mn in organelles (mitochondria, nuclei) and whole motor neuron cell cultured in vitro. We performed experiments using two ways to access oxidative stress: cell treatments with H 2 O 2 or Aβ-42 peptide in its oligomeric form. Both treatments caused accumulation of markers of oxidative stress, such as oxidized proteins and lipids, and alteration in DNA. Regarding trace elements, cells treated with H 2 O 2 showed higher levels of Zn and lower levels of Ca in nuclei when compared to control cells with no oxidative treatments. On the other hand, cells treated with Aβ-42 peptide in its oligomeric form showed higher levels of Mg, Ca, Fe and Zn in nuclei when compared to control cells. These differences showed that metal flux in cell organelles during an intrinsic external oxidative condition (H 2 O 2 treatment) are different from an intrinsic external neurodegenerative treatment.

Interplay between microorganisms and geochemistry in geological carbon storage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altman, Susan J.; Kirk, Matthew Fletcher; Santillan, Eugenio-Felipe U.

Researchers at the Center for Frontiers of Subsurface Energy Security (CFSES) have conducted laboratory and modeling studies to better understand the interplay between microorganisms and geochemistry for geological carbon storage (GCS). We provide evidence of microorganisms adapting to high pressure CO 2 conditions and identify factors that may influence survival of cells to CO 2 stress. Factors that influenced the ability of cells to survive exposure to high-pressure CO 2 in our experiments include mineralogy, the permeability of cell walls and/or membranes, intracellular buffering capacity, and whether cells live planktonically or within biofilm. Column experiments show that, following exposure tomore » acidic water, biomass can remain intact in porous media and continue to alter hydraulic conductivity. Our research also shows that geochemical changes triggered by CO 2 injection can alter energy available to populations of subsurface anaerobes and that microbial feedbacks on this effect can influence carbon storage. Our research documents the impact of CO 2 on microorganisms and in turn, how subsurface microorganisms can influence GCS. Furthermore, we conclude that microbial presence and activities can have important implications for carbon storage and that microorganisms should not be overlooked in further GCS research.« less

Interplay between microorganisms and geochemistry in geological carbon storage

DOE PAGES

Altman, Susan J.; Kirk, Matthew Fletcher; Santillan, Eugenio-Felipe U.; ...

2016-02-28

Researchers at the Center for Frontiers of Subsurface Energy Security (CFSES) have conducted laboratory and modeling studies to better understand the interplay between microorganisms and geochemistry for geological carbon storage (GCS). We provide evidence of microorganisms adapting to high pressure CO 2 conditions and identify factors that may influence survival of cells to CO 2 stress. Factors that influenced the ability of cells to survive exposure to high-pressure CO 2 in our experiments include mineralogy, the permeability of cell walls and/or membranes, intracellular buffering capacity, and whether cells live planktonically or within biofilm. Column experiments show that, following exposure tomore » acidic water, biomass can remain intact in porous media and continue to alter hydraulic conductivity. Our research also shows that geochemical changes triggered by CO 2 injection can alter energy available to populations of subsurface anaerobes and that microbial feedbacks on this effect can influence carbon storage. Our research documents the impact of CO 2 on microorganisms and in turn, how subsurface microorganisms can influence GCS. Furthermore, we conclude that microbial presence and activities can have important implications for carbon storage and that microorganisms should not be overlooked in further GCS research.« less

Cell lineage tracing in the developing enteric nervous system: superstars revealed by experiment and simulation

PubMed Central

Cheeseman, Bevan L.; Zhang, Dongcheng; Binder, Benjamin J.; Newgreen, Donald F.; Landman, Kerry A.

2014-01-01

Cell lineage tracing is a powerful tool for understanding how proliferation and differentiation of individual cells contribute to population behaviour. In the developing enteric nervous system (ENS), enteric neural crest (ENC) cells move and undergo massive population expansion by cell division within self-growing mesenchymal tissue. We show that single ENC cells labelled to follow clonality in the intestine reveal extraordinary and unpredictable variation in number and position of descendant cells, even though ENS development is highly predictable at the population level. We use an agent-based model to simulate ENC colonization and obtain agent lineage tracing data, which we analyse using econometric data analysis tools. In all realizations, a small proportion of identical initial agents accounts for a substantial proportion of the total final agent population. We term these individuals superstars. Their existence is consistent across individual realizations and is robust to changes in model parameters. This inequality of outcome is amplified at elevated proliferation rate. The experiments and model suggest that stochastic competition for resources is an important concept when understanding biological processes which feature high levels of cell proliferation. The results have implications for cell-fate processes in the ENS. PMID:24501272

Effect of promoter architecture on the cell-to-cell variability in gene expression.

PubMed

Sanchez, Alvaro; Garcia, Hernan G; Jones, Daniel; Phillips, Rob; Kondev, Jané

2011-03-01

According to recent experimental evidence, promoter architecture, defined by the number, strength and regulatory role of the operators that control transcription, plays a major role in determining the level of cell-to-cell variability in gene expression. These quantitative experiments call for a corresponding modeling effort that addresses the question of how changes in promoter architecture affect variability in gene expression in a systematic rather than case-by-case fashion. In this article we make such a systematic investigation, based on a microscopic model of gene regulation that incorporates stochastic effects. In particular, we show how operator strength and operator multiplicity affect this variability. We examine different modes of transcription factor binding to complex promoters (cooperative, independent, simultaneous) and how each of these affects the level of variability in transcriptional output from cell-to-cell. We propose that direct comparison between in vivo single-cell experiments and theoretical predictions for the moments of the probability distribution of mRNA number per cell can be used to test kinetic models of gene regulation. The emphasis of the discussion is on prokaryotic gene regulation, but our analysis can be extended to eukaryotic cells as well.

Effect of Promoter Architecture on the Cell-to-Cell Variability in Gene Expression

PubMed Central

Sanchez, Alvaro; Garcia, Hernan G.; Jones, Daniel; Phillips, Rob; Kondev, Jané

2011-01-01

According to recent experimental evidence, promoter architecture, defined by the number, strength and regulatory role of the operators that control transcription, plays a major role in determining the level of cell-to-cell variability in gene expression. These quantitative experiments call for a corresponding modeling effort that addresses the question of how changes in promoter architecture affect variability in gene expression in a systematic rather than case-by-case fashion. In this article we make such a systematic investigation, based on a microscopic model of gene regulation that incorporates stochastic effects. In particular, we show how operator strength and operator multiplicity affect this variability. We examine different modes of transcription factor binding to complex promoters (cooperative, independent, simultaneous) and how each of these affects the level of variability in transcriptional output from cell-to-cell. We propose that direct comparison between in vivo single-cell experiments and theoretical predictions for the moments of the probability distribution of mRNA number per cell can be used to test kinetic models of gene regulation. The emphasis of the discussion is on prokaryotic gene regulation, but our analysis can be extended to eukaryotic cells as well. PMID:21390269

Cellular structure of lean hydrogen flames in microgravity

NASA Technical Reports Server (NTRS)

Patnaik, G.; Kailasanath, K.

1990-01-01

Detailed, time-dependent, two-dimensional numerical simulations of premixed laminar flames have been used to study the initiation and subsequent development of cellular structures in lean hydrogen-air flames. The model includes detailed hydrogen-oxygen combustion with 24 elementary reactions of eight reactive species and a nitrogen diluent, molecular diffusion of all species, thermal conduction, viscosity, and convection. This model has been used to study the nonlinear evolution of cellular flame structure and shows that cell splitting, as observed in experiments, can be predicted numerically for sufficiently reactive mixtures. The structures that evolved also resembled the cellular structures observed in experiments. The present study shows that the 'cell-split limit' postulated from experimental observations is an intrinsic property of the mixture and that external factors such as heat losses are not necessary to cause this limit.

Anti-tumor effect of in vivo IL-2 and GM-CSF electrogene therapy in murine hepatoma model.

PubMed

Chi, Chau-Hwa; Wang, Yu-Shan; Lai, Yen-Shuae; Chi, Kwan-Hwa

2003-01-01

We evaluated the effect of in vivo electrogene therapy (EGT), a newly-developed gene transfer method using electroporation on the induction of anti-cancer immunity. The in vivo EGT was carried out by direct injection of plasmid DNAs encoding mouse interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in a subcutaneous murine hepatoma model of 1MEA.7R.1 cells. Six electric pulses were generated in situ from a square-wave electroporator fitted with a circular, six-needle electrode array. 1MEA.7R1 cells in vitro were modified to secret IL-2 (1MEA.7R.1/IL-2 cells). The 1MEA.7R.1/IL-2 cells had a similar cell doubling-time as their parent cells but showed a much slower growth rate on Balb/C mice. One, or 3 rounds of single gene EGT with IL-2 gene showed a dose-responsive effect of growth retardation. Co-administration of 3 rounds of IL-2/GM-CSF double genes EGT had a stronger growth inhibition effect than 3 rounds of IL-2 single gene EGT. Three rounds of IL-2/GM-CSF EGT rendered the tumor to a growth rate of stably transfected 1MEA.7R.1/IL-2 cells. Seven rounds of IL-2/GM-CSF EGT markedly inhibited the tumor growth. Reverse transciptase-polymerase chain reaction confirmed the expression of IL-2, GM-CSF and interferon-gamma within treated tumors. Systemic inhibitory effects can be demonstrated from tumor-re-challenged experiments on mice which received 3 rounds of double-gene EGT. The T cell proliferation assay revealed an increased T cell proliferation in double-gene EGT-treated mice. This experiment showed that partial systemic immunity can be provoked by IL-2/GM-CSF double-gene EGT. These findings suggest that our immuno-gene therapy protocol has the potential for future clinical applications.

Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties.

PubMed

Chiba, Tetsuhiro; Kita, Kaoru; Zheng, Yun-Wen; Yokosuka, Osamu; Saisho, Hiromitsu; Iwama, Atsushi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2006-07-01

Recent advances in stem cell biology enable us to identify cancer stem cells in solid tumors as well as putative stem cells in normal solid organs. In this study, we applied side population (SP) cell analysis and sorting to established hepatocellular carcinoma (HCC) cell lines to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis. Among four cell lines analyzed, SP cells were detected in Huh7 (0.25%) and PLC/PRF/5 cells (0.80%), but not in HepG2 and Huh6 cells. SP cells demonstrated high proliferative potential and anti-apoptotic properties compared with those of non-SP cells. Immunocytochemistry examination showed that SP fractions contain a large number of cells presenting characteristics of both hepatocyte and cholangiocyte lineages. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that only 1 x 10(3) SP cells were sufficient for tumor formation, whereas an injection of 1 x 10(6) non-SP cells did not initiate tumors. Re-analysis of SP cell-derived tumors showed that SP cells generated both SP and non-SP cells and tumor-initiating potential was maintained only in SP cells in serial transplantation. Microarray analysis discriminated a differential gene expression profile between SP and non-SP cells, and several so-called "stemness genes" were upregulated in SP cells in HCC cells. In conclusion, we propose that a minority population, detected as SP cells in HCC cells, possess extreme tumorigenic potential and provide heterogeneity to the cancer stem cell system characterized by distinct hierarchy.

Intracellular Pressure Dynamics in Blebbing Cells

PubMed Central

Strychalski, Wanda; Guy, Robert D.

2016-01-01

Blebs are pressure-driven protrusions that play an important role in cell migration, particularly in three-dimensional environments. A bleb is initiated when the cytoskeleton detaches from the cell membrane, resulting in the pressure-driven flow of cytosol toward the area of detachment and local expansion of the cell membrane. Recent experiments involving blebbing cells have led to conflicting hypotheses regarding the timescale of intracellular pressure propagation. The interpretation of one set of experiments supports a poroelastic model of the cytoplasm that leads to slow pressure equilibration when compared to the timescale of bleb expansion. A different study concludes that pressure equilibrates faster than the timescale of bleb expansion. To address this discrepancy, a dynamic computational model of the cell was developed that includes mechanics of and the interactions among the cytoplasm, the actin cortex, the cell membrane, and the cytoskeleton. The model results quantify the relationship among cytoplasmic rheology, pressure, and bleb expansion dynamics, and provide a more detailed picture of intracellular pressure dynamics. This study shows the elastic response of the cytoplasm relieves pressure and limits bleb size, and that both permeability and elasticity of the cytoplasm determine bleb expansion time. Our model with a poroelastic cytoplasm shows that pressure disturbances from bleb initiation propagate faster than the timescale of bleb expansion and that pressure equilibrates slower than the timescale of bleb expansion. The multiple timescales in intracellular pressure dynamics explain the apparent discrepancy in the interpretation of experimental results. PMID:26958893

A thiazolo[4,5-b]pyridine-based fluorescent probe for detection of zinc ions and application for in vitro and in vivo bioimaging.

PubMed

Gong, Jiao; Li, Yuan-Heng; Zhang, Chan-Juan; Huang, Jian; Sun, Qi

2018-08-01

2-HPTP, a novel thiazolo [4, 5-b] pyridine-based Zn 2+ selective fluorescent probe has been synthesized and investigated. This probe exhibited a high selectivity towards Zn 2+ over other biologically essential cations such as Na + , K + , Ca 2+ , or Mg 2+ . 2-HPTP formed a 1:1 complex with Zn 2+ and showed a fluorescent enhancement with a long emission wavelength red-shift (85 nm) upon complex formation with Zn 2+ . The detection limit and association constant were calculated as 3.48 × 10 -7 M and 2.40 × 10 6 M -1 by a fluorescence titration experiment. Furthermore, the live cell imaging experiment showed that 2-HPTP was membrane permeable and photostable, and hence, could be used to monitor the concentration changes of intracellular Zn 2+ . The co-staining experiments in the cells demonstrated that 2-HPTP possessed high lysosomal selectivity in living cells. Finally, using the nematode C. elegans as an experimental model, we established that 2-HPTP could be successful in imaging Zn 2+ concentration changes in living tissues. Therefore, this molecule should be useful for studies on the biological functions of Zn 2+ . Copyright © 2018 Elsevier B.V. All rights reserved.

Transplantation of Scaffold-Free Cartilage-Like Cell-Sheets Made from Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair: A Preclinical Study.

PubMed

Itokazu, Maki; Wakitani, Shigeyuki; Mera, Hisashi; Tamamura, Yoshihiro; Sato, Yasushi; Takagi, Mutsumi; Nakamura, Hiroaki

2016-10-01

The object of this study was to determine culture conditions that create stable scaffold-free cartilage-like cell-sheets from human bone marrow-derived mesenchymal stem cells (hBMSCs) and to assess their effects after transplantation into osteochondral defects in nude rats. (Experiment 1) The hBMSCs were harvested from 3 males, the proliferative and chondrogenic capacities were assessed at passage 1, and the cells were expanded in 3 different culture conditions: (1) 5% fetal bovine serum (FBS), (2) 10% FBS, and (3) 5% FBS with fibroblast growth factor 2 (FGF-2). The cells were harvested and made chondrogenic pellet culture. The cell proliferation rate, glycosaminoglycan/DNA ratio, and safranin-O staining intensity of pellets cultured condition 3 were higher than those of conditions 1 and 2. (Experiment 2) The hBMSCs were expanded and passaged 3 times under culture condition 3, and fabricate the cell-sheets in chondrogenic medium either with or without FBS. The cell-sheets fabricated with FBS maintained their size with flat edges. (Experiment 3) The cell-sheets were transplanted into osteochondral defects in nude rats. Histological analysis was performed at 2, 4, and 12 weeks after surgery. The osteochondral repair was better after sheet transplantation than in the control group and significantly improved Wakitani score. Immunostaining with human-specific vimentin antibody showed that the transplanted cells became fewer and disappeared at 12 weeks. These results indicate that culture with FGF-2 may help to quickly generate sufficient numbers of cells to create stable and reliable scaffold-free cartilage-like cell-sheets, which contribute to the regeneration of osteochondral defects.

In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images.

PubMed

Christiansen, Eric M; Yang, Samuel J; Ando, D Michael; Javaherian, Ashkan; Skibinski, Gaia; Lipnick, Scott; Mount, Elliot; O'Neil, Alison; Shah, Kevan; Lee, Alicia K; Goyal, Piyush; Fedus, William; Poplin, Ryan; Esteva, Andre; Berndl, Marc; Rubin, Lee L; Nelson, Philip; Finkbeiner, Steven

2018-04-19

Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire. Copyright © 2018 Elsevier Inc. All rights reserved.

NMR quantification of diffusional exchange in cell suspensions with relaxation rate differences between intra and extracellular compartments.

PubMed

Eriksson, Stefanie; Elbing, Karin; Söderman, Olle; Lindkvist-Petersson, Karin; Topgaard, Daniel; Lasič, Samo

2017-01-01

Water transport across cell membranes can be measured non-invasively with diffusion NMR. We present a method to quantify the intracellular lifetime of water in cell suspensions with short transverse relaxation times, T2, and also circumvent the confounding effect of different T2 values in the intra- and extracellular compartments. Filter exchange spectroscopy (FEXSY) is specifically sensitive to exchange between compartments with different apparent diffusivities. Our investigation shows that FEXSY could yield significantly biased results if differences in T2 are not accounted for. To mitigate this problem, we propose combining FEXSY with diffusion-relaxation correlation experiment, which can quantify differences in T2 values in compartments with different diffusivities. Our analysis uses a joint constrained fitting of the two datasets and considers the effects of diffusion, relaxation and exchange in both experiments. The method is demonstrated on yeast cells with and without human aquaporins.

NMR quantification of diffusional exchange in cell suspensions with relaxation rate differences between intra and extracellular compartments

PubMed Central

Eriksson, Stefanie; Elbing, Karin; Söderman, Olle; Lindkvist-Petersson, Karin; Topgaard, Daniel

2017-01-01

Water transport across cell membranes can be measured non-invasively with diffusion NMR. We present a method to quantify the intracellular lifetime of water in cell suspensions with short transverse relaxation times, T2, and also circumvent the confounding effect of different T2 values in the intra- and extracellular compartments. Filter exchange spectroscopy (FEXSY) is specifically sensitive to exchange between compartments with different apparent diffusivities. Our investigation shows that FEXSY could yield significantly biased results if differences in T2 are not accounted for. To mitigate this problem, we propose combining FEXSY with diffusion-relaxation correlation experiment, which can quantify differences in T2 values in compartments with different diffusivities. Our analysis uses a joint constrained fitting of the two datasets and considers the effects of diffusion, relaxation and exchange in both experiments. The method is demonstrated on yeast cells with and without human aquaporins. PMID:28493928

Tafazzin (TAZ) promotes the tumorigenicity of cervical cancer cells and inhibits apoptosis.

PubMed

Chen, Mei; Zhang, Yuan; Zheng, Peng-Sheng

2017-01-01

Tafazzin (TAZ) is often aberrantly expressed in some cancers, including rectal cancer and thyroid neoplasms. However, the function of TAZ in cervical cancer cells remains unknown. This study aims to explore the expression and function of TAZ in cervical cancer cells. Here, we determined the expression of TAZ protein in normal cervical tissue (NC, n = 27), high-grade squamous intraepithelial lesions (HSIL, n = 26) and squamous cervical carcinoma (SCC, n = 41) by immunohistochemistry, the expression of TAZ protein gradually increased from NC to HSIL to SCC. TAZ was overexpressed or down-regulated in cervical cancer cells by stably transfecting a TAZ-expressing plasmid or a shRNA plasmid targeting TAZ. In vitro, the cell growth curves and MTT assays showed that TAZ may promote the growth and viability of cervical cancer cells. In vivo, xenografts experiment showed that TAZ may increase tumor-forming ability. The percentage of apoptosis cells analyzed by FACS and TUNEL assays consistently showed that TAZ inhibits apoptosis in cervical cancer cells. Furthermore, the Cleaved Caspase 9 and Cleaved Caspase 3 were down-regulated by TAZ in cervical cancer cells. Taken together, this study demonstrated that TAZ is overexpressed in cervical cancer and may promote tumorigenicity of cervical cancer cells and inhibit apoptosis.

Tafazzin (TAZ) promotes the tumorigenicity of cervical cancer cells and inhibits apoptosis

PubMed Central

Chen, Mei; Zhang, Yuan; Zheng, Peng-Sheng

2017-01-01

Tafazzin (TAZ) is often aberrantly expressed in some cancers, including rectal cancer and thyroid neoplasms. However, the function of TAZ in cervical cancer cells remains unknown. This study aims to explore the expression and function of TAZ in cervical cancer cells. Here, we determined the expression of TAZ protein in normal cervical tissue (NC, n = 27), high-grade squamous intraepithelial lesions (HSIL, n = 26) and squamous cervical carcinoma (SCC, n = 41) by immunohistochemistry, the expression of TAZ protein gradually increased from NC to HSIL to SCC. TAZ was overexpressed or down-regulated in cervical cancer cells by stably transfecting a TAZ-expressing plasmid or a shRNA plasmid targeting TAZ. In vitro, the cell growth curves and MTT assays showed that TAZ may promote the growth and viability of cervical cancer cells. In vivo, xenografts experiment showed that TAZ may increase tumor-forming ability. The percentage of apoptosis cells analyzed by FACS and TUNEL assays consistently showed that TAZ inhibits apoptosis in cervical cancer cells. Furthermore, the Cleaved Caspase 9 and Cleaved Caspase 3 were down-regulated by TAZ in cervical cancer cells. Taken together, this study demonstrated that TAZ is overexpressed in cervical cancer and may promote tumorigenicity of cervical cancer cells and inhibit apoptosis. PMID:28489874

Epithelial stratification and placode invagination are separable functions in early morphogenesis of the molar tooth

PubMed Central

Li, Jingjing; Chatzeli, Lemonia; Panousopoulou, Eleni; Tucker, Abigail S.; Green, Jeremy B. A.

2016-01-01

Ectodermal organs, which include teeth, hair follicles, mammary ducts, and glands such as sweat, mucous and sebaceous glands, are initiated in development as placodes, which are epithelial thickenings that invaginate and bud into the underlying mesenchyme. These placodes are stratified into a basal and several suprabasal layers of cells. The mechanisms driving stratification and invagination are poorly understood. Using the mouse molar tooth as a model for ectodermal organ morphogenesis, we show here that vertical, stratifying cell divisions are enriched in the forming placode and that stratification is cell division dependent. Using inhibitor and gain-of-function experiments, we show that FGF signalling is necessary and sufficient for stratification but not invagination as such. We show that, instead, Shh signalling is necessary for, and promotes, invagination once suprabasal tissue is generated. Shh-dependent suprabasal cell shape suggests convergent migration and intercalation, potentially accounting for post-stratification placode invagination to bud stage. We present a model in which FGF generates suprabasal tissue by asymmetric cell division, while Shh triggers cell rearrangement in this tissue to drive invagination all the way to bud formation. PMID:26755699

Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers.

PubMed

Min, Kyoung Ah; Rosania, Gus R; Kim, Chong-Kook; Shin, Meong Cheol

2016-03-01

To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies.

Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers

PubMed Central

Min, Kyoung Ah; Rosania, Gus R.; Kim, Chong-Kook; Shin, Meong Cheol

2016-01-01

To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies. PMID:26746641

Collective dynamics of cell migration and cell rearrangements

NASA Astrophysics Data System (ADS)

Kabla, Alexandre

Understanding multicellular processes such as embryo development or cancer metastasis requires to decipher the contributions of local cell autonomous behaviours and long range interactions with the tissue environment. A key question in this context concerns the emergence of large scale coordination in cell behaviours, a requirement for collective cell migration or convergent extension. I will present a few examples where physical and mechanical aspects play a significant role in driving tissue scale dynamics.

Rapid isolation of cancer cells using microfluidic deterministic lateral displacement structure.

PubMed

Liu, Zongbin; Huang, Fei; Du, Jinghui; Shu, Weiliang; Feng, Hongtao; Xu, Xiaoping; Chen, Yan

2013-01-01

This work reports a microfluidic device with deterministic lateral displacement (DLD) arrays allowing rapid and label-free cancer cell separation and enrichment from diluted peripheral whole blood, by exploiting the size-dependent hydrodynamic forces. Experiment data and theoretical simulation are presented to evaluate the isolation efficiency of various types of cancer cells in the microfluidic DLD structure. We also demonstrated the use of both circular and triangular post arrays for cancer cell separation in cell solution and blood samples. The device was able to achieve high cancer cell isolation efficiency and enrichment factor with our optimized design. Therefore, this platform with DLD structure shows great potential on fundamental and clinical studies of circulating tumor cells.

Electron Radiation Damage of (alga) As-gaas Solar Cells

NASA Technical Reports Server (NTRS)

Loo, R.; Kamath, G. S.; Knechtli, R.

1979-01-01

Solar cells (2 cm by 2 cm (AlGa) As-GaAs cells) were fabricated and then subjected to irradiation at normal incidence by electrons. The influence of junction depth and n-type buffer layer doping level on the cell's resistance to radiation damage was investigated. The study shows that (1) a 0.3 micrometer deep junction results in lower damage to the cells than does a 0.5 micrometer junction, and (2) lowering the n buffer layer doping density does not improve the radiation resistance of the cell. Rather, lowering the doping density decreases the solar cell's open circuit voltage. Some preliminary thermal annealing experiments in vacuum were performed on the (AlGa)As-GaAs solar cells damaged by 1-MeV electron irradiation. The results show that cell performance can be expected to partially recover at 200 C with more rapid and complete recovery occurring at higher temperature. For a 0.5hr anneal at 400 C, 90% of the initial power is recovered. The characteristics of the (AlGa)As-GaAs cells both before and after irradiation are described.

Quantitative and Functional Requirements for Bioluminescent Cancer Models.

PubMed

Feys, Lynn; Descamps, Benedicte; Vanhove, Christian; Vermeulen, Stefan; Vandesompele, J O; Vanderheyden, Katrien; Messens, Kathy; Bracke, Marc; De Wever, Olivier

2016-01-01

Bioluminescent cancer models are widely used but detailed quantification of the luciferase signal and functional comparison with a non-transfected control cell line are generally lacking. In the present study, we provide quantitative and functional tests for luciferase-transfected cells. We quantified the luciferase expression in BLM and HCT8/E11 transfected cancer cells, and examined the effect of long-term luciferin exposure. The present study also investigated functional differences between parental and transfected cancer cells. Our results showed that quantification of different single-cell-derived populations are superior with droplet digital polymerase chain reaction. Quantification of luciferase protein level and luciferase bioluminescent activity is only useful when there is a significant difference in copy number. Continuous exposure of cell cultures to luciferin leads to inhibitory effects on mitochondrial activity, cell growth and bioluminescence. These inhibitory effects correlate with luciferase copy number. Cell culture and mouse xenograft assays showed no significant functional differences between luciferase-transfected and parental cells. Luciferase-transfected cells should be validated by quantitative and functional assays before starting large-scale experiments. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Pancreatic β-Cells Express the Fetal Islet Hormone Gastrin in Rodent and Human Diabetes.

PubMed

Dahan, Tehila; Ziv, Oren; Horwitz, Elad; Zemmour, Hai; Lavi, Judith; Swisa, Avital; Leibowitz, Gil; Ashcroft, Frances M; In't Veld, Peter; Glaser, Benjamin; Dor, Yuval

2017-02-01

β-Cell failure in type 2 diabetes (T2D) was recently proposed to involve dedifferentiation of β-cells and ectopic expression of other islet hormones, including somatostatin and glucagon. Here we show that gastrin, a stomach hormone typically expressed in the pancreas only during embryogenesis, is expressed in islets of diabetic rodents and humans with T2D. Although gastrin in mice is expressed in insulin + cells, gastrin expression in humans with T2D occurs in both insulin + and somatostatin + cells. Genetic lineage tracing in mice indicates that gastrin expression is turned on in a subset of differentiated β-cells after exposure to severe hyperglycemia. Gastrin expression in adult β-cells does not involve the endocrine progenitor cell regulator neurogenin3 but requires membrane depolarization, calcium influx, and calcineurin signaling. In vivo and in vitro experiments show that gastrin expression is rapidly eliminated upon exposure of β-cells to normal glucose levels. These results reveal the fetal hormone gastrin as a novel marker for reversible human β-cell reprogramming in diabetes. © 2017 by the American Diabetes Association.

Pancreatic β-Cells Express the Fetal Islet Hormone Gastrin in Rodent and Human Diabetes

PubMed Central

Dahan, Tehila; Ziv, Oren; Horwitz, Elad; Zemmour, Hai; Lavi, Judith; Swisa, Avital; Leibowitz, Gil; Ashcroft, Frances M.; In’t Veld, Peter

2017-01-01

β-Cell failure in type 2 diabetes (T2D) was recently proposed to involve dedifferentiation of β-cells and ectopic expression of other islet hormones, including somatostatin and glucagon. Here we show that gastrin, a stomach hormone typically expressed in the pancreas only during embryogenesis, is expressed in islets of diabetic rodents and humans with T2D. Although gastrin in mice is expressed in insulin+ cells, gastrin expression in humans with T2D occurs in both insulin+ and somatostatin+ cells. Genetic lineage tracing in mice indicates that gastrin expression is turned on in a subset of differentiated β-cells after exposure to severe hyperglycemia. Gastrin expression in adult β-cells does not involve the endocrine progenitor cell regulator neurogenin3 but requires membrane depolarization, calcium influx, and calcineurin signaling. In vivo and in vitro experiments show that gastrin expression is rapidly eliminated upon exposure of β-cells to normal glucose levels. These results reveal the fetal hormone gastrin as a novel marker for reversible human β-cell reprogramming in diabetes. PMID:27864307

Use of Protein Biotinylation In Vivo for Immunoelectron Microscopic Localization of a Specific Protein Isoform

PubMed Central

Viens, Antoine; Harper, Francis; Pichard, Evelyne; Comisso, Martine; Pierron, Gérard; Ogryzko, Vasily

2008-01-01

Tagging of proteins in vivo by covalent attachment of a biotin moiety has emerged as a new prospective tool for protein detection and purification. Previously, we established a strategy for expression of in vivo biotinylated proteins in mammalian cells. It is based on coexpression of the protein of interest fused to a short biotin acceptor peptide and biotin ligase BirA cloned in the same vector. We show here that the in vivo biotinylation can be used for immunogold postembedding labeling in immunoelectron microscopy experiments. We show that immunoelectron microscopy with biotinylated nuclear proteins is compatible with a wide range of postembedding methods, facilitating combination of morphological and localization studies in a single experiment. We also show that the method works in both transient transfection and stable cell line expression protocols and can be used for colocalization studies. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 56:911–919, 2008) PMID:18574249

Direct Ca2+-dependent Heterophilic Interaction between Desmosomal Cadherins, Desmoglein and Desmocollin, Contributes to Cell–Cell Adhesion

PubMed Central

Chitaev, Nikolai A.; Troyanovsky, Sergey M.

1997-01-01

Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg1-Thr170) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca2+-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca2+-dependent manner. The contribution of the Dsg/ Dsc interaction to cell–cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell–cell adhesion. PMID:9214392

Receptor-mediated binding of IgE-sensitized rat basophilic leukemia cells to antigen-coated substrates under hydrodynamic flow.

PubMed Central

Tempelman, L A; Hammer, D A

1994-01-01

The physiological function of many cells is dependent on their ability to adhere via receptors to ligand-coated surfaces under fluid flow. We have developed a model experimental system to measure cell adhesion as a function of cell and surface chemistry and fluid flow. Using a parallel-plate flow chamber, we measured the binding of rat basophilic leukemia cells preincubated with anti-dinitrophenol IgE antibody to polyacrylamide gels covalently derivatized with 2,4-dinitrophenol. The rat basophilic leukemia cells' binding behavior is binary: cells are either adherent or continue to travel at their hydrodynamic velocity, and the transition between these two states is abrupt. The spatial location of adherent cells shows cells can adhere many cell diameters down the length of the gel, suggesting that adhesion is a probabilistic process. The majority of experiments were performed in the excess ligand limit in which adhesion depends strongly on the number of receptors but weakly on ligand density. Only 5-fold changes in IgE surface density or in shear rate were necessary to change adhesion from complete to indistinguishable from negative control. Adhesion showed a hyperbolic dependence on shear rate. By performing experiments with two IgE-antigen configurations in which the kinetic rates of receptor-ligand binding are different, we demonstrate that the forward rate of reaction of the receptor-ligand pair is more important than its thermodynamic affinity in the regulation of binding under hydrodynamic flow. In fact, adhesion increases with increasing receptor-ligand reaction rate or decreasing shear rate, and scales with a single dimensionless parameter which compares the relative rates of reaction to fluid shear. Images FIGURE 2 FIGURE 3 FIGURE 6 FIGURE 8 FIGURE 10 PMID:8038394

A gas trapping method for high-throughput metabolic experiments.

PubMed

Krycer, James R; Diskin, Ciana; Nelson, Marin E; Zeng, Xiao-Yi; Fazakerley, Daniel J; James, David E

2018-01-01

Research into cellular metabolism has become more high-throughput, with typical cell-culture experiments being performed in multiwell plates (microplates). This format presents a challenge when trying to collect gaseous products, such as carbon dioxide (CO2), which requires a sealed environment and a vessel separate from the biological sample. To address this limitation, we developed a gas trapping protocol using perforated plastic lids in sealed cell-culture multiwell plates. We used this trap design to measure CO2 production from glucose and fatty acid metabolism, as well as hydrogen sulfide production from cysteine-treated cells. Our data clearly show that this gas trap can be applied to liquid and solid gas-collection media and can be used to study gaseous product generation by both adherent cells and cells in suspension. Since our gas traps can be adapted to multiwell plates of various sizes, they present a convenient, cost-effective solution that can accommodate the trend toward high-throughput measurements in metabolic research.

Using a combined computational-experimental approach to predict antibody-specific B cell epitopes.

PubMed

Sela-Culang, Inbal; Benhnia, Mohammed Rafii-El-Idrissi; Matho, Michael H; Kaever, Thomas; Maybeno, Matt; Schlossman, Andrew; Nimrod, Guy; Li, Sheng; Xiang, Yan; Zajonc, Dirk; Crotty, Shane; Ofran, Yanay; Peters, Bjoern

2014-04-08

Antibody epitope mapping is crucial for understanding B cell-mediated immunity and required for characterizing therapeutic antibodies. In contrast to T cell epitope mapping, no computational tools are in widespread use for prediction of B cell epitopes. Here, we show that, utilizing the sequence of an antibody, it is possible to identify discontinuous epitopes on its cognate antigen. The predictions are based on residue-pairing preferences and other interface characteristics. We combined these antibody-specific predictions with results of cross-blocking experiments that identify groups of antibodies with overlapping epitopes to improve the predictions. We validate the high performance of this approach by mapping the epitopes of a set of antibodies against the previously uncharacterized D8 antigen, using complementary techniques to reduce method-specific biases (X-ray crystallography, peptide ELISA, deuterium exchange, and site-directed mutagenesis). These results suggest that antibody-specific computational predictions and simple cross-blocking experiments allow for accurate prediction of residues in conformational B cell epitopes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mammalian skin cell biology: at the interface between laboratory and clinic.

PubMed

Watt, Fiona M

2014-11-21

Mammalian skin research represents the convergence of three complementary disciplines: cell biology, mouse genetics, and dermatology. The skin provides a paradigm for current research in cell adhesion, inflammation, and tissue stem cells. Here, I discuss recent insights into the cell biology of skin. Single-cell analysis has revealed that human epidermal stem cells are heterogeneous and differentiate in response to multiple extrinsic signals. Live-cell imaging, optogenetics, and cell ablation experiments show skin cells to be remarkably dynamic. High-throughput, genome-wide approaches have yielded unprecedented insights into the circuitry that controls epidermal stem cell fate. Last, integrative biological analysis of human skin disorders has revealed unexpected functions for elements of the skin that were previously considered purely structural. Copyright © 2014, American Association for the Advancement of Science.

Systems biology approach to transplant tolerance: proof of concept experiments using RNA interference (RNAi) to knock down hub genes in Jurkat and HeLa cells in vitro.

PubMed

Lwin, Wint Wah; Park, Ken; Wauson, Matthew; Gao, Qin; Finn, Patricia W; Perkins, David; Khanna, Ajai

2012-07-01

Systems biology is gaining importance in studying complex systems such as the functional interconnections of human genes [1]. To investigate the molecular interactions involved in T cell immune responses, we used databases of physical gene-gene interactions to constructed molecular interaction networks (interconnections) with R language algorithms. This helped to identify highly interconnected "hub" genes AT(1)P5C1, IL6ST, PRKCZ, MYC, FOS, JUN, and MAPK1. We hypothesized that suppression of these hub genes in the gene network would result in significant phenotypic effects on T cells and examined this in vitro. The molecular interaction networks were then analyzed and visualized with Cytoscape. Jurkat and HeLa cells were transfected with siRNA for the selected hub genes. Cell proliferation was measured using ATP luminescence and BrdU labeling, which were measured 36, 72, and 96 h after activation. Following T cell stimulation, we found a significant decrease in ATP production (P < 0.05) when the hub genes ATP5C1 and PRKCZ were knocked down using siRNA transfection, whereas no difference in ATP production was observed in siRNA transfected HeLa cells. However, HeLa cells showed a significant (P < 0.05) decrease in cell proliferation when the genes MAPK1, IL6ST, ATP5C1, JUN, and FOS were knocked down. In both Jurkat and HeLa cells, targeted gene knockdown using siRNA showed decreased cell proliferation and ATP production in both Jurkat and HeLa cells. However, Jurkat T cells and HELA cells use different hub genes to regulate activation responses. This experiment provides proof of principle of applying siRNA knockdown of T cell hub genes to evaluate their proliferative capacity and ATP production. This novel concept outlines a systems biology approach to identify hub genes for targeted therapeutics. Published by Elsevier Inc.

Interplay of cell dynamics and epithelial tension during morphogenesis of the Drosophila pupal wing

PubMed Central

Etournay, Raphaël; Popović, Marko; Merkel, Matthias; Nandi, Amitabha; Blasse, Corinna; Aigouy, Benoît; Brandl, Holger; Myers, Gene; Salbreux, Guillaume; Jülicher, Frank; Eaton, Suzanne

2015-01-01

How tissue shape emerges from the collective mechanical properties and behavior of individual cells is not understood. We combine experiment and theory to study this problem in the developing wing epithelium of Drosophila. At pupal stages, the wing-hinge contraction contributes to anisotropic tissue flows that reshape the wing blade. Here, we quantitatively account for this wing-blade shape change on the basis of cell divisions, cell rearrangements and cell shape changes. We show that cells both generate and respond to epithelial stresses during this process, and that the nature of this interplay specifies the pattern of junctional network remodeling that changes wing shape. We show that patterned constraints exerted on the tissue by the extracellular matrix are key to force the tissue into the right shape. We present a continuum mechanical model that quantitatively describes the relationship between epithelial stresses and cell dynamics, and how their interplay reshapes the wing. DOI: http://dx.doi.org/10.7554/eLife.07090.001 PMID:26102528

Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells.

PubMed

Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

2014-02-18

In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca(2+)] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells.

Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells

PubMed Central

Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

2014-01-01

In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca2+] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells. PMID:24535122

Effects of soya fatty acids on cassava ethanol fermentation.

PubMed

Xiao, Dongguang; Wu, Shuai; Zhu, Xudong; Chen, Yefu; Guo, Xuewu

2010-01-01

Ethanol tolerance is a key trait of microbes in bioethanol production. Previous studies have shown that soya flour contributed to the increase of ethanol tolerance of yeast cells. In this paper, the mechanism of this ethanol tolerance improvement was investigated in cassava ethanol fermentation supplemented with soya flour or defatted soya flour, respectively. Experiment results showed that ethanol tolerance of cells from soya flour supplemented medium increased by 4-6% (v/v) than the control with defatted soya flour. Microscopic observation found that soya flour can retain the cell shape while dramatic elongations of cells were observed with the defatted soya flour supplemented medium. Unsaturated fatty acids (UFAs) compositions of cell membrane were analyzed and the UFAs amounts increased significantly in all tested strains grown in soya flour supplemented medium. Growth study also showed that soya flour stimulated the cell growth rate by approximately tenfolds at 72-h fermentation. All these results suggested that soya fatty acids play an important role to protect yeast cells from ethanol stress during fermentation process.

[Individual variation in the frequency of chromosome aberrations under the influence of chemical mutagens. I. Inter-cultural and inter-individual variations in the effect of mutagens on human lymphocytes].

PubMed

Iakovenko, K N; Tarusina, T O

1976-01-01

The study of the distribution law of human peripheral blood cultures for the sensitivity to thiophosphamide was performed. In the first experiment the blood from one person was used, in the second one the blood was used from different persons. "The percent of aberrant cells" and "the number of chromosome breaks per 100 cells" were scored. The distribution law of the cultures in all the experiments was found to be normal. Analysis of the variances on the percent of aberrant cells showed that the distribution law of the cultures received from one donor corresponded to the binomial one, and that of the cultures received from different donors--to the Poisson's one.

Some karyological observations on plants grown in space

NASA Technical Reports Server (NTRS)

Krikorian, A. D.; Oconnor, S. A.

1982-01-01

Experiments were conducted to assess whether cell division in a plant root would be affected by prolonged exposure to microgravity. Root materials from sunflower, oat, and mung bean plants grown on STS-2 and STS-3 were utilized for the experiments. It is found that all oat, sunflower, and mung seedlings showed a reduced number of cells in division as they went through their first cell division cycle on earth when compared to their ground controls. A significant number of oat, mung, and sunflower plantlets exhibited random root orientation and the lack of strictly orthotropic growth of their shoot systems in the flight samples. In addition, it is found that the mung roots were apparently least affected in terms of their cytology despite the fact that their roots were often randomly oriented.

The effect of microgravity on the in vitro NK cell function during six International Space Station Missions

NASA Astrophysics Data System (ADS)

Buravkova, L. B.; Grigorieva, V.; Rykova, M. P.

2007-09-01

The level of natural killer (NK) cytotoxic activity was measured during co-cultivation of human lymphocytes and target cells (K-562) in microgravity. Flight experiments were carried out using special instrumentation, the "Fibroblast-1 " cassettes, in the frame of Russian scientific program during six ISS missions. Lymphocyte suspensions from human venous blood were used in experiments during short-term flights on six ISS missions (7-12). Russian space crew members performed the experiments after Soyuz docking. The first step was mixing lymphocytes and3H-labeled K-562 cells and their incubation at 37°C during 24 hs; the second step was filtration of the cell suspension. The frozen medium and filters were analyzed for the cytokine level and cytotoxic activity after landing. It was found that lymphocytes with different basal levels of cytotoxic activity kept the ability of recognizing and lysing malignant cells. In microgravity, cytotoxity increased to 160% of the basal levels. Donor individual features modulated the magnitude of the increase. The measurement of interleukin levels (TNF-α, IL-1, IL-2) in medium showed that synthesis of TNF-α increased during cell co-cultivation in microgravity. The level of IL-2 was very low inflight and ground control samples. The production of IL-1 by lymphocytes decreased after in-flight incubation. The results indicate that microgravity did not disturb the cytotoxic function of immune cells in vitro during 24 h incubation with specific target cells.

Stock culture heterogeneity rather than new mutational variation complicates short-term cell physiology studies of Escherichia coli K-12 MG1655 in continuous culture.

PubMed

Nahku, Ranno; Peebo, Karl; Valgepea, Kaspar; Barrick, Jeffrey E; Adamberg, Kaarel; Vilu, Raivo

2011-09-01

Nutrient-limited continuous cultures in chemostats have been used to study microbial cell physiology for over 60 years. Genome instability and genetic heterogeneity are possible uncontrolled factors in continuous cultivation experiments. We investigated these issues by using high-throughput (HT) DNA sequencing to characterize samples from different phases of a glucose-limited accelerostat (A-stat) experiment with Escherichia coli K-12 MG1655 and a duration regularly used in cell physiology studies (20 generations of continuous cultivation). Seven consensus mutations from the reference sequence and five subpopulations characterized by different mutations were detected in the HT-sequenced samples. This genetic heterogeneity was confirmed to result from the stock culture by Sanger sequencing. All the subpopulations in which allele frequencies increased (betA, cspG/cspH, glyA) during the experiment were also present at the end of replicate A-stats, indicating that no new subpopulations emerged during our experiments. The fact that ~31 % of the cells in our initial cultures obtained directly from a culture stock centre were mutants raises concerns that even if cultivations are started from single colonies, there is a significant chance of picking a mutant clone with an altered phenotype. Our results show that current HT DNA sequencing technology allows accurate subpopulation analysis and demonstrates that a glucose-limited E. coli K-12 MG1655 A-stat experiment with a duration of tens of generations is suitable for studying cell physiology and collecting quantitative data for metabolic modelling without interference from new mutations.

Stock culture heterogeneity rather than new mutational variation complicates short-term cell physiology studies of Escherichia coli K-12 MG1655 in continuous culture

PubMed Central

Nahku, Ranno; Peebo, Karl; Valgepea, Kaspar; Barrick, Jeffrey E.; Adamberg, Kaarel

2011-01-01

Nutrient-limited continuous cultures in chemostats have been used to study microbial cell physiology for over 60 years. Genome instability and genetic heterogeneity are possible uncontrolled factors in continuous cultivation experiments. We investigated these issues by using high-throughput (HT) DNA sequencing to characterize samples from different phases of a glucose-limited accelerostat (A-stat) experiment with Escherichia coli K-12 MG1655 and a duration regularly used in cell physiology studies (20 generations of continuous cultivation). Seven consensus mutations from the reference sequence and five subpopulations characterized by different mutations were detected in the HT-sequenced samples. This genetic heterogeneity was confirmed to result from the stock culture by Sanger sequencing. All the subpopulations in which allele frequencies increased (betA, cspG/cspH, glyA) during the experiment were also present at the end of replicate A-stats, indicating that no new subpopulations emerged during our experiments. The fact that ~31 % of the cells in our initial cultures obtained directly from a culture stock centre were mutants raises concerns that even if cultivations are started from single colonies, there is a significant chance of picking a mutant clone with an altered phenotype. Our results show that current HT DNA sequencing technology allows accurate subpopulation analysis and demonstrates that a glucose-limited E. coli K-12 MG1655 A-stat experiment with a duration of tens of generations is suitable for studying cell physiology and collecting quantitative data for metabolic modelling without interference from new mutations. PMID:21700661

Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

NASA Astrophysics Data System (ADS)

Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

2015-11-01

Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in hypergravity conditions does not affect the normal development and actin filament structures of mouse embryos.

The origin of mesoderm in phoronids

NASA Technical Reports Server (NTRS)

Freeman, Gary; Martindale, Mark Q.

2002-01-01

Descriptive studies of phoronid development have concluded that the mesoderm of these animals originates from the endoderm during gastrulation. This interpretation has been tested by labeling one blastomere of 4- through 16-cell embryos and examining the position and germ layers occupied by the labeled clones of cells in the larva. No 2 injections gave rise to identical clones of cells, suggesting that the cleavage program does not generate cells of unique identity and that cell fates are established at later developmental time points. In many cases, a relatively large sector composed of ectodermal cells was labeled. When these labeled cells were adjacent to the mouth or anus of the larva, muscle and mesenchyme cells originated from the labeled clones. Under these circumstances, nerve cells also originated from these labeled sectors. These labeling studies also showed that endodermal cells can give rise to mesodermal and neural cells. These results suggest that nerve and muscle cells are induced to form at ectodermal-endodermal boundaries from both germ layers. These marking experiments also confirmed the observation that nerve cells originate both from the apical organ and the trunk region and show for the first time that the intestine originates by ingression of posterior ectoderm.

Application of the thiocarbohydrazide method for vicinal glycol group detection to the study of gastric mucosa endocrine cells.

PubMed

Lefranc, G; Chung, Y T; Barrière, P; Pradal, G

1980-01-01

The thiocarbohydrazide-silver proteinate (TCH SP) method was applied to the study of cat, rabbit and mouse gastric mucosa endocrine cells. After 24-h treatment with thiocarbohydrazide (TCH), glycogen was seen in the hyaloplasm of X, D, P, A and O cells but not in EC, EC-like or D1 cells. With flotation times as short as 30 to 40 min glycogen was readily detected in X cells. Secretory granules of EC cells were constantly stained, while those of D1 cells failed to react. In most experiments granules of X, A and O cells showed peripheral "staining", while in others staining of variable intensity affected the entire granular cross-section in X, D and P cells. With 72-h exposure to TCH, EC and EC-like cells showed particles resembling glycogen, even staining or only peripheral staining of certain EC cell granules. From the results of this and previous studies, EC cell staining is believed to be due wholly or partly, according to exposure times, to the action of silver proteinate, while that of certain non-EC cells is probably a specific indicator of complexed carbohydrates.

A non-invasive measure of minerals and electrolytes in tissue

NASA Technical Reports Server (NTRS)

Arnaud, Sara B.; Silver, Burton

1991-01-01

A system for collecting epithelial cells from the oral mucosa for the determination of ion concentration is discussed with application to the study of man's adaptation to microgravity. A number of characteristics of these cells influenced the choice for clinical testing. They are non-cornified epithelial cells located on the inferior aspect of the tongue; therefore, they are well protected from trauma. They have the capability of reflecting relatively recent physiologic changes since they are renewed every three days and have aerobic metabolism. Most importantly, they are easily accessible and can be removed by a wooden applicator stick with minimum discomfort. Smears of cells removed in this manner show predominantly individual cells rather than sheets of contiuous cells. This facilitates the visual isolation of single cells with the electron microscope for analysis. NASA's principle effort in the development of a test to measure the ion concentration in sublingual cells has been research by the biomedical program carried out by scientists with expertise in skeletal metabolism. These efforts were directed toward determining the biological meaning and deviations in interacellular ions in nonhuman primates and in male volunteers for experiments in a model for weightlessness. A brief one page summary of the experiments and results are presented.

Manganese Transport and Toxicity in Polarized WIF-B Hepatocytes.

PubMed

Thompson, Khristy J; Hein, Jennifer; Baez, Andrew; Sosa, Jose Carlo; Wessling-Resnick, Marianne

2018-05-24

Mn toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to determine the cellular distribution of Mn transporters in polarized hepatocytes, to establish an in vitro assay for hepatocyte Mn efflux, and to examine possible roles the Mn transporters would play in metal import and export. For these experiments, hepatocytoma WIF-B cells were grown for 12-14 days to achieve maximal polarity. Immunoblots showed that Mn transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14 were present. Indirect immunofluorescence microscopy localized Fpn and ZIP14 to WIF-B cell basolateral domains while ZnT10 and ZIP8 associated with intracellular vesicular compartments. ZIP8-positive structures were distributed uniformly throughout the cytoplasm, but ZnT10-positive vesicles were adjacent to apical bile compartments. WIF-B cells were sensitive to Mn toxicity, showing decreased viability after 16 h exposure to > 250 M MnCl2. However, the hepatocytes were resistant to 4 h exposures of up to 500 M MnCl2 despite 50-fold increased Mn content. Washout experiments showed time-dependent efflux with 80% Mn released after a 4 h chase period. Hepcidin reduced levels of Fpn in WIF-B cells, clearing Fpn from the cell surface, but Mn efflux was unaffected. The secretory inhibitor brefeldin A did block release of Mn from WIF-B cells, suggesting vesicle fusion may be involved in export. These results point to a possible role of ZnT10 to import Mn into vesicles that subsequently fuse with the apical membrane and empty their contents into bile.

Protein Transfer Into Human Cells by VSV-G-induced Nanovesicles

PubMed Central

Mangeot, Philippe-Emmanuel; Dollet, Sandra; Girard, Mathilde; Ciancia, Claire; Joly, Stéphane; Peschanski, Marc; Lotteau, Vincent

2011-01-01

Identification of new techniques to express proteins into mammal cells is of particular interest for both research and medical purposes. The present study describes the use of engineered vesicles to deliver exogenous proteins into human cells. We show that overexpression of the spike glycoprotein of the vesicular stomatitis virus (VSV-G) in human cells induces the release of fusogenic vesicles named gesicles. Biochemical and functional studies revealed that gesicles incorporated proteins from producer cells and could deliver them to recipient cells. This protein-transduction method allows the direct transport of cytoplasmic, nuclear or surface proteins in target cells. This was demonstrated by showing that the TetR transactivator and the receptor for the murine leukemia virus (MLV) envelope [murine cationic amino acid transporter-1 (mCAT-1)] were efficiently delivered by gesicles in various cell types. We further shows that gesicle-mediated transfer of mCAT-1 confers to human fibroblasts a robust permissiveness to ecotropic vectors, allowing the generation of human-induced pluripotent stem cells in level 2 biosafety facilities. This highlights the great potential of mCAT-1 gesicles to increase the safety of experiments using retro/lentivectors. Besides this, gesicles is a versatile tool highly valuable for the nongenetic delivery of functions such as transcription factors or genome engineering agents. PMID:21750535

Energy output of a single outer hair cell: Effect of resonance

NASA Astrophysics Data System (ADS)

Iwasa, Kuni H.

2018-05-01

The ability of the mammalian ear in processing high frequency sounds, up to ˜100 kHz, is based on the capability of outer hair cells (OHCs) in responding to stimulation at high frequencies. These cells show a unique motility in their cell body coupled with charge movement. With this motile element, voltage changes generated by stimuli at their hair bundles drive the cell body and that, in turn, amplifies the signal. In vitro experiments show that the movement of these charges significantly increases the membrane capacitance, limiting the motile activity by an additional attenuation of voltage changes. It was found, however, that such an effect is due to the absence of mechanical load. In the presence of mechanical load, particularly inertial load, such as under in vivo conditions, the movement of motile charges should reduce the membrane capacitance, enhancing the mechanical power output.

Synthesis and characterization of 2-substituted benzimidazoles and their evaluation as anticancer agent.

PubMed

Azam, Mohammad; Khan, Azmat Ali; Al-Resayes, Saud I; Islam, Mohammad Shahidul; Saxena, Ajit Kumar; Dwivedi, Sourabh; Musarrat, Javed; Trzesowska-Kruszynska, Agata; Kruszynski, Rafal

2015-05-05

In this work, we report a series of benzimidazole derivatives synthesized from benzene-1,2-diamine and aryl-aldehydes at room temperature. The synthesized compounds have been characterized on the basis of elemental analysis and various spectroscopic studies viz., IR, (1)H- and (13)C-NMR, ESI-MS as well by X-ray single X-ray crystallographic study. Interaction of these compounds with CT-DNA has been examined with fluorescence experiments and showed significant binding ability. All the synthesized compounds have been screened for their antitumor activities against various human cancer cell lines viz., Human breast adenocarcinoma cell line (MCF-7), Human leukemia cell line (THP-1), Human prostate cancer cell lines (PC-3) and adenocarcinomic human alveolar basal epithelial cell lines (A-549). Interestingly, all the compounds showed significant anticancer activity. Copyright © 2015 Elsevier B.V. All rights reserved.

Nickel-Hydrogen Cell Testing Experience, NASA/Goddard Space Flight Center

NASA Technical Reports Server (NTRS)

Rao, Gopalakrishna M.

1999-01-01

The objectives of the project were to test the Nickel-Hydrogen Cell to: (1) verify the Aerospace Cell Flight Worthiness, (2) Elucidate the Aerospace Cell Thermal Behavior, (3) Develop the Aerospace Battery Assembly Design(s) and In-orbit Battery Management plan(s) and (4) Understand the Aerospace Cell Failure Mechanism. The tests included the LEO and GEO Life cycle tests, Calorimetric Analysis, Destructive Physical analysis, and special tests. Charts show the Mission Profile Cycling Data, Stress Cycling Data. The test data complies with the mission requirements, validating the flight worthiness of batteries. The nominal stress and mission profile cycling performance test shows the charge voltage as high as 1.60V and recharge ratio greater than 1.05. It is apparent that the electrochemical signatures alone do not provide conclusive proof for Nickel precharge. The researchers recommend a gas and positive plate analyses for further confirmation.

Pharbinilic acid, an allogibberic acid from morning glory (Pharbitis nil).

PubMed

Kim, Ki Hyun; Choi, Sang Un; Son, Mi Won; Choi, Sang Zin; Clardy, Jon; Lee, Kang Ro

2013-07-26

Pharbinilic acid (1), the first naturally occurring allogibberic acid, was isolated from ethanol extracts of morning glory (Pharbitis nil) seeds. Its absolute configuration was determined by NOESY NMR and ECD experiments. Compound 1 showed weak cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cells and weakly inhibited nitric oxide production in lipopolysaccharide-activated BV-2 microglia cells.

Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

PubMed

Jarukanont, Daungruthai; Bonifas Arredondo, Imelda; Femat, Ricardo; Garcia, Martin E

2015-01-01

Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that including the fusion-time statistics in our model does not produce any significant changes on the results. These findings indicate that the motion of the whole ensemble of vesicles towards the membrane is directed and reflected in the amperometric signals. Our results confirm the conclusions of previous imaging studies performed on single vesicles that vesicles' motion underneath plasma membranes is not purely random, but biased towards the membrane.

Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements

PubMed Central

Jarukanont, Daungruthai; Bonifas Arredondo, Imelda; Femat, Ricardo; Garcia, Martin E.

2015-01-01

Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles’ arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that including the fusion-time statistics in our model does not produce any significant changes on the results. These findings indicate that the motion of the whole ensemble of vesicles towards the membrane is directed and reflected in the amperometric signals. Our results confirm the conclusions of previous imaging studies performed on single vesicles that vesicles’ motion underneath plasma membranes is not purely random, but biased towards the membrane. PMID:26675312

Collective Signal Processing in Cluster Chemotaxis: Roles of Adaptation, Amplification, and Co-attraction in Collective Guidance

PubMed Central

Camley, Brian A.; Zimmermann, Juliane; Levine, Herbert; Rappel, Wouter-Jan

2016-01-01

Single eukaryotic cells commonly sense and follow chemical gradients, performing chemotaxis. Recent experiments and theories, however, show that even when single cells do not chemotax, clusters of cells may, if their interactions are regulated by the chemoattractant. We study this general mechanism of “collective guidance” computationally with models that integrate stochastic dynamics for individual cells with biochemical reactions within the cells, and diffusion of chemical signals between the cells. We show that if clusters of cells use the well-known local excitation, global inhibition (LEGI) mechanism to sense chemoattractant gradients, the speed of the cell cluster becomes non-monotonic in the cluster’s size—clusters either larger or smaller than an optimal size will have lower speed. We argue that the cell cluster speed is a crucial readout of how the cluster processes chemotactic signals; both amplification and adaptation will alter the behavior of cluster speed as a function of size. We also show that, contrary to the assumptions of earlier theories, collective guidance does not require persistent cell-cell contacts and strong short range adhesion. If cell-cell adhesion is absent, and the cluster cohesion is instead provided by a co-attraction mechanism, e.g. chemotaxis toward a secreted molecule, collective guidance may still function. However, new behaviors, such as cluster rotation, may also appear in this case. Co-attraction and adaptation allow for collective guidance that is robust to varying chemoattractant concentrations while not requiring strong cell-cell adhesion. PMID:27367541

Blue light inhibits proliferation of melanoma cells

NASA Astrophysics Data System (ADS)

Becker, Anja; Distler, Elisabeth; Klapczynski, Anna; Arpino, Fabiola; Kuch, Natalia; Simon-Keller, Katja; Sticht, Carsten; van Abeelen, Frank A.; Gretz, Norbert; Oversluizen, Gerrit

2016-03-01

Photobiomodulation with blue light is used for several treatment paradigms such as neonatal jaundice, psoriasis and back pain. However, little is known about possible side effects concerning melanoma cells in the skin. The aim of this study was to assess the safety of blue LED irradiation with respect to proliferation of melanoma cells. For that purpose we used the human malignant melanoma cell line SK-MEL28. Cell proliferation was decreased in blue light irradiated cells where the effect size depended on light irradiation dosage. Furthermore, with a repeated irradiation of the melanoma cells on two consecutive days the effect could be intensified. Fluorescence-activated cell sorting with Annexin V and Propidium iodide labeling did not show a higher number of dead cells after blue light irradiation compared to non-irradiated cells. Gene expression analysis revealed down-regulated genes in pathways connected to anti-inflammatory response, like B cell signaling and phagosome. Most prominent pathways with up-regulation of genes were cytochrome P450, steroid hormone biosynthesis. Furthermore, even though cells showed a decrease in proliferation, genes connected to the cell cycle were up-regulated after 24h. This result is concordant with XTT test 48h after irradiation, where irradiated cells showed the same proliferation as the no light negative control. In summary, proliferation of melanoma cells can be decreased using blue light irradiation. Nevertheless, the gene expression analysis has to be further evaluated and more studies, such as in-vivo experiments, are warranted to further assess the safety of blue light treatment.

Interactions between macrophage/Kupffer cells and hepatocytes in surgical sepsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

West, M.A.

Experiments were performed to investigate the role of Kupffer cell/macrophage interactions with hepatocytes in modulating liver function during infections using direct in vitro cocultivation of rat macrophages or Kupffer cells with rat hepatocytes. Protein synthesis was assayed as a sensitive indicator of integrated hepatocellular function by measuring {sup 3}H-leucine incorporation into hepatocyte protein. Septic stimuli such as lipoploysaccharide and killed bacteria were added to cocultures of hepatocytes and macrophages or Kupffer cells and the responses compared to hepatocytes alone. Information about the types of proteins synthesized by hepatocytes under various culture conditions was determined using polyacrylamide gel electrophoresis and autoradiography.more » These experiments showed that septic stimuli alter the amount and type of protein synthesized by hepatocytes and had no direct effect on hepatocytes in the absence of macrophages or Kupffer cells. The mediator(s) appears to be a heat labile, soluble monokine(s) which is distinct from interleukin-1 or tumor necrosis factor. The important role of Kupffer cells/macrophages in mediating alterations in hepatocellular function in sepsis may ultimately improve patient care.« less

Automated analysis of time-lapse fluorescence microscopy images: from live cell images to intracellular foci.

PubMed

Dzyubachyk, Oleh; Essers, Jeroen; van Cappellen, Wiggert A; Baldeyron, Céline; Inagaki, Akiko; Niessen, Wiro J; Meijering, Erik

2010-10-01

Complete, accurate and reproducible analysis of intracellular foci from fluorescence microscopy image sequences of live cells requires full automation of all processing steps involved: cell segmentation and tracking followed by foci segmentation and pattern analysis. Integrated systems for this purpose are lacking. Extending our previous work in cell segmentation and tracking, we developed a new system for performing fully automated analysis of fluorescent foci in single cells. The system was validated by applying it to two common tasks: intracellular foci counting (in DNA damage repair experiments) and cell-phase identification based on foci pattern analysis (in DNA replication experiments). Experimental results show that the system performs comparably to expert human observers. Thus, it may replace tedious manual analyses for the considered tasks, and enables high-content screening. The described system was implemented in MATLAB (The MathWorks, Inc., USA) and compiled to run within the MATLAB environment. The routines together with four sample datasets are available at http://celmia.bigr.nl/. The software is planned for public release, free of charge for non-commercial use, after publication of this article.

Spacelab 1 hematology experiment (INS103): Influence of space flight on erythrokinetics in man

NASA Technical Reports Server (NTRS)

Leach, C. S.; Chen, J. P.; Crosby, W.; Dunn, C. D. R.; Johnson, P. C.; Lange, R. D.; Larkin, E.; Tavassoli, M.

1985-01-01

An experiment conducted on the 10-day Spacelab 1 mission aboard the ninth Space Shuttle flight in November to December 1983 was designed to measure factors involved in the control of erythrocyte turnover that might be altered during weightlessness. Blood samples were collected before, during, and after the flight. Immediately after landing, red cell mass showed a mean decrease of 9.3 percent in the four astronauts. Neither hyperoxia nor an increase in blood phosphate was a cause of the decrease. Red cell survival time and iron incorporation postflight were not significantly different from their preflight levels. Serum haptoglobin did not decrease, indicating that intravascular hemolysis was not a major cause of red cell mass change. An increase in serum ferritin after the second day of flight may have been caused by red cell breakdown early in flight. Erythropoietin levels decreased during and after flight, but preflight levels were high and the decrease was not significant. The space flight-induced decrease in red cell mass may result from a failure of erythropoiesis to replace cells destroyed by the spleen soon after weightlessness is attained.

Dynamic Nuclear Polarization NMR in Human Cells Using Fluorescent Polarizing Agents.

PubMed

Albert, Brice J; Gao, Chukun; Sesti, Erika L; Saliba, Edward P; Alaniva, Nicholas; Scott, Faith J; Sigurdsson, Snorri Th; Barnes, Alexander B

2018-06-20

Solid-state nuclear magnetic resonance (NMR) enables atomic resolution characterization of molecular structure and dynamics within complex heterogeneous samples, but it is typically insensitive. Dynamic nuclear polarization (DNP) increases NMR signal intensity by orders of magnitude and can be performed in combination with magic angle spinning (MAS) for sensitive, high-resolution spectroscopy. Here we report MAS DNP experiments, for the first time, within intact human cells with >40-fold DNP enhancement and a sample temperature below 6 K. In addition to cryogenic MAS results below 6 K, we also show in-cell DNP enhancements of 57-fold at 90 K. In-cell DNP is demonstrated using biradicals and sterically-shielded monoradicals as polarizing agents. A novel trimodal polarizing agent is introduced for DNP, which contains a nitroxide biradical, a targeting peptide for cell penetration, and a fluorophore for subcellular localization with confocal microscopy. The fluorescent polarizing agent provides in-cell DNP enhancements of 63-fold at a concentration of 2.7 mM. These experiments pave the way for structural characterization of biomolecules in an endogenous cellular context.

A bacteria antibiotic system in space (23-F ANTIBIO)

NASA Technical Reports Server (NTRS)

Tixador, Rene; Gasset, G.; Eche, B.; Moatti, N.; Lapchine, L.; Woldringh, C.; Toorop, P.; Moatti, J. P.; Delmotte, F.; Tap, G.

1995-01-01

In order to evaluate the effects of weightlessness and cosmic radiations on the bacteria resistance to antibiotics, the Antibio 23F experiment was undertaken onboard Discovery during the 1st International Microgravity Laboratory (IML-1) mission. The effects of various antibiotic concentrations (dihydrostreptomycin) on Escherichia coli growth and cell division behavior were studied. The antibiotic binding was investigated using a radioactive tracer (tritium). The results showed that microgravity did not affect E. coli cells in regards the growth and the cell division. The antibiotic added to the culture medium induced an inhibition of the cultures both in the flight and ground controls. However, the antibiotic was less efficient in flight. The behavior of bacteria was modified, and the exponential growth rate was increased in flight. The incorporation of radioactive antibiotics in flight was comparatively different to ground incorporation, which indicated some perturbations in antibiotic binding. The experiments performed in the 1 g centrifuge did not show any difference in the cultures developed on the static rack, and could support a radiative effect of cosmic radiation to explain the results.

Identification of HECT E3 ubiquitin ligase family genes involved in stem cell regulation and regeneration in planarians.

PubMed

Henderson, Jordana M; Nisperos, Sean V; Weeks, Joi; Ghulam, Mahjoobah; Marín, Ignacio; Zayas, Ricardo M

2015-08-15

E3 ubiquitin ligases constitute a large family of enzymes that modify specific proteins by covalently attaching ubiquitin polypeptides. This post-translational modification can serve to regulate protein function or longevity. In spite of their importance in cell physiology, the biological roles of most ubiquitin ligases remain poorly understood. Here, we analyzed the function of the HECT domain family of E3 ubiquitin ligases in stem cell biology and tissue regeneration in planarians. Using bioinformatic searches, we identified 17 HECT E3 genes that are expressed in the Schmidtea mediterranea genome. Whole-mount in situ hybridization experiments showed that HECT genes were expressed in diverse tissues and most were expressed in the stem cell population (neoblasts) or in their progeny. To investigate the function of all HECT E3 ligases, we inhibited their expression using RNA interference (RNAi) and determined that orthologs of huwe1, wwp1, and trip12 had roles in tissue regeneration. We show that huwe1 RNAi knockdown led to a significant expansion of the neoblast population and death by lysis. Further, our experiments showed that wwp1 was necessary for both neoblast and intestinal tissue homeostasis as well as uncovered an unexpected role of trip12 in posterior tissue specification. Taken together, our data provide insights into the roles of HECT E3 ligases in tissue regeneration and demonstrate that planarians will be a useful model to evaluate the functions of E3 ubiquitin ligases in stem cell regulation. Copyright © 2015 Elsevier Inc. All rights reserved.

Increased cellular levels of spermidine or spermine are required for optimal DNA synthesis in lymphocytes activated by concanavalin A.

PubMed Central

Fillingame, R H; Jorstad, C M; Morris, D R

1975-01-01

There are large increases in cellular levels of the polyamines spermidine and spermine in lymphocytes induced to transform by concanavalin A. The anti-leukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) blocks synthesis of these polyamines by inhibiting S-adenosylmethionine decarboxylase. Previous results showed that when cells are activated in the presence of MGBG the synthesis and processing of RNA, as well as protein synthesis, proceed as in the absence of the drug. In contrast, the incorporation of [methyl-3H]thymidine into DNA and the rate of entry of the cells into mitosis are inhibited by 60% in the presence of MGBG. Several experiments suggest that MGBG inhibits cell proliferation by directly blocking polyamine synthesis and not by an unrelated pharmacological effect: (1) the inhibitory action of MGBG is reversed by exogenously added spermidine or spermine; (2) inhibition of DNA synthesis by MGBG shows the same dose-response curve as does inhibition of spermidine and spermine synthesis; and (3) if MGBG is added to cells which have been allowed to accumulate their maximum complement of polyamines, there is no inhibition of thymidine incorporation. MGBG-treated and control cultures initiate DNA synthesis at the same time and show the same percentage of labeled cells by autoradiography. Therefore, it appears that in the absence of increased cellular levels of polyamines, lymphocytes progress normally from G0 through G1 and into S-phase. Furthermore, these experiments suggest that the increased levels of spermidine and spermine generally seen in rapidly proliferating eukaryotic systems are necessary for enhanced rates of DNA replication. PMID:1060087

Cellular Metabolic Activity and the Oxygen and Hydrogen Stable Isotope Composition of Intracellular Water and Metabolites

NASA Astrophysics Data System (ADS)

Kreuzer-Martin, H. W.; Hegg, E. L.

2008-12-01

Intracellular water is an important pool of oxygen and hydrogen atoms for biosynthesis. Intracellular water is usually assumed to be isotopically identical to extracellular water, but an unexpected experimental result caused us to question this assumption. Heme O isolated from Escherichia coli cells grown in 95% H218O contained only a fraction of the theoretical value of labeled oxygen at a position where the O atom was known to be derived from water. In fact, fewer than half of the oxygen atoms were labeled. In an effort to explain this surprising result, we developed a method to determine the isotope ratios of intracellular water in cultured cells. The results of our experiments showed that during active growth, up to 70% of the oxygen atoms and 50% of the hydrogen atoms in the intracellular water of E. coli are generated during metabolism and can be isotopically distinct from extracellular water. The fraction of isotopically distinct atoms was substantially less in stationary phase and chilled cells, consistent with our hypothesis that less metabolically-generated water would be present in cells with lower metabolic activity. Our results were consistent with and explained the result of the heme O labeling experiment. Only about 40% of the O atoms on the heme O molecule were labeled because, presumably, only about 40% of the water inside the cells was 18O water that had diffused in from the culture medium. The rest of the intracellular water contained 16O atoms derived from either nutrients or atmospheric oxygen. To test whether we could also detect metabolically-derived hydrogen atoms in cellular constituents, we isolated fatty acids from log-phase and stationary phase E. coli and determined the H isotope ratios of individual fatty acids. The results of these experiments showed that environmental water contributed more H atoms to fatty acids isolated in stationary phase than to the same fatty acids isolated from log-phase cells. Stable isotope analyses of biomass of Bacillus subtilis, a Gram-positive bacterium, showed the same pattern. Rapidly-dividing cells derived fewer of their O and H atoms from environmental water than did more slowly-growing cells and spores. To test whether a eukaryotic cell, surrounded by only a membrane, would also maintain an isotopic gradient and a detectable percentage of metabolic water, we applied our approach to cultured rat fibroblasts. Preliminary results showed that approximately 50% of the O and H atoms in exponentially growing cells were derived from metabolic activity. In quiescent cells, metabolic activity generated approximately 25% of the O and H atoms in intracellular water. Thus far, the data we have obtained is consistent with the following model: (1) Intracellular water is composed of water that diffuses in from the extracellular environment and water that is created as a result of metabolic activity. (2) The relative amounts of environmental and metabolic water inside a cell are a function of the cell's metabolic activity. (3) The oxygen and hydrogen isotope ratios of cellular metabolites are a function of those of intracellular water, and therefore reflect the metabolic activity of the cell at the time of biosynthesis.

Drug adsorption to plastic containers and retention of drugs in cultured cells under in vitro conditions.

PubMed

Palmgrén, Joni J; Mönkkönen, Jukka; Korjamo, Timo; Hassinen, Anssi; Auriola, Seppo

2006-11-01

Loss of drug content during cell culture transport experiment can lead to misinterpretations in permeability analysis. This study analyses drug adsorption to various plastic containers and drug retention in cultured cells under in vitro conditions. The loss of various drugs to polystyrene tubes and well plates was compared to polypropylene and glass tubes both in deionised water and buffer solution. In cellular uptake experiments, administered drugs were obtained from cultured cells by liquid extraction. Samples were collected at various time points and drug concentrations were measured by a new HPLC-MS/MS method. Acidic drugs (hydrochlorothiazide, naproxen, probenicid, and indomethacin) showed little if any sorption to all tested materials in either water or buffer. In the case of basic drugs, substantial loss to polystyrene tubes and well plates was observed. After 4.5 h, the relative amount remaining in aqueous test solution stored in polystyrene tubes was 64.7 +/- 6.8%, 38.4 +/- 9.1%, 31.9 +/- 6.7%, and 23.5 +/- 6.1% for metoprolol, medetomidine, propranolol, and midazolam, respectively. Interestingly, there was no significant loss of drugs dissolved in buffer to any of the tested materials indicating that buffer reduced surficial interaction. The effect of drug concentration to sorption was also tested. Results indicated that the higher the concentration in the test solution the lower the proportional drug loss, suggesting that the polystyrene contained a limited amount of binding sites. Cellular uptake studies showed considerable retention of drugs in cultured cells. The amounts of absorbed drugs in cellular structures were 0.45%, 4.88%, 13.15%, 43.80%, 23.57% and 11.22% for atenolol, metoprolol, medetomidine, propranolol, midazolam, and diazepam, respectively. Overall, these findings will benefit development and validation of further in vitro drug permeation experiments.

Nuclear Synthesis of Cytoplasmic Ribonucleic Acid in Amoeba proteus

PubMed Central

Prescott, David M.

1959-01-01

The enucleation technique has been applied to Amoeba proteus by several laboratories in attempts to determine whether the cytoplasm is capable of nucleus-independent ribonucleic acid synthesis. This cell is very convenient for micrurgy, but its use requires a thorough starvation period to eliminate the possibility of metabolic influence by food vacuoles and frequent washings and medium renewal to maintain asepsis. In the experiments described here, amoebae were starved for periods of 24 to 96 hours, cut into nucleated and enucleated halves, and exposed to either C-14 uracil, C-14 adenine, C-14 orotic acid, or a mixture of all three. When the starvation period was short (less than 72 hours), organisms (especially yeast cells) contained within amoeba food vacuoles frequently showed RNA synthesis in both nucleated and enucleated amoebae. When the preperiod of starvation was longer than 72 hours, food vacuole influence was apparently negligible, and a more meaningful comparison between enucleated and nucleated amoebae was possible. Nucleated cells incorporated all three precursors into RNA; enucleated cells were incapable of such incorporation. The experiments indicate a complete dependence on the nucleus for RNA synthesis. The conflict with the experimental results of others on this problem could possibly stem from differences in culture conditions, starvation treatment, or experimental conditions. For an unequivocal answer in experiments of this design, ideally the cells should be capable of growth on an entirely synthetic medium under aseptic conditions. The use of a synthetic medium (experiments with A. proteus are done under starvation conditions) would permit, moreover, a more realistic comparison of metabolic capacities of nucleated and enucleated cells. PMID:14434750

Nuclear synthesis of cytoplasmic ribonucleic acid in Amoeba proteus.

PubMed

PRESCOTT, D M

1959-10-01

The enucleation technique has been applied to Amoeba proteus by several laboratories in attempts to determine whether the cytoplasm is capable of nucleus-independent ribonucleic acid synthesis. This cell is very convenient for micrurgy, but its use requires a thorough starvation period to eliminate the possibility of metabolic influence by food vacuoles and frequent washings and medium renewal to maintain asepsis. In the experiments described here, amoebae were starved for periods of 24 to 96 hours, cut into nucleated and enucleated halves, and exposed to either C-14 uracil, C-14 adenine, C-14 orotic acid, or a mixture of all three. When the starvation period was short (less than 72 hours), organisms (especially yeast cells) contained within amoeba food vacuoles frequently showed RNA synthesis in both nucleated and enucleated amoebae. When the preperiod of starvation was longer than 72 hours, food vacuole influence was apparently negligible, and a more meaningful comparison between enucleated and nucleated amoebae was possible. Nucleated cells incorporated all three precursors into RNA; enucleated cells were incapable of such incorporation. The experiments indicate a complete dependence on the nucleus for RNA synthesis. The conflict with the experimental results of others on this problem could possibly stem from differences in culture conditions, starvation treatment, or experimental conditions. For an unequivocal answer in experiments of this design, ideally the cells should be capable of growth on an entirely synthetic medium under aseptic conditions. The use of a synthetic medium (experiments with A. proteus are done under starvation conditions) would permit, moreover, a more realistic comparison of metabolic capacities of nucleated and enucleated cells.

Long non-coding RNA BANCR regulates cancer stem cell markers in papillary thyroid cancer via the RAF/MEK/ERK signaling pathway.

PubMed

Wang, Yuanyuan; Lin, Xiangde; Fu, Xinghao; Yan, Wei; Lin, Fusheng; Kuang, Penghao; Luo, Yezhe; Lin, Ende; Hong, Xiaoquan; Wu, Guoyang

2018-06-18

Thyroid cancer is one of the most common malignant tumors of the endocrine system. Among all thyroid cancers, papillary thyroid carcinoma (PTC) is the most common type. The BRAF-activated non-coding RNA (BANCR) is a 693-bp nucleotide transcript which was first identified in melanoma. However, the role of BANCR in the development of thyroid cancer remains unclear. Therefore, the present study investigated the potential involvement of BANCR in the development of thyroid cancer in vitro using patient tissue samples and a panel of thyroid cancer cell lines, and in vivo using a xenograft mouse model. We observed that BANCR was expressed at a higher level in human thyroid tumor tissues than that noted in the adjacent normal tissues. The expression level of BANCR differed between cultured thyroid cancer cell lines; BANCR expression was lower in the BCPAP cell line than that observed in the CAL-62, WRO and FTC-133 cell lines. Western blot analysis and flow cytometry revealed that overexpression of BANCR in the BCPAP cell line resulted in increased expression of the cancer stem cell markers, LGR5 and EpCAM. Single-clone formation experiments showed that upregulated expression of BANCR in the BCPAP cell line promoted an increase in the number of clones formed. Similarly, in microsphere formation experiments, overexpression of BANCR resulted in increased number and size of microspheres compared with the control cell line. Western blotting experiments showed that BANCR overexpression in BCPAP upregulated the expression of phosphorylated c-Raf, MEK1/2 and ERK1/2. Inhibition of c-Raf via U0126 decreased the expression of LGR5 and EpCAM, as well as phosphorylated levels of c-Raf, MEK1/2 and ERK1/2 in the BCPAP cells, compared to levels in the DMSO controls. In the xenograft mouse model, BANCR overexpression in the thyroid cancer cells significantly increased tumor growth. Taken together, these results suggest that BANCR plays a role in PTC development by regulating the expression of cancer stem cell markers LGR5 and EpCAM via the c-Raf/MEK/ERK signaling pathway. Therefore, BANCR may be used as a novel prognostic marker for PTC.

Development of an efficient Procedure for Resist Wall Space Experiment

NASA Astrophysics Data System (ADS)

Matsumoto, Shouhei; Kumasaki, Saori; Higuchi, Sayoko; Kirihata, Kuniaki; Inoue, Yasue; Fujie, Miho; Soga, Kouichi; Wakabayashi, Kazuyuki; Hoson, Takayuki

The Resist Wall space experiment aims to examine the role of the cortical microtubule-plasma membrane-cell wall continuum in plant resistance to the gravitational force, thereby clarifying the mechanism of gravity resistance. For this purpose, we will cultivate Arabidopsis mutants defective in organization of cortical microtubules (tua6 ) or synthesis of membrane sterols (hmg1 ) as well as the wild type under microgravity and 1 g conditions in the European Modular Cultivation System on the International Space Station up to reproductive stage, and compare phenotypes on growth and development. We will also analyze cell wall properties and gene expression levels using collected materials. However, the amounts of materials collected will be severely limited, and we should develop an efficient procedure for this space experiment. In the present study, we examined the possibility of analyzing various parameters successively using the identical material. On orbit, plant materials will be fixed with RNAlater solution, kept at 4° C for several days and then frozen in a freezer at -20° C. We first examined whether the cell wall extensibility of inflorescence stems can be measured after RNAlater fixation. The gradient of the cell wall extensibility along inflorescence stems was detected in RNAlater-fixed materials as in methanol-killed ones. The sufficient amounts of RNA to analyze the gene expression were also obtained from the materials after measurement of the cell wall extensibility. Furthermore, the levels and composition of cell wall polysaccharides could be measured using the materials after extraction of RNA. These results show that we can analyze the physical and chemical properties of the cell wall as well as gene expression using the identical material obtained in the space experiments.

Cellular immunity in vitro. Clonal proliferation of antigen-stimulated lymphocytes.

PubMed

Marshall, W H; Valentine, F T; Lawrence, H S

1969-08-01

When sensitive lymphocytes are cultured with the appropriate antigen, lymphoblasts appear after 24-48 hr of incubation and the number of these increases steadily from the 2nd to the 6th or 7th day. Our problem was to discover, at a cellular level, how this increase takes place; whether it is a massive response of many cells, stepwise recruitment of cells into the lymphoblast class, or simply repeated division of a few cells to form clones. In these experiments lymphocytes were incubated with antigen in culture tubes for 2-4 days and then a few cells, usually less than 200, were transferred to special microchambers for further culture. In these microchambers the cells could be viewed continually with a microscope and their fate recorded over the next 3-5 days by time-lapse cinemicrography. Examination of the film produced in this way showed that lymphoblasts divided and redivided to produce clones of 64 cells or more. It was possible to measure generation times from the film for 301 cells; the majority were between 8 and 13 hr but the range was 7.5-38.0 hr. There was no clear difference between generation times of human lymphocytes stimulated with tuberculin, streptokinase-streptodrnase, extract of the American pokeweed, or in the mixed leukocyte reaction. Similar times were also found for rat cells in the mixed leukocyte reaction. While these observations show that clonal proliferation does occur and could reasonably account for all the increase of lymphoblasts in lymphocyte cultures, the experiments, because of their design, do not exclude the possibility that other mechanisms such as recruitment may play a role as well, particularly during the first 48 hr after contact between sensitive cells and antigens.

Influence on cell death of high frequency motion of magnetic nanoparticles during magnetic hyperthermia experiments

NASA Astrophysics Data System (ADS)

Hallali, N.; Clerc, P.; Fourmy, D.; Gigoux, V.; Carrey, J.

2016-07-01

Studies with transplanted tumors in animals and clinical trials have provided the proof-of-concept of magnetic hyperthermia (MH) therapy of cancers using iron oxide nanoparticles. Interestingly, in several studies, the application of an alternating magnetic field (AMF) to tumor cells having internalized and accumulated magnetic nanoparticles (MNPs) into their lysosomes can induce cell death without detectable temperature increase. To explain these results, among other hypotheses, it was proposed that cell death could be due to the high-frequency translational motion of MNPs under the influence of the AMF gradient generated involuntarily by most inductors. Such mechanical actions of MNPs might cause cellular damages and participate in the induction of cell death under MH conditions. To test this hypothesis, we developed a setup maximizing this effect. It is composed of an anti-Helmholtz coil and two permanent magnets, which produce an AMF gradient and a superimposed static MF. We have measured the MNP heating power and treated tumor cells by a standard AMF and by an AMF gradient, on which was added or not a static magnetic field. We showed that the presence of a static magnetic field prevents MNP heating and cell death in standard MH conditions. The heating power of MNPs in an AMF gradient is weak, position-dependent, and related to the presence of a non-zero AMF. Under an AMF gradient and a static field, no MNP heating and cell death were measured. Consequently, the hypothesis that translational motions could be involved in cell death during MH experiments is ruled out by our experiments.

Calreticulin is expressed on the cell surface of activated human peripheral blood T lymphocytes in association with major histocompatibility complex class I molecules.

PubMed

Arosa, F A; de Jesus, O; Porto, G; Carmo, A M; de Sousa, M

1999-06-11

Calreticulin is an endoplasmic reticulum resident molecule known to be involved in the folding and assembly of major histocompatibility complex (MHC) class I molecules. In the present study, expression of calreticulin was analyzed in human peripheral blood T lymphocytes. Pulse-chase experiments in [35S]methionine-labeled T cell blasts showed that calreticulin was associated with several proteins in the endoplasmic reticulum and suggested that it was expressed at the cell surface. Indeed, the 60-kDa calreticulin was labeled by cell surface biotinylation and precipitated from the surface of activated T cells together with a protein with an apparent molecular mass of 46 kDa. Cell surface expression of calreticulin by activated T lymphocytes was further confirmed by immunofluorescence and flow cytometry, studies that showed that both CD8+ and CD4+ T cells expressed calreticulin in the plasma membrane. Low amounts of cell surface calreticulin were detected in resting T lymphocytes. By sequential immunoprecipitation using the conformation independent monoclonal antibody HC-10, we provided evidence that the cell surface 46-kDa protein co-precipitated with calreticulin is unfolded MHC I. These results show for the first time that after T cell activation, significant amounts of calreticulin are expressed on the T cell surface, where they are found in physical association with a pool of beta2-free MHC class I molecules.

PEG-detachable lipid-polymer hybrid nanoparticle for delivery of chemotherapy drugs to cancer cells.

PubMed

Du, Jiang-bo; Song, Yan-feng; Ye, Wei-liang; Cheng, Ying; Cui, Han; Liu, Dao-zhou; Liu, Miao; Zhang, Bang-le; Zhou, Si-yuan

2014-08-01

The experiment aimed to increase the drug-delivery efficiency of poly-lactic-co-glycolic acid (PLGA) nanoparticles. Lipid-polymer hybrid nanoparticles (LPNs-1) were prepared using PLGA as a hydrophobic core and FA-PEG-hyd-DSPE as an amphiphilic shell. Uniform and spherical nanoparticles with an average size of 185 nm were obtained using the emulsification solvent evaporation method. The results indicated that LPNs-1 showed higher drug loading compared with naked PLGA nanoparticles (NNPs). Drug release from LPNs-1 was faster in an acidic environment than in a neutral environment. LPNs-1 showed higher cytotoxicity on KB cells, A549 cells, MDA-MB-231 cells, and MDA-MB-231/ADR cells compared with free doxorubicin (DOX) and NNPs. The results also showed that, compared with free DOX and NNPs, LPNs-1 delivered more DOX to the nuclear of KB cells and MDA-MB-231/ADR cells. LPNs-1 induced apoptosis in KB cells and MDA-MB-231/ADR cells in a dose-dependent manner. The above data indicated that DOX-loaded LPNs-1 could kill not only normal tumor cells but also drug-resistant tumor cells. These results indicated that modification of PLGA nanoparticles with FA-PEG-hyd-DSPE could considerably increase the drug-delivery efficiency and LPNs-1 had potential in the delivery of chemotherapeutic agents in the treatment of cancer.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Essig, Stephanie; Geisz, John F.; Steiner, Myles A.

Dual-junction solar cells consisting of rear-heterojunction GaInP top cells and back-junction, back-contacted crystalline Si bottom cells were fabricated and characterized. Our calculations show that theoretical efficiencies up to 38.9% can be achieved with Si-based tandem devices. In our experiments, the two subcells were fabricated separately and stacked with an index matching fluid. In contrast to conventional mechanically stacked solar cells, that contain two metal grids at the interface, our concept includes a fully back contacted bottom cell which reduces the shadow losses in the device. A 1-sun AM1.5g cumulative efficiency of (26.2 +/- 0.6)% has been achieved with this novelmore » GaInP/Si 4-terminal tandem solar cell.« less

Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells.

PubMed

Ganguly, Abira; Bhattacharjee, Chumki; Bhave, Madhura; Kailaje, Vaishali; Jain, Bhawik K; Sengupta, Isha; Rangarajan, Annapoorni; Bhattacharyya, Dibyendu

2016-03-01

Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation. © 2016 Federation of European Biochemical Societies.

Follow-the-leader cell migration requires biased cell-cell contact and local microenvironmental signals

NASA Astrophysics Data System (ADS)

Wynn, Michelle L.; Rupp, Paul; Trainor, Paul A.; Schnell, Santiago; Kulesa, Paul M.

2013-06-01

Directed cell migration often involves at least two types of cell motility that include multicellular streaming and chain migration. However, what is unclear is how cell contact dynamics and the distinct microenvironments through which cells travel influence the selection of one migratory mode or the other. The embryonic and highly invasive neural crest (NC) are an excellent model system to study this question since NC cells have been observed in vivo to display both of these types of cell motility. Here, we present data from tissue transplantation experiments in chick and in silico modeling that test our hypothesis that cell contact dynamics with each other and the microenvironment promote and sustain either multicellular stream or chain migration. We show that when premigratory cranial NC cells (at the pre-otic level) are transplanted into a more caudal region in the head (at the post-otic level), cells alter their characteristic stream behavior and migrate in chains. Similarly, post-otic NC cells migrate in streams after transplantation into the pre-otic hindbrain, suggesting that local microenvironmental signals dictate the mode of NC cell migration. Simulations of an agent-based model (ABM) that integrates the NC cell behavioral data predict that chain migration critically depends on the interplay of biased cell-cell contact and local microenvironment signals. Together, this integrated modeling and experimental approach suggests new experiments and offers a powerful tool to examine mechanisms that underlie complex cell migration patterns.

The Determination of the pH of Standard Buffer Solution: A Laboratory Experiment.

ERIC Educational Resources Information Center

Harris, K. R.

1985-01-01

Describes an experiment which shows: (1) how measurements of the reaction electromotive force for the cell (Pt/glass/NaCl(aq,m),buffer/AgCl/Ag/Pt) can be utilized in determining the absolute pH of the buffer; and (2) the demonstration of the use of the Debye-Huckel model of an electrolyte solution in solving an important electrochemical problem.…

Timation 3 satellite. [artificial satellite for navigation, space radiation, and time transfer applications

NASA Technical Reports Server (NTRS)

Bartholomew, C. A.

1972-01-01

The characteristics of the Timation 3 satellite are discussed. A diagram of the basic structure is provide to show the solar panels, navigation and telemetry antennas, gravity gradient booms, and solar cell experiments. The specific application of the satellite for time management or time transfer for navigation purposes is reported. Various measurements and experiments conducted by the satellite are described.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish.

PubMed

Moore, Finola E; Garcia, Elaine G; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C; Garcia, Amaris J; Mylvaganam, Ravi; Yoder, Jeffrey A; Blackburn, Jessica S; Sadreyev, Ruslan I; Ceol, Craig J; North, Trista E; Langenau, David M

2016-05-30

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4(+) cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2(E450fs) mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4(+) cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2(E450fs) mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4(+)/CD8(+) cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. © 2016 Moore et al.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish

PubMed Central

Garcia, Elaine G.; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C.; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C.; Garcia, Amaris J.; Mylvaganam, Ravi; Yoder, Jeffrey A.; Blackburn, Jessica S.; Sadreyev, Ruslan I.; Ceol, Craig J.; North, Trista E.

2016-01-01

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4+ cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2E450fs mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4+ cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2E450fs mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4+/CD8+ cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb. In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. PMID:27139488

[The cultivation and identification of lacrimal gland adenoid cystic cancer stem cells].

PubMed

Lyu, Jianmei; He, Yanjin; Xie, Lianfeng; Liu, Xun; Zhu, Limin

2015-10-01

To isolate and cultivate the Lacrimal gland Adenoid Cystic Carcinoma cells line, study Cancer Stem Cells properties. Experimental study. Lacrimal gland adenoid cystic carcinoma cancer stem cells were cultivated in serum-free suspension culture and the morphological changes were observed. Cells were divided into two groups, the LACC-CSC experimental group and the LACC control group. The flow cytometry instrument was used to detect the expression of classical stem cell markers CD133 and ABCG2. Transwell chamber was used to detect the cancer stem cell aggressivity and differentiated into the vascular endothelial cells. The tumorigenic force in vitro xenotransplantation were applied. LACC cells can grow suspensively after vaccinated in serum free medium and form tumor microspheres after 10-12 days. Flow cytometry experiments showed that the expression ratio of stem cell markers CD133 in LACC-CSC was (35.67 ± 6.86)%, significantly different to LACC with (0.46 ± 0.48)%, (t = 8.867, P < 0.05). Similarly, the expression ratio of stem cell marker ABCG2 in LACC-CSC was (39.99 ± 4.54)%, significantly different to LACC with (6.75 ± 1.34)%, (t = -9.932, P < 0.05). In vitro experiment of Matrigel invasion, LACC-CSC went through the matrigel basement membrane averagely (32.60 ± 8.79)/HP contrary to LACC with average (10.20 ± 2.77)/HP after 24 hours, showing statistically significance (t = 5.433, P < 0.05) between the two groups. After training for 48 hours, the difference between two groups was still obvious (t = 5.779, P < 0.05) with LACC-CSC average (62.60 ± 4.83)/HP to LACC (44.00 ± 5.34)/HP. When induced by serum medium containing VEGF and bFGF, LACC-CSC grew adherent gradually and cell morphological changes occurred after continuous induction to long spindle cells. When cultured into three-dimensional matrix structure they formed vessel samples and expressed vascular endothelial marker CD31 and CD34. Transplanted tumor in vitro experiment, mice of LACC-CSC group grew tumors in 9 days with 100% tumorigenic rate, whereas LACC group 12 days with 100% tumorigenic rate. LACC-CSC can be obtained through serum-free culture method. LACC-CSC grew suspensively and expressed classical stem cell markers. LACC-CSC were identified as cancer stem cells with stronger migration and invasion. LACC-CSC have tumorigenic force and multi-directional differentiation potential with general characteristics of the stem cell.

The application of digital image plane holography technology to identify Chinese herbal medicine

NASA Astrophysics Data System (ADS)

Wang, Huaying; Guo, Zhongjia; Liao, Wei; Zhang, Zhihui

2012-03-01

In this paper, the imaging technology of digital image plane holography to identify the Chinese herbal medicine is studied. The optical experiment system of digital image plane holography which is the special case of pre-magnification digital holography was built. In the record system, one is an object light by using plane waves which illuminates the object, and the other one is recording hologram by using spherical light wave as reference light. There is a Micro objective lens behind the object. The second phase factor which caus ed by the Micro objective lens can be eliminated by choosing the proper position of the reference point source when digital image plane holography is recorded by spherical light. In this experiment, we use the Lygodium cells and Onion cells as the object. The experiment results with Lygodium cells and Onion cells show that digital image plane holography avoid the process of finding recording distance by using auto-focusing approach, and the phase information of the object can be reconstructed more accurately. The digital image plane holography is applied to the microscopic imaging of cells more effectively, and it is suit to apply for the identify of Chinese Herbal Medicine. And it promotes the application of digital holographic in practice.

Substrate stiffness-modulated registry phase correlations in cardiomyocytes map structural order to coherent beating

NASA Astrophysics Data System (ADS)

Dasbiswas, K.; Majkut, S.; Discher, D. E.; Safran, Samuel A.

2015-01-01

Recent experiments show that both striation, an indication of the structural registry in muscle fibres, as well as the contractile strains produced by beating cardiac muscle cells can be optimized by substrate stiffness. Here we show theoretically how the substrate rigidity dependence of the registry data can be mapped onto that of the strain measurements. We express the elasticity-mediated structural registry as a phase-order parameter using a statistical physics approach that takes the noise and disorder inherent in biological systems into account. By assuming that structurally registered myofibrils also tend to beat in phase, we explain the observed dependence of both striation and strain measurements of cardiomyocytes on substrate stiffness in a unified manner. The agreement of our ideas with experiment suggests that the correlated beating of heart cells may be limited by the structural order of the myofibrils, which in turn is regulated by their elastic environment.

Characterization of two new plasmid DNAs found in mitochondria of wild-type Neurospora intermedia strains.

PubMed Central

Stohl, L L; Collins, R A; Cole, M D; Lambowitz, A M

1982-01-01

Mitochondria from two Neurospora intermedia strains (P4O5-Labelle and Fiji N6-6) were found to contain plasmid DNAs in addition to the standard mitochondrial DNA species. The plasmid DNAs consist of monomeric circles (4.1-4.3 kbp and 5.2-5.3 kbp for Labelle and Fiji, respectively) and oligomers in which monomers are organized as head-to-tail repeats. DNA-DNA hybridization experiments showed that the plasmids have no substantial sequence homology to mtDNA, to each other, or to a previously characterized mitochondrial plasmid from N. crassa strain Mauriceville-lc (Collins et al. Cell 24, 443-452, 1981). The intramitochondrial location of the plasmids was established by cell fractionation and nuclease protection experiments. In sexual crosses, the plasmids showed strict maternal inheritance, the same as Neurospora mitochondrial DNA. The plasmids may represent a novel class of mitochondrial genetic elements. Images PMID:6280144

Dynamics of Cell Area and Force during Spreading

PubMed Central

Brill-Karniely, Yifat; Nisenholz, Noam; Rajendran, Kavitha; Dang, Quynh; Krishnan, Ramaswamy; Zemel, Assaf

2014-01-01

Experiments on human pulmonary artery endothelial cells are presented to show that cell area and the force exerted on a substrate increase simultaneously, but with different rates during spreading; rapid-force increase systematically occurred several minutes past initial spreading. We examine this theoretically and present three complementary mechanisms that may accompany the development of lamellar stress during spreading and underlie the observed behavior. These include: 1), the dynamics of cytoskeleton assembly at the cell basis; 2), the strengthening of acto-myosin forces in response to the generated lamellar stresses; and 3), the passive strain-stiffening of the cytoskeleton. PMID:25517168

Distortion product otoacoustic emission (2f1-f2) amplitude growth in human adults and neonates.

PubMed

Abdala, C

2000-01-01

Distortion product otoacoustic emissions (DPOAEs) are thought to be by-products of an active amplification process in the cochlea and thus serve as a metric for evaluating the integrity of this process. Because the cochlear amplifier functions in a level-dependent fashion, DPOAEs recorded as a function of stimulus level (i.e., a DPOAE growth function) may provide important information about the range and operational characteristics of the cochlear amplifier. The DPOAE growth functions recorded in human adults and neonates may provide information about the maturation of these active cochlear processes. Two experiments were conducted. Experiment I included normal-hearing adults and term-born neonates. The 2f1-f2 DPOAE growth functions were recorded for both age groups at three f2 frequencies. Experiment II was an extension of the first experiment but added a subject group of premature neonates. The results of these studies indicate that DPOAE growth functions most often show amplitude saturation and nonmonotonic growth for all age groups. However, premature neonates show monotonic growth and the absence of amplitude saturation more often than adults. Those premature neonates who do show saturation also show an elevated threshold for amplitude saturation relative to adults. In contrast, term neonates are adultlike for most measures except that they show a larger percentage of nonsaturating growth functions than adults. These results may indicate immaturity in cochlear amplifier function prior to term birth in humans. Outer hair cell function and/or efferent regulation of outer hair cell function are hypothesized sources of this immaturity, although some contribution from the immature middle ear cannot be ruled out.

Cytotoxicity, genotoxicity and oxidative stress of malachite green on the kidney and gill cell lines of freshwater air breathing fish Channa striata.

PubMed

Majeed, S Abdul; Nambi, K S N; Taju, G; Vimal, S; Venkatesan, C; Hameed, A S Sahul

2014-12-01

The cytotoxicity, genotoxicity and oxidative stress of malachite green (MG) was investigated using the fish Channa striata kidney (CSK) and Channa striata gill (CSG) cell lines. Five concentrations ranging from 0.001 to 10 μg mL(-1) were tested in three independent experiments. Cytotoxicity was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Rhodamine 123 and Alamar Blue. The mitochondrial changes and apoptosis of MG-exposed cells were observed by Rhodamine 123 and acridine orange/ethidium bromide (AO/EB) staining, respectively. In vitro potential DNA damaging effect of MG was tested using comet assay. Mitochondrial damage, apoptosis and DNA fragmentation increased in a concentration-dependent manner. Additionally, DNA electrophoretic mobility experiments were carried out to study the binding effect of MG to double-stranded DNA (dsDNA) of cells. DNA shift mobility experiments showed that MG is capable of strongly binding to linear dsDNA causing its degradation. Biochemical parameters such as lipid peroxidation (MDA), catalase (CAT) activity and reduced glutathione (GSH) levels were evaluated after exposure to MG. In CSK and CSG cell lines exposed to MG for 48 h, a significant increase in lipid peroxidation, which might be associated with decreased levels of reduced glutathione and catalase activity in these cell lines (p < 0.001), was observed.

Development of Multiple Antibiotic Resistance in Bacillus subtilis Cells Exposed to Microgravity: the BRIC-18 Experiment to the International Space Station

NASA Astrophysics Data System (ADS)

Fajardo-Cavazos, Patricia; Moeller, Ralf; Nicholson, Wayne; Narvel, Raed

Increased pathogenicity of opportunistic bacteria during long-term spaceflight is considered an astronaut risk. Because only a limited pharmacy can be carried on long-duration missions, the development of resistance to multiple antibiotics is a concern for mission planning. In support of the BRIC-18 experiment to the ISS, we have performed ground-based experiments to address the question whether simulated microgravity affects the frequency of resistance to the model antibiotics rifampicin (RFM) and trimethoprim (TMP). In these experiments, the model bacteria Bacillus subtilis and Staphylococcus epidermidis were cultivated for 6 days at ISS ambient temperature in 10-ml High Aspect Ratio Vessels (HARVs) on two 4-place clinostats (Synthecon) oriented either vertically (V) or horizontally (H). Cells were harvested, enumerated and plated onto medium containing RFM (5 micrograms/ml). The frequency of mutation to RFM resistance was calculated, and RFM-resistant mutants were plated onto medium containing the second antibiotic, TMP (5 micrograms/ml) to determine the frequency of mutation to double (RFM+TMP) resistance. After 6 days of cultivation, V-cultures showed higher cell densities and than H-cultures for both bacteria. However, only in B. subtilis did V-cultures show higher frequencies of mutation to RFM resistance than H-cultures. Launch of BRIC-18 to the ISS is currently scheduled for March 16, 2014 and return 30 days later. Results from both the spaceflight and ground control experiments will be presented. Supported by NASA-SAIP fellowship to R.N. and NASA grant (NNX12AN70G) to P.F.-C., R.M., and W.L.N.

In vitro test of external Qigong

PubMed Central

Yount, Garret; Solfvin, Jerry; Moore, Dan; Schlitz, Marilyn; Reading, Melissa; Aldape, Ken; Qian, Yifang

2004-01-01

Background Practitioners of the alternative medical practice 'external Qigong' generally claim the ability to emit or direct "healing energy" to treat patients. We investigated the ability of experienced Qigong practitioners to enhance the healthy growth of cultured human cells in a series of studies, each following a rigorously designed protocol with randomization, blinding and controls for variability. Methods Qigong practitioners directed healing intentionality toward normal brain cell cultures in a basic science laboratory. Qigong treatments were delivered for 20 minutes from a minimum distance of 10 centimeters. Cell proliferation was measured by a standard colony-forming efficiency (CFE) assay and a CFE ratio (CFE for treated samples/CFE for sham samples) was the dependent measure for each experiment. Results During a pilot study (8 experiments), a trend of increased cell proliferation in Qigong-treated samples (CFE Qigong/sham ratios > 1.0) was observed (P = 0.162). In a formal study (28 experiments), a similar trend was observed, with Qigong-treated samples showing on average more colony formation than sham samples (P = 0.036). In a replication study (60 experiments), no significant difference between Qigong-treated samples and sham samples was observed (P = 0.465). Conclusion We observed an apparent increase in the proliferation of cultured cells following external Qigong treatment by practitioners under strictly controlled conditions, but we did not observe this effect in a replication study. These results suggest the need for more controlled and thorough investigation of external Qigong before scientific validation is claimed. PMID:15102336

Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells.

PubMed

Miranda, Moysés S; Nascimento, Hamilton S; Costa, Mayra P R; Costa, Nathália N; Brito, Karynne N L; Lopes, Cinthia T A; Santos, Simone S D; Cordeiro, Marcela S; Ohashi, Otávio M

2016-10-01

Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 10 3 and 10 4 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 10 4 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). In experiment 1, co-culture with 10 4 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p < 0.05). Despite that it did not show difference with 10 3 b-ATMSCs/mL (p = 0.051), group 10 4 b-ATMSCs/mL yielded higher results of blastocyst production. In experiment 2, when compared to group Gran, co-culture with 10 4 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p < 0.05). Co-culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine and other species.

Experiment K-6-22. Growth hormone regulation, synthesis and secretion in microgravity. Part 1: Somatotroph physiology. Part 2: Immunohistochemical analysis of hypothalamic hormones. Part 3: Plasma analysis

NASA Technical Reports Server (NTRS)

Grindeland, R.; Vale, W.; Hymer, W.; Sawchenko, P.; Vasques, M.; Krasnov, I.; Kaplanski, A.; Victorov, I.

1990-01-01

The objectives of the 1887 mission were: (1) to determine if the results of the SL-3 pituitary gland experiment (1) were repeatable; and (2) to determine what effect a longer mission would have on the rat pituitary gland growth hormone (GH) system. In the 1887 experiment two issues were considered especially important. First, it was recognized that cells prepared from individual rat pituitary glands should be considered separately so that the data from the 5 glands could be analyzed in a statistically meaningful way. Second, results of the SL-3 flight involving the hollow fiber implant and HPLC GH-variant experiments suggested that the biological activity of the hormone had been negatively affected by flight. The results of the 1887 experiment documented the wisdom of addressing both issues in the protocol. Thus, the reduction in secretory capacity of flight cells during subsequent extended cell culture on Earth was documented statistically, and thereby established the validity of the SL-3 result. The results of both flight experiments thus support the contention that there is a secretory lesion in pituitary GH cells of flight animals. The primary objective of both missions was a clear definition of the effect of spaceflight on the GH cell system. There can no longer be any reasonable doubt that this system is affected in microgravity. One explanation for the reason(s) underlying the better known effects of spaceflight on organisms, viz. changes in bone, muscle and immune systems may very well rest with such changes in bGH. In spite of the fact that rats in the Cosmos 1887 flight were on Earth for two days after flight, the data show that the GH system had still not recovered from the effects of flight. Many questions remain. One of the more important concerns the GRF responsiveness of somatotrophs after flight. This will be tested in an upcoming experiment.

Pressure profiles in detonation cells with rectangular and diagonal structures

NASA Astrophysics Data System (ADS)

Hanana, M.; Lefebvre, M. H.

Experimental results presented in this work enable us to classify the three-dimensional structure of the detonation into two fundamental types: a rectangular structure and a diagonal structure. The rectangular structure is well documented in the literature and consists of orthogonal waves travelling independently from each another. The soot record in this case shows the classical diamond detonation cell exhibiting `slapping waves'. The experiments indicate that the diagonal structure is a structure with the triple point intersections moving along the diagonal line of the tube cross section. The axes of the transverse waves are canted at 45 degrees to the wall, accounting for the lack of slapping waves. It is possible to reproduce these diagonal structures by appropriately controlling the experimental ignition procedure. The characteristics of the diagonal structure show some similarities with detonation structure in round tube. Pressure measurements recorded along the central axis of the cellular structure show a series of pressure peaks, depending on the type of structure and the position inside the detonation cell. Pressure profiles measured for the whole length of the two types of detonation cells show that the intensity of the shock front is higher and the length of the detonation cell is shorter for the diagonal structures.

Clonal tracing of Sox9+ liver progenitors in oval cell injury

PubMed Central

Tarlow, Branden D.; Finegold, Milton J.; Grompe, Markus

2014-01-01

Proliferating ducts, termed “oval cells”, have long thought to be bipotential, i.e. produce both biliary ducts and hepatocytes during chronic liver injury. The precursor to oval cells is considered to be a facultative liver stem cell (LSC). Recent lineage tracing experiments indicated that the LSC is Sox9+ and can replace the bulk of hepatocyte mass in several settings. However, no clonal relationship between Sox9+ cells and the two epithelial liver lineages was established. We labeled Sox9+ mouse liver cells at low density with a multicolor fluorescent confetti reporter. Organoid formation validated the progenitor activity of the labeled population. Sox9+ cells were traced in multiple oval cell injury models using both histology and FACS. Surprisingly, only rare clones containing both hepatocytes and oval cells were found in any experiment. Quantitative analysis showed that Sox9+ cells contributed only minimally (<1%) to the hepatocyte pool, even in classic oval cell injury models. In contrast, clonally marked mature hepatocytes demonstrated the ability to self-renew in all classic mouse oval cell activation injuries. A hepatocyte chimera model to trace hepatocytes and non-parenchymal cells also demonstrated the prevalence of hepatocyte-driven regeneration in mouse oval cell injury models. Conclusion Sox9+ ductal progenitor cells give rise to clonal oval cell proliferation and bipotential organoids but rarely produce hepatocytes in vivo. Hepatocytes themselves are the predominant source of new parenchyma cells in prototypical mouse models of oval cell activation. PMID:24700457

Mechanism of cell alignment in groups of Myxococcus xanthus bacteria

NASA Astrophysics Data System (ADS)

Balgam, Rajesh; Igoshin, Oleg

2015-03-01

Myxococcus xanthus is a model for studying self-organization in bacteria. These flexible cylindrical bacteria move along. In groups, M. xanthus cells align themselves into dynamic cell clusters but the mechanism underlying their formation is unknown. It has been shown that steric interactions can cause alignment in self-propelled hard rods but it is not clear how flexibility and reversals affect the alignment and cluster formation. We have investigated cell alignment process using our biophysical model of M. xanthus cell in an agent-based simulation framework under realistic cell flexibility values. We observed that flexible model cells can form aligned cell clusters when reversals are suppressed but these clusters disappeared when reversals frequency becomes similar to the observed value. However, M. xanthus cells follow slime (polysaccharide gel like material) trails left by other cells and we show that implementing this into our model rescues cell clustering for reversing cells. Our results show that slime following along with periodic cell reversals act as positive feedback to reinforce existing slime trails and recruit more cells. Furthermore, we have observed that mechanical cell alignment combined with slime following is sufficient to explain the distinct clustering patterns of reversing and non-reversing cells as observed in recent experiments. This work is supported by NSF MCB 0845919 and 1411780.

Cellular volume regulation and substrate stiffness modulate the detachment dynamics of adherent cells

NASA Astrophysics Data System (ADS)

Yang, Yuehua; Jiang, Hongyuan

2018-03-01

Quantitative characterizations of cell detachment are vital for understanding the fundamental mechanisms of cell adhesion. Experiments have found that cell detachment shows strong rate dependence, which is mostly attributed to the binding-unbinding kinetics of receptor-ligand bond. However, our recent study showed that the cellular volume regulation can significantly regulate the dynamics of adherent cell and cell detachment. How this cellular volume regulation contributes to the rate dependence of cell detachment remains elusive. Here, we systematically study the role of cellular volume regulation in the rate dependence of cell detachment by investigating the cell detachments of nonspecific adhesion and specific adhesion. We find that the cellular volume regulation and the bond kinetics dominate the rate dependence of cell detachment at different time scales. We further test the validity of the traditional Johnson-Kendall-Roberts (JKR) contact model and the detachment model developed by Wyart and Gennes et al (W-G model). When the cell volume is changeable, the JKR model is not appropriate for both the detachments of convex cells and concave cells. The W-G model is valid for the detachment of convex cells but is no longer applicable for the detachment of concave cells. Finally, we show that the rupture force of adherent cells is also highly sensitive to substrate stiffness, since an increase in substrate stiffness will lead to more associated bonds. These findings can provide insight into the critical role of cell volume in cell detachment and might have profound implications for other adhesion-related physiological processes.

Kinetic and functional analysis of transient, persistent and resurgent sodium currents in rat cerebellar granule cells in situ: an electrophysiological and modelling study

PubMed Central

Magistretti, Jacopo; Castelli, Loretta; Forti, Lia; D'Angelo, Egidio

2006-01-01

Cerebellar neurones show complex and differentiated mechanisms of action potential generation that have been proposed to depend on peculiar properties of their voltage-dependent Na+ currents. In this study we analysed voltage-dependent Na+ currents of rat cerebellar granule cells (GCs) by performing whole-cell, patch-clamp experiments in acute rat cerebellar slices. A transient Na+ current (INaT) was always present and had the properties of a typical fast-activating/inactivating Na+ current. In addition to INaT, robust persistent (INaP) and resurgent (INaR) Na+ currents were observed. INaP peaked at ∼−40 mV, showed half-maximal activation at ∼−55 mV, and its maximal amplitude was about 1.5% of that of INaT. INaR was elicited by repolarizing pulses applied following step depolarizations able to activate/inactivate INaT, and showed voltage- and time-dependent activation and voltage-dependent decay kinetics. The conductance underlying INaR showed a bell-shaped voltage dependence, with peak at −35 mV. A significant correlation was found between GC INaR and INaT peak amplitudes; however, GCs expressing INaT of similar size showed marked variability in terms of INaR amplitude, and in a fraction of cells INaR was undetectable. INaT, INaP and INaR could be accounted for by a 13-state kinetic scheme comprising closed, open, inactivated and blocked states. Current-clamp experiments carried out to identify possible functional correlates of INaP and/or INaR revealed that in GCs single action potentials were followed by depolarizing afterpotentials (DAPs). In a majority of cells, DAPs showed properties consistent with INaR playing a role in their generation. Computer modelling showed that INaR promotes DAP generation and enhances high-frequency firing, whereas INaP boosts near-threshold firing activity. Our findings suggest that special properties of voltage-dependent Na+ currents provides GCs with mechanisms suitable for shaping activity patterns, with potentially important consequences for cerebellar information transfer and computation. PMID:16527854

Conditions Determining Initiation of DNA Synthesis in 3T3 Cells*

PubMed Central

Dulbecco, R.; Stoker, M. G. P.

1970-01-01

Experiments were designed to discriminate between inhibition of growth due to contacts or exhaustion of serum factors. The cell layer was wounded and the migrating cells were followed by time-lapse cinematography; DNA synthesis in the same cells was recognized by means of 3H-thymidine labeling and radioautography. In this way, the complete history of individual cells migrating to the wound could be described. The results show that topographical relationships between cells play an important role in controlling initiation of DNA synthesis. It is still unclear whether initiation is promoted by release from contacts or by the increased ability of the cells to utilize serum factors because of their changes in shapes and activities. PMID:5273897

On Leakage Current Measured at High Cell Voltages in Lithium-Ion Batteries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vadivel, Nicole R.; Ha, Seungbum; He, Meinan

2017-01-01

In this study, parasitic side reactions in lithium-ion batteries were examined experimentally using a potentiostatic hold at high cell voltage. The experimental leakage current measured during the potentiostatic hold was compared to the Tafel expression and showed poor agreement with the expected transfer coefficient values, indicating that a more complicated expression could be needed to accurately capture the physics of this side reaction. Here we show that cross-talk between the electrodes is the primary contribution to the observed leakage current after the relaxation of concentration gradients has ceased. This cross-talk was confirmed with experiments using a lithium-ion conducting glass ceramicmore » (LICGC) separator, which has high conductance only for lithium cations. The cells with LICGC separators showed significantly less leakage current during the potentiostatic hold test compared to cells with standard microporous separators where cross-talk is present. In addition, direct-current pulse power tests show an impedance rise for cells held at high potentials and for cells held at high temperatures, which could be attributed to film formation from the parasitic side reaction. Based on the experimental findings, a phenomenological mechanism is proposed for the parasitic side reaction which accounts for cross-talk and mass transport of the decomposition products across the separator.« less

The Flavonoid Jaceosidin from Artemisia princeps Induces Apoptotic Cell Death and Inhibits the Akt Pathway in Oral Cancer Cells.

PubMed

Han, Hye-Yeon; Kim, Hyung Joon; Jeong, Seung-Hwa; Kim, Jiyeon; Jeong, Sung-Hee; Kim, Gyoo Cheon; Hwang, Dae-Seok; Kim, Uk-Kyu; Ryu, Mi Heon

2018-01-01

Jaceosidin is a single compound from the Japanese mugwort Artemisia princeps , which is used as a food and a traditional medicinal herb. A. princeps extracts and flavonoid components have been shown to have antihyperglycaemic, antioxidant, and anti-inflammatory properties. Although the anticancer properties of these extracts were recently demonstrated, the related mechanisms have not been characterised. In this study, we investigated the effects of jaceosidin in oral squamous cell carcinoma (OSCC) cells and initially showed selective suppression of proliferation (IC 50 = 82.1  μ M in HSC-3 cells and 97.5  μ M in Ca9.22 cells) and accumulation of cells at the sub-G1 stage of the cell cycle. In addition, jaceosidin increased cleavage of caspase-9 and caspase-3 in OSCC cells, although caspase-8 was not detected. In further experiments, jaceosidin downregulated Akt phosphorylation and ectopic activation of Akt blocked the antiproliferative effects of jaceosidin. Finally, we showed that jaceosidin has no effects on HaCaT normal epithelial cell viability, indicating selective chemotherapeutic potential of jaceosidin and that tumour-specific downregulation of Akt increases apoptosis and inhibits growth in OSCC cells.

The Flavonoid Jaceosidin from Artemisia princeps Induces Apoptotic Cell Death and Inhibits the Akt Pathway in Oral Cancer Cells

PubMed Central

Han, Hye-Yeon; Kim, Hyung Joon; Jeong, Seung-Hwa; Kim, Jiyeon; Jeong, Sung-Hee; Kim, Gyoo Cheon; Hwang, Dae-Seok; Kim, Uk-Kyu

2018-01-01

Jaceosidin is a single compound from the Japanese mugwort Artemisia princeps, which is used as a food and a traditional medicinal herb. A. princeps extracts and flavonoid components have been shown to have antihyperglycaemic, antioxidant, and anti-inflammatory properties. Although the anticancer properties of these extracts were recently demonstrated, the related mechanisms have not been characterised. In this study, we investigated the effects of jaceosidin in oral squamous cell carcinoma (OSCC) cells and initially showed selective suppression of proliferation (IC50 = 82.1 μM in HSC-3 cells and 97.5 μM in Ca9.22 cells) and accumulation of cells at the sub-G1 stage of the cell cycle. In addition, jaceosidin increased cleavage of caspase-9 and caspase-3 in OSCC cells, although caspase-8 was not detected. In further experiments, jaceosidin downregulated Akt phosphorylation and ectopic activation of Akt blocked the antiproliferative effects of jaceosidin. Finally, we showed that jaceosidin has no effects on HaCaT normal epithelial cell viability, indicating selective chemotherapeutic potential of jaceosidin and that tumour-specific downregulation of Akt increases apoptosis and inhibits growth in OSCC cells. PMID:29861773

Effects of Heavy Metals from Soil and Dust Source on DNA Damage of the Leymus chinensis Leaves in Coal-Mining Area in Northwest China

PubMed Central

Li, Tianxin; Zhang, Minjie; Lu, Zhongming; Herman, Uwizeyimana; Mumbengegwi, Dzivaidzo; Crittenden, John

2016-01-01

Air and soil pollution from mining activities has been considered as a critical issue to the health of living organisms. However, few efforts have been made in distinguishing the main pathway of organism genetic damage by heavy metals related to mining activities. Therefore, we investigated the genetic damage of Leymus chinensis leaf cells, the air particulate matter (PM) contents, and concentrations of the main heavy metals (Pb, Cd, Cr, Hg) in soil and foliar dust samples collected from seven experiment points at the core mining area and one control point 20 kilometers away from the core mining area in Inner Mongolia in 2013. Comet assay was used to test the genetic damage of the Leymus chinensis leaf cells; the Tail DNA% and Tail Moment were used to characterize the genetic damage degree of the plant cells. The comet assay results showed that the cell genetic damage ratio was up to 77.0% in experiment points but was only 35.0% in control point. The control point also had the slight Tail DNA% and Tail Moment values than other experiment groups. The cell damage degree of the control group was 0.935 and experiment groups were 1.299–1.815. The geo-accumulation index and comperehensive pollution index(CPI) were used to characterize heavy metal pollution in foliar dust samples, and single factor pollution index and CPI were used to characterize the heavy metal pollution in soil samples. The CPIfoliar dust of control group was 0.36 and experiment groups were 1.45–2.57; the CPIsoil of control group was 0.04 and experiment groups were 0.07–0.12. The results of correlation analyze showed that Air Quality Index (AQI) -CPIfoliar dust(r = 0.955**)>Damage degree-CPIfoliar dust(r = 0.923**)>Damage degree-AQI(r = 0.908**)>Damage degree-CPIsoil (r = 0.824*). The present research proved that mining activity had a high level of positive correlation with organism genetic damage caused by heavy metals through comparing with the control point; soil and atmosphere were both the important action pathway for heavy metal induced genetic damage in mining area. Furthermore, heavy metal contents in foliar dust showed a higher positive correlation with genetic damage than when compared with soil. This means the heavy metal contents that L.chinensis absorbed through respiration from the atmosphere could make more serious genetic damage than when absorbed by root systems from soil in the mining area. This study can provide theoretical support for research on plant genetic damage mechanisms and exposure pathways induced by environmental pollution. PMID:27935969

Effects of Heavy Metals from Soil and Dust Source on DNA Damage of the Leymus chinensis Leaves in Coal-Mining Area in Northwest China.

PubMed

Li, Tianxin; Zhang, Minjie; Lu, Zhongming; Herman, Uwizeyimana; Mumbengegwi, Dzivaidzo; Crittenden, John

2016-01-01

Air and soil pollution from mining activities has been considered as a critical issue to the health of living organisms. However, few efforts have been made in distinguishing the main pathway of organism genetic damage by heavy metals related to mining activities. Therefore, we investigated the genetic damage of Leymus chinensis leaf cells, the air particulate matter (PM) contents, and concentrations of the main heavy metals (Pb, Cd, Cr, Hg) in soil and foliar dust samples collected from seven experiment points at the core mining area and one control point 20 kilometers away from the core mining area in Inner Mongolia in 2013. Comet assay was used to test the genetic damage of the Leymus chinensis leaf cells; the Tail DNA% and Tail Moment were used to characterize the genetic damage degree of the plant cells. The comet assay results showed that the cell genetic damage ratio was up to 77.0% in experiment points but was only 35.0% in control point. The control point also had the slight Tail DNA% and Tail Moment values than other experiment groups. The cell damage degree of the control group was 0.935 and experiment groups were 1.299-1.815. The geo-accumulation index and comperehensive pollution index(CPI) were used to characterize heavy metal pollution in foliar dust samples, and single factor pollution index and CPI were used to characterize the heavy metal pollution in soil samples. The CPIfoliar dust of control group was 0.36 and experiment groups were 1.45-2.57; the CPIsoil of control group was 0.04 and experiment groups were 0.07-0.12. The results of correlation analyze showed that Air Quality Index (AQI) -CPIfoliar dust(r = 0.955**)>Damage degree-CPIfoliar dust(r = 0.923**)>Damage degree-AQI(r = 0.908**)>Damage degree-CPIsoil (r = 0.824*). The present research proved that mining activity had a high level of positive correlation with organism genetic damage caused by heavy metals through comparing with the control point; soil and atmosphere were both the important action pathway for heavy metal induced genetic damage in mining area. Furthermore, heavy metal contents in foliar dust showed a higher positive correlation with genetic damage than when compared with soil. This means the heavy metal contents that L.chinensis absorbed through respiration from the atmosphere could make more serious genetic damage than when absorbed by root systems from soil in the mining area. This study can provide theoretical support for research on plant genetic damage mechanisms and exposure pathways induced by environmental pollution.

Overexpression of phosphoserine aminotransferase PSAT1 stimulates cell growth and increases chemoresistance of colon cancer cells

PubMed Central

Vié, Nadia; Copois, Virginie; Bascoul-Mollevi, Caroline; Denis, Vincent; Bec, Nicole; Robert, Bruno; Fraslon, Caroline; Conseiller, Emmanuel; Molina, Franck; Larroque, Christian; Martineau, Pierre; Del Rio, Maguy; Gongora, Céline

2008-01-01

Background Colorectal cancer (CRC) is one of the most common causes of cancer death throughout the world. In this work our aim was to study the role of the phosphoserine aminotransferase PSAT1 in colorectal cancer development. Results We first observed that PSAT1 is overexpressed in colon tumors. In addition, we showed that after drug treatment, PSAT1 expression level in hepatic metastases increased in non responder and decreased in responder patients. In experiments using human cell lines, we showed that ectopic PSAT1 overexpression in colon carcinoma SW480 cell line resulted in an increase in its growth rate and survival. In addition, SW480-PSAT1 cells presented a higher tumorigenic potential than SW480 control cells in xenografted mice. Moreover, the SW480-PSAT1 cell line was more resistant to oxaliplatin treatment than the non-transfected SW480 cell line. This resistance resulted from a decrease in the apoptotic response and in the mitotic catastrophes induced by the drug treatment. Conclusion These results show that an enzyme playing a role in the L-serine biosynthesis could be implicated in colon cancer progression and chemoresistance and indicate that PSAT1 represents a new interesting target for CRC therapy. PMID:18221502

Human mammary microenvironment better regulates the biology of human breast cancer in humanized mouse model.

PubMed

Zheng, Ming-Jie; Wang, Jue; Xu, Lu; Zha, Xiao-Ming; Zhao, Yi; Ling, Li-Jun; Wang, Shui

2015-02-01

During the past decades, many efforts have been made in mimicking the clinical progress of human cancer in mouse models. Previously, we developed a human breast tissue-derived (HB) mouse model. Theoretically, it may mimic the interactions between "species-specific" mammary microenvironment of human origin and human breast cancer cells. However, detailed evidences are absent. The present study (in vivo, cellular, and molecular experiments) was designed to explore the regulatory role of human mammary microenvironment in the progress of human breast cancer cells. Subcutaneous (SUB), mammary fat pad (MFP), and HB mouse models were developed for in vivo comparisons. Then, the orthotopic tumor masses from three different mouse models were collected for primary culture. Finally, the biology of primary cultured human breast cancer cells was compared by cellular and molecular experiments. Results of in vivo mouse models indicated that human breast cancer cells grew better in human mammary microenvironment. Cellular and molecular experiments confirmed that primary cultured human breast cancer cells from HB mouse model showed a better proliferative and anti-apoptotic biology than those from SUB to MFP mouse models. Meanwhile, primary cultured human breast cancer cells from HB mouse model also obtained the migratory and invasive biology for "species-specific" tissue metastasis to human tissues. Comprehensive analyses suggest that "species-specific" mammary microenvironment of human origin better regulates the biology of human breast cancer cells in our humanized mouse model of breast cancer, which is more consistent with the clinical progress of human breast cancer.

Targeting Src and tubulin in mucinous ovarian carcinoma

PubMed Central

Liu, Tao; Hu, Wei; Dalton, Heather J.; Choi, Hyun Jin; Huang, Jie; Kang, Yu; Pradeep, Sunila; Miyake, Takahito; Song, Jian H.; Wen, Yunfei; Lu, Chunhua; Pecot, Chad V.; Bottsford-Miller, Justin; Zand, Behrouz; Jennings, Nicholas B; Ivan, Cristina; Gallick, Gary E.; Baggerly, Keith A; Hangauer, David G.; Coleman, Robert L.; Frumovitz, Michael; Sood, Anil K.

2013-01-01

Purpose To investigate the antitumor effects of targeting Src and tubulin in mucinous ovarian carcinoma. Experimental design The in vitro and in vivo effects and molecular mechanisms of KX-01, which inhibits Src pathway and tubulin polymerization, were examined in mucinous ovarian cancer models. Results In vitro studies using RMUG-S and RMUG-L cell lines showed that KX-01 inhibited cell proliferation, induced apoptosis, arrested the cell cycle at the G2/M phase, and enhanced the cytotoxicity of oxaliplatin in the KX-01-sensitive cell line, RMUG-S. In vivo studies showed that KX-01 significantly decreased tumor burden in RMUG-S and RMUG-L mouse models relative to untreated controls, and the effects were greater when KX-01 was combined with oxaliplatin. KX-01 alone and in combination with oxaliplatin significantly inhibited tumor growth by reducing cell proliferation and inducing apoptosis in vivo. PTEN knock-in experiments in RMUG-L cells showed improved response to KX-01. Reverse phase protein array analysis showed that in addition to blocking downstream molecules of Src family kinases, KX-01 also activated acute stress-inducing molecules. Conclusion Our results showed that targeting both the Src pathway and tubulin with KX-01 significantly inhibited tumor growth in preclinical mucinous ovarian cancer models, suggesting that this may be a promising therapeutic approach for patients with mucinous ovarian carcinoma. PMID:24100628

A novel concentration and viability detection method for Brettanomyces using the Cellometer image cytometry.

PubMed

Martyniak, Brian; Bolton, Jason; Kuksin, Dmitry; Shahin, Suzanne M; Chan, Leo Li-Ying

2017-01-01

Brettanomyces spp. can present unique cell morphologies comprised of excessive pseudohyphae and budding, leading to difficulties in enumerating cells. The current cell counting methods include manual counting of methylene blue-stained yeasts or measuring optical densities using a spectrophotometer. However, manual counting can be time-consuming and has high operator-dependent variations due to subjectivity. Optical density measurement can also introduce uncertainties where instead of individual cells counted, an average of a cell population is measured. In contrast, by utilizing the fluorescence capability of an image cytometer to detect acridine orange and propidium iodide viability dyes, individual cell nuclei can be counted directly in the pseudohyphae chains, which can improve the accuracy and efficiency of cell counting, as well as eliminating the subjectivity from manual counting. In this work, two experiments were performed to demonstrate the capability of Cellometer image cytometer to monitor Brettanomyces concentrations, viabilities, and budding/pseudohyphae percentages. First, a yeast propagation experiment was conducted to optimize software counting parameters for monitoring the growth of Brettanomyces clausenii, Brettanomyces bruxellensis, and Brettanomyces lambicus, which showed increasing cell concentrations, and varying pseudohyphae percentages. The pseudohyphae formed during propagation were counted either as multiple nuclei or a single multi-nuclei organism, where the results of counting the yeast as a single multi-nuclei organism were directly compared to manual counting. Second, a yeast fermentation experiment was conducted to demonstrate that the proposed image cytometric analysis method can monitor the growth pattern of B. lambicus and B. clausenii during beer fermentation. The results from both experiments displayed different growth patterns, viability, and budding/pseudohyphae percentages for each Brettanomyces species. The proposed Cellometer image cytometry method can improve efficiency and eliminate operator-dependent variations of cell counting compared with the traditional methods, which can potentially improve the quality of beverage products employing Brettanomyces yeasts.

Migration of lymphocytes on fibronectin-coated surfaces: temporal evolution of migratory parameters

NASA Technical Reports Server (NTRS)

Bergman, A. J.; Zygourakis, K.; McIntire, L. V. (Principal Investigator)

1999-01-01

Lymphocytes typically interact with implanted biomaterials through adsorbed exogenous proteins. To provide a more complete characterization of these interactions, analysis of lymphocyte migration on adsorbed extracellular matrix proteins must accompany the commonly performed adhesion studies. We report here a comparison of the migratory and adhesion behavior of Jurkat cells (a T lymphoblastoid cell line) on tissue culture treated and untreated polystyrene surfaces coated with various concentrations of fibronectin. The average speed of cell locomotion showed a biphasic response to substrate adhesiveness for cells migrating on untreated polystyrene and a monotonic decrease for cells migrating on tissue culture-treated polystyrene. A modified approach to the persistent random walk model was implemented to determine the time dependence of cell migration parameters. The random motility coefficient showed significant increases with time when cells migrated on tissue culture-treated polystyrene surfaces, while it remained relatively constant for experiments with untreated polystyrene plates. Finally, a cell migration computer model was developed to verify our modified persistent random walk analysis. Simulation results suggest that our experimental data were consistent with temporally increasing random motility coefficients.

[Studies on red orpiment induction of NB4 and HL-60 cell apoptosis].

PubMed

Bai, Y; Huang, S

1998-09-01

To study the possible mechanism of red orpiment, which is main component of composite indigo naturalis tablets, in the treatment of acute promyelocytic leukemia(APL). The effect of red orpiment on induction of APL cell line NB4 and HL-60 apoptosis were studied by cell morphology, DNA gel electrophoresis and flow cytometry assay. Red orpiment induced NB4 and HL-60 cell apoptosis. When treated with different concentration of red orpiment(25-200 micrograms/ml) for 16 hours, both NB4 and HL-60 cells showed typical apoptosis features. If decreased the concentration of red orpiment to 12.5 micrograms/ml, the NB4 cell still showed apoptosis features while the HL-60 cell did not when cultured for 72 hours. Arsenic disulfide(As2S2) had the same effect as red orpiment did under the same experiment condition. It is the main component, As2S2 of the red orpiment that can induces NB4 and HL-60 cell apoptosis. and the red orpiment is responsible for the high CR rate of APL induced by the composite indigo naturalis tablets.

Fluorescent pH-Sensing Probe Based on Biorefinery Wood Lignosulfonate and Its Application in Human Cancer Cell Bioimaging.

PubMed

Xue, Yuyuan; Liang, Wanshan; Li, Yuan; Wu, Ying; Peng, Xinwen; Qiu, Xueqing; Liu, Jinbin; Sun, Runcang

2016-12-28

A water-soluble, ratiometric fluorescent pH probe, L-SRhB, was synthesized via grafting spirolactam Rhodamine B (SRhB) to lignosulfonate (LS). As the ring-opening product of L-SRhB, FL-SRhB was also prepared. The pH-response experiment indicated that L-SRhB showed a rapid response to pH changes from 4.60 to 6.20 with a pK a of 5.35, which indicated that L-SRhB has the potential for pH detection of acidic organelle. In addition, the two probes were internalized successfully by living cells through the endocytosis pathway and could distinguish normal cells from cancer cells by different cell staining rates. In addition, L-SRhB showed obvious cytotoxicity to cancer cells, whereas it was nontoxic to normal cells in the same condition. L-SRhB might have potential in cancer therapy. L-SRhB might be a promising ratiometric fluorescent pH sensor and bioimaging dye for the recognition of cancer cells. The results also provided a new perspective to the high-value utilization of lignin.

Increasing the efficacy of antitumor glioma vaccines by photodynamic therapy and local injection of allogeneic glioma cells

NASA Astrophysics Data System (ADS)

Christie, Catherine E.; Peng, Qian; Madsen, Steen J.; Uzal, Francisco A.; Hirschberg, Henry

2016-03-01

Immunotherapy of brain tumors involves the stimulation of an antitumor immune response. This type of therapy can be targeted specifically to tumor cells thus sparing surrounding normal brain. Due to the presence of the blood-brain barrier, the brain is relatively isolated from the systemic circulation and, as such, the initiation of significant immune responses is more limited than other types of cancers. The purpose of this study was to show that the efficacy of tumor primed antigen presenting macrophage vaccines could be increased by: (1) PDT of the priming tumor cells, and (2) injection of allogeneic glioma cells directly into brain tumors. Experiments were conducted in an in vivo brain tumor model using Fisher rats and BT4C (allogeneic) and F98 (syngeneic) glioma cells. Preliminary results showed that vaccination alone had significantly less inhibitory effect on F98 tumor growth compared to the combination of vaccination and allogeneic cell (BT4C) injection.

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

PubMed

Tran, Thai Q; Ishak Gabra, Mari B; Lowman, Xazmin H; Yang, Ying; Reid, Michael A; Pan, Min; O'Connor, Timothy R; Kong, Mei

2017-11-01

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes

PubMed Central

Tran, Thai Q.; Ishak Gabra, Mari B.; Lowman, Xazmin H.; Yang, Ying; Reid, Michael A.; Pan, Min; O’Connor, Timothy R.

2017-01-01

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer. PMID:29107960

Distinct mechanisms underlie oral vs aboral regeneration in the cnidarian Hydractinia echinata.

PubMed

Bradshaw, Brian; Thompson, Kerry; Frank, Uri

2015-04-17

Cnidarians possess remarkable powers of regeneration, but the cellular and molecular mechanisms underlying this capability are unclear. Studying the hydrozoan Hydractinia echinata we show that a burst of stem cell proliferation occurs following decapitation, forming a blastema at the oral pole within 24 hr. This process is necessary for head regeneration. Knocking down Piwi1, Vasa, Pl10 or Ncol1 expressed by blastema cells inhibited regeneration but not blastema formation. EdU pulse-chase experiments and in vivo tracking of individual transgenic Piwi1(+) stem cells showed that the cellular source for blastema formation is migration of stem cells from a remote area. Surprisingly, no blastema developed at the aboral pole after stolon removal. Instead, polyps transformed into stolons and then budded polyps. Hence, distinct mechanisms act to regenerate different body parts in Hydractinia. This model, where stem cell behavior can be monitored in vivo at single cell resolution, offers new insights for regenerative biology.

Effects of spaceflight on levels and activity of immune cells

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald; Berry, Wallace D.; Mandel, Adrian D.; Konstantinova, Irena V.; Taylor, Gerald R.

1990-01-01

Experiments were carried out on cells from rats that had been flown on Soviet Biosputnik Cosmos 1887 to explore the effects of speceflight on immune responses. Rat bone marrow cells were examined for their response to colony stimulating factor-M. Rat spleen and bone marrow cells were stained with antibodies directed against cell surface antigenic markers. The results of the studies indicate that bone marrow cells from flown rats showed a decreased response to colony stimulating factor. There was a higher percentage of spleen cells from flown rats staining positively for pan-T-cell, suppressor-T-cell, and interleukin-2 receptor cell surface antigens. A small increase in the percentage of cells staining positively for helper-T-cell antigens was also noted. In addition, a higher percentage of cells that appeared to be part of the myelogenous population of bone marrow cells from flown rats stained positively for surface immunoglobulin.

Prevalence of human cell material: DNA and RNA profiling of public and private objects and after activity scenarios.

PubMed

van den Berge, M; Ozcanhan, G; Zijlstra, S; Lindenbergh, A; Sijen, T

2016-03-01

Especially when minute evidentiary traces are analysed, background cell material unrelated to the crime may contribute to detectable levels in the genetic analyses. To gain understanding on the composition of human cell material residing on surfaces contributing to background traces, we performed DNA and mRNA profiling on samplings of various items. Samples were selected by considering events contributing to cell material deposits in exemplary activities (e.g. dragging a person by the trouser ankles), and can be grouped as public objects, private samples, transfer-related samples and washing machine experiments. Results show that high DNA yields do not necessarily relate to an increased number of contributors or to the detection of other cell types than skin. Background cellular material may be found on any type of public or private item. When a major contributor can be deduced in DNA profiles from private items, this can be a different person than the owner of the item. Also when a specific activity is performed and the areas of physical contact are analysed, the "perpetrator" does not necessarily represent the major contributor in the STR profile. Washing machine experiments show that transfer and persistence during laundry is limited for DNA and cell type dependent for RNA. Skin conditions such as the presence of sebum or sweat can promote DNA transfer. Results of this study, which encompasses 549 samples, increase our understanding regarding the prevalence of human cell material in background and activity scenarios. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Genotoxicity of tungsten carbide-cobalt (WC-Co) nanoparticles in vitro: mechanisms-of-action studies.

PubMed

Moche, Hélène; Chevalier, Dany; Vezin, Hervé; Claude, Nancy; Lorge, Elisabeth; Nesslany, Fabrice

2015-02-01

We showed previously that tungsten carbide-cobalt (WC-Co) nanoparticles (NP) can be used as a nanoparticulate positive control in some in vitro mammalian genotoxicity assays. Here, we investigate the mechanisms of action involved in WC-Co NP genotoxicity in L5178Y mouse lymphoma cells and primary human lymphocytes, in vitro. Data from the micronucleus assay coupled with centromere staining and from the chromosome-aberration assay show the involvement of both clastogenic and aneugenic events. Experiments with the formamidopyrimidine DNA glycosylase (FPG)-modified comet assay showed a slight (non-significant) increase in FPG-sensitive sites in the L5178Y mouse lymphoma cells but not in the human lymphocytes. Electron paramagnetic resonance spin-trapping results showed the presence of hydroxyl radicals (•OH) in WC-Co NP suspensions, with or without cells, but with time-dependent production in the presence of cells. However, a significant difference in •OH production was observed between human lymphocytes from two different donors. Using H2O2, we showed that WC-Co NP can participate in Fenton-like reactions. Thus, •OH might be produced either via intrinsic generation by WC-Co NP or through a Fenton-like reaction in the presence of cells. Copyright © 2015 Elsevier B.V. All rights reserved.

MicroRNA-199 suppresses cell proliferation, migration and invasion by downregulating RGS17 in hepatocellular carcinoma.

PubMed

Zhang, Wei; Qian, Sheng; Yang, Guowei; Zhu, Liang; Zhou, Bo; Wang, Jianhua; Liu, Rong; Yan, Zhiping; Qu, Xudong

2018-06-15

Hepatocellular carcinoma (HCC), the most common primary tumor of the liver, has a poor prognosis and shows rapid progression. MicroRNAs (miRNAs) play important roles in carcinogenesis and tumor progression. Regulators of G-protein signaling (RGS) are critical for defining G-protein-dependent signal fidelity. RGS17 plays an important role in the regulation of cancer cell proliferation, migration and invasion. Here, we showed that miR-199 was downregulated in a hepatocarcinoma cell line. Overexpression of miR-199 significantly suppressed HCC cell proliferation, migration, and invasion in vitro. RGS17 overexpression promoted HCC cell proliferation, migration, and invasion, and reversed the miR-199 mediated inhibition of proliferation, migration, and invasion. Dual-fluorescence reporter experiments confirmed that miR-199 downregulated RGS17 by direct interaction with the 3'-UTR of RGS17 mRNA. In vivo studies showed that miR-199 overexpression significantly inhibited the growth of tumors. Taken together, the results suggested that miR-199 inhibited tumor growth and metastasis by targeting RGS17. Published by Elsevier B.V.

Antitumor effects of Marginisporum crassissimum (Rhodophyceae), a marine red alga.

PubMed

Hiroishi, S; Sugie, K; Yoshida, T; Morimoto, J; Taniguchi, Y; Imai, S; Kurebayashi, J

2001-06-26

Marginisporum crassissimum (Yendo) Ganesan, a marine red alga found in the ordinal coastal sea around Japan, revealed antitumor (antimetastatic) effects in vitro and in vivo. In in vitro experiments, extracts of this alga inhibited not only the growth of several tumor cell lines, such as B16-BL6 (a mouse melanoma cell line), JYG-B (a mouse mammary carcinoma cell line) and KPL-1 (a human mammary carcinoma cell line), but also invasion of B16-BL6 cells in a culture system. In in vivo experiments, the lung metastasis of B16-BL6 cells inoculated to the tail vein of B57BL/6J mice was inhibited by intraperitoneal administration of an extract from the alga. In addition, life prolongation of B57BL/6J mice inoculated with B16-BL6 cells was also observed by the intraperitoneal administration of the extract. An effective substance showing B16-BL6 growth inhibition in vitro was partially purified by filtration and hydrophobic column chromatography, and was revealed to be sensitive to trypsin-digestion and heat-treatment. The molecular weight of the substance was greater than 100 kDa. This is the first study demonstrating antitumor (antimetastatic) effects of M. crassissimum.

Mobile phone radiation causes changes in gene and protein expression in human endothelial cell lines and the response seems to be genome- and proteome-dependent.

PubMed

Nylund, Reetta; Leszczynski, Dariusz

2006-09-01

We have examined in vitro cell response to mobile phone radiation (900 MHz GSM signal) using two variants of human endothelial cell line: EA.hy926 and EA.hy926v1. Gene expression changes were examined in three experiments using cDNA Expression Arrays and protein expression changes were examined in ten experiments using 2-DE and PDQuest software. Obtained results show that gene and protein expression were altered, in both examined cell lines, in response to one hour mobile phone radiation exposure at an average specific absorption rate of 2.8 W/kg. However, the same genes and proteins were differently affected by the exposure in each of the cell lines. This suggests that the cell response to mobile phone radiation might be genome- and proteome-dependent. Therefore, it is likely that different types of cells and from different species might respond differently to mobile phone radiation or might have different sensitivity to this weak stimulus. Our findings might also explain, at least in part, the origin of discrepancies in replication studies between different laboratories.

Adipocytes impair efficacy of antiretroviral therapy.

PubMed

Couturier, Jacob; Winchester, Lee C; Suliburk, James W; Wilkerson, Gregory K; Podany, Anthony T; Agarwal, Neeti; Xuan Chua, Corrine Ying; Nehete, Pramod N; Nehete, Bharti P; Grattoni, Alessandro; Sastry, K Jagannadha; Fletcher, Courtney V; Lake, Jordan E; Balasubramanyam, Ashok; Lewis, Dorothy E

2018-06-01

Adequate distribution of antiretroviral drugs to infected cells in HIV patients is critical for viral suppression. In humans and primates, HIV- and SIV-infected CD4 T cells in adipose tissues have recently been identified as reservoirs for infectious virus. To better characterize adipose tissue as a pharmacological sanctuary for HIV-infected cells, in vitro experiments were conducted to assess antiretroviral drug efficacy in the presence of adipocytes, and drug penetration in adipose tissue cells (stromal-vascular-fraction cells and mature adipocytes) was examined in treated humans and monkeys. Co-culture experiments between HIV-1-infected CD4 T cells and primary human adipocytes showed that adipocytes consistently reduced the antiviral efficacy of the nucleotide reverse transcriptase inhibitor tenofovir and its prodrug forms tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF). In HIV-infected persons, LC-MS/MS analysis of intracellular lysates derived from adipose tissue stromal-vascular-fraction cells or mature adipocytes suggested that integrase inhibitors penetrate adipose tissue, whereas penetration of nucleoside/nucleotide reverse transcriptase inhibitors such as TDF, emtricitabine, abacavir, and lamivudine is restricted. The limited distribution and functions of key antiretroviral drugs within fat depots may contribute to viral persistence in adipose tissue. Copyright © 2018 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2017-09-01

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

Adipocytes Impair Efficacy of Antiretroviral Therapy

PubMed Central

Couturier, Jacob; Winchester, Lee C.; Suliburk, James W.; Wilkerson, Gregory K.; Podany, Anthony T.; Agarwal, Neeti; Chua, Corrine Ying Xuan; Nehete, Pramod N.; Nehete, Bharti P.; Grattoni, Alessandro; Sastry, K. Jagannadha; Fletcher, Courtney V.; Lake, Jordan E.; Balasubramanyan, Ashok; Lewis, Dorothy E.

2018-01-01

Adequate distribution of antiretroviral drugs to infected cells in HIV patients is critical for viral suppression. In humans and primates, HIV- and SIV-infected CD4 T cells in adipose tissues have recently been identified as reservoirs for infectious virus. To better characterize adipose tissue as a pharmacological sanctuary for HIV-infected cells, in vitro experiments were conducted to assess antiretroviral drug efficacy in the presence of adipocytes, and drug penetration in adipose tissue cells (stromal-vascular-fraction cells and mature adipocytes) was examined in treated humans and monkeys. Co-culture experiments between HIV-1-infected CD4 T cells and primary human adipocytes showed that adipocytes consistently reduced the antiviral efficacy of the nucleotide reverse transcriptase inhibitor tenofovir and its prodrug forms tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF). In HIV-infected persons, LC-MS/MS analysis of intracellular lysates derived from adipose tissue stromal-vascular-fraction cells or mature adipocytes suggested that integrase inhibitors penetrate adipose tissue, whereas penetration of nucleoside/nucleotide reverse transcriptase inhibitors such as TDF, emtricitabine, abacavir, and lamivudine is restricted. The limited distribution and functions of key antiretroviral drugs within fat depots may contribute to viral persistence in adipose tissue. PMID:29630975

Differences In Early T-Cell Signaling In Cultures Grown In a Rotating Clinostat vs. Static Controls

NASA Technical Reports Server (NTRS)

Alexamder. M.; Nelman-Gonzales, M.; Penkala, J.; Sams, C.

1999-01-01

Altered gravity has previously been demonstrated to be a stress that can influence components of the immune system. Specifically, T-cell activation has been shown to be affected by changes in gravity, exhibiting a decrease in proliferative response to in vitro stimulation in microgravity. Subsequent ground based studies utilizing a rotating clinostat to model some of the effects of microgravity have been consistent with earlier flight based experiments. These ground and flight experiments have examined T-cell activation by measuring various responses including production of cytokines, DNA synthesis and the production of various cell surface activation markers. These indicators of T-cell activation were measured anywhere from 4 to 72 hours after stimulation. Prior to the work described here, the initial signaling events in T-cell activation had not been directly examined. The goal of this project was to determine how the process of early signal transduction was affected by growth in a rotating clinostat. Here we directly show a defect in signaling from TCR to MAPK in purified peripheral T-cells activated in the clinostat by OKT3/antiCD28 coated microbeads as compared to static controls.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nielsen, Nathalie; Laustsen, Christoffer; Bertelsen, Lotte Bonde, E-mail: Lotte@clin.au.dk

Endothelial progenitor cells (EPCs) represent a heterogeneous cell population that is believed to be involved in vasculogenesis. With the purpose of enhancing endothelial repair, EPCs could have a potential for future cell therapies. Due to the low amount of EPCs in the peripheral circulating blood, in vitro expansion is needed before administration to recipients and the effects of in vitro culturing is still an under-evaluated field with little knowledge of how the cells change over time in culture. The aim of this study was to use hyperpolarised carbon-13 magnetic resonance spectroscopy to profile important metabolic pathways in a population ofmore » progenitor cells and to show that cell culturing in 3D scaffolds seem to block the metabolic processes that leads to cell senescence. The metabolic breakdown of hyperpolarized [1-{sup 13}C]pyruvate was followed after injection of the substrate to a bioreactor system with EPCs either adhered to 3D printed scaffolds or kept in cell suspension. The pyruvate-to-lactate conversion was elevated in suspension of EPCs compared to the EPCs adhered to scaffolds. Furthermore in the setup with EPCs in suspension, an increase in lactate production was seen over time indicating that the older the cultures of EPCs was before using the cells for cell suspension experiments, the more lactate they produce, compared to a constant lactate level in the cells adhered to scaffolds. It could therefore be stated that cells grown first in 2D culture and subsequent prepared for cell suspension show a metabolism with higher lactate production consistent with cells senescence processes compared to cells grown first at 2D culture and subsequent in the 3D printed scaffolds, where metabolism shows no sign of metabolic shifting during the monitored period. - Highlights: • Hyperpolarized 13C MRS detects EPCs metabolic changes associated with ageing and cultivating conditions. • Increased lactate production in EPC’s correlates positively with aging. • 2D cultivation show an increased lactate production, while 3D cultivation show a maintained lactate production.« less

Numerical simulations of thermal convection on a hemisphere

NASA Astrophysics Data System (ADS)

Bruneau, C.-H.; Fischer, P.; Xiong, Y.-L.; Kellay, H.; Cyclobulle Collaboration

2018-04-01

In this paper we present numerical simulations of two-dimensional turbulent convection on a hemisphere. Recent experiments on a half soap bubble located on a heated plate have shown that such a configuration is ideal for studying thermal convection on a curved surface. Thermal convection and fluid flows on curved surfaces are relevant to a variety of situations, notably for simulating atmospheric and geophysical flows. As in experiments, our simulations show that the gradient of temperature between the base and the top of the hemisphere generates thermal plumes at the base that move up from near the equator to the pole. The movement of these plumes gives rise to a two-dimensional turbulent thermal convective flow. Our simulations turn out to be in qualitative and quantitative agreement with experiments and show strong similarities with Rayleigh-Bénard convection in classical cells where a fluid is heated from below and cooled from above. To compare to results obtained in classical Rayleigh-Bénard convection in standard three-dimensional cells (rectangular or cylindrical), a Nusselt number adapted to our geometry and a Reynolds number are calculated as a function of the Rayleigh number. We find that the Nusselt and Reynolds numbers verify scaling laws consistent with turbulent Rayleigh-Bénard convection: Nu∝Ra0.31 and Re∝Ra1/2 . Further, a Bolgiano regime is found with the Bolgiano scale scaling as Ra-1/4. All these elements show that despite the significant differences in geometry between our simulations and classical 3D cells, the scaling laws of thermal convection are robust.

miR-203a is involved in HBx-induced inflammation by targeting Rap1a

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, AiRong; Chen, Huo; Xu, ChunFang

Hepatitis B virus (HBV) causes acute and chronic hepatitis, and is one of the major causes of cirrhosis and hepatocellular carcinoma. Accumulating evidence suggests that inflammation is the key factor for liver cirrhosis and hepatocellular carcinoma. MicroRNAs play important roles in many biological processes. Here, we aim to explore the function of microRNAs in the HBX-induced inflammation. First, microarray experiment showed that HBV{sup +} liver samples expressed higher level of miR-203a compared to HBV{sup -} liver samples. To verify these alterations, HBx-coding plasmid was transfected into HepG2 cells to overexpress HBx protein. The real-time PCR results suggested that over-expression ofmore » HBx could induce up-regulation of miR-203a. To define how up-regulation of miR-203a can induce liver cells inflammation, we over-expressed miR-203a in HepG2 cells. Annexin V staining and BrdU staining suggested that overexpression of miR-203a significantly increased the cell apoptosis and proliferation, meanwhile, over-expression of miR-203a could lead to a decrease in G0/G1 phase cells and an increase in G2/M phase cells. Some cytokines production including IL-6 and IL-8 were significantly increased, but TGFβ and IFNγ were decreased in miR-203a over-expressed HepG2 cells. Luciferase reporter assay experiments, protein mass-spectrum assay and real-time PCR all together demonstrated that Rap1a was the target gene of miR-203a. Further experiments showed that these alterations were modulated through PI3K/ERK/p38/NFκB pathways. These data suggested that HBV-infection could up-regulate the expression of miR-203a, thus down regulated the expression of Rap1a and affected the PI3K/ERK/p38/NFκB pathways, finally induced the hepatitis inflammation. - Highlights: • HBX induces the over-expression of miR-203a in HepG2 cells. • miR-203a targets Rap1a to induce the inflammation in HepG2 cells. • miR-203a regulates the apoptosis and cell cycles of HepG2 cells. • miR-203a alters the MAPK signaling pathway by down-regulating the Rap1a.« less

Origin and evolution of circular waves and spirals in Dictyostelium discoideum territories.

PubMed

Pálsson, E; Cox, E C

1996-02-06

Randomly distributed Dictyostelium discoideum cells form cooperative territories by signaling to each other with cAMP. Cells initiate the process by sending out pulsatile signals, which propagate as waves. With time, circular and spiral patterns form. We show that by adding spatial and temporal noise to the levels of an important regulator of external cAMP levels, the cAMP phosphodiesterase inhibitor, we can explain the natural progression of the system from randomly firing cells to circular waves whose symmetries break to form double- and single- or multi-armed spirals. When phosphodiesterase inhibitor is increased with time, mimicking experimental data, the wavelength of the spirals shortens, and a proportion of them evolve into pairs of connected spirals. We compare these results to recent experiments, finding that the temporal and spatial correspondence between experiment and model is very close.

High-pressure resistivity technique for quasi-hydrostatic compression experiments.

PubMed

Rotundu, C R; Ćuk, T; Greene, R L; Shen, Z-X; Hemley, Russell J; Struzhkin, V V

2013-06-01

Diamond anvil cell techniques are now well established and powerful methods for measuring materials properties to very high pressure. However, high pressure resistivity measurements are challenging because the electrical contacts attached to the sample have to survive to extreme stress conditions. Until recently, experiments in a diamond anvil cell were mostly limited to non-hydrostatic or quasi-hydrostatic pressure media other than inert gases. We present here a solution to the problem by using focused ion beam ultrathin lithography for a diamond anvil cell loaded with inert gas (Ne) and show typical resistivity data. These ultrathin leads are deposited on the culet of the diamond and are attaching the sample to the anvil mechanically, therefore allowing for measurements in hydrostatic or nearly hydrostatic conditions of pressure using noble gases like Ne or He as pressure transmitting media.

Ultimobranchial body and parathyroid gland of the parrot Psittacula psittacula in response to experimental hypercalcemia.

PubMed

Swarup, K; Tewari, N P; Srivastav, A K

Psittacula psittacula when subjected to long term hypercalcemia by intramuscular injections of vitamin D2 (20,000 I.U.) on alternate days and by increasing dietary calcium, exhibit a rise in the serum calcium level after 10, 20 and 30 days of treatment as compared to their corresponding controls. The ultimobranchial cells show progressive hypertrophy up to 20th day of the treatment. From 20th day till the end of the experiment (30 days) these cells show feeble staining response. The parathyroid glands suffer from degenerative changes due to its inactivity under chronic hypercalcemia.

Alignment of cellular motility forces with tissue flow as a mechanism for efficient wound healing

PubMed Central

Basan, Markus; Elgeti, Jens; Hannezo, Edouard; Rappel, Wouter-Jan; Levine, Herbert

2013-01-01

Recent experiments have shown that spreading epithelial sheets exhibit a long-range coordination of motility forces that leads to a buildup of tension in the tissue, which may enhance cell division and the speed of wound healing. Furthermore, the edges of these epithelial sheets commonly show finger-like protrusions whereas the bulk often displays spontaneous swirls of motile cells. To explain these experimental observations, we propose a simple flocking-type mechanism, in which cells tend to align their motility forces with their velocity. Implementing this idea in a mechanical tissue simulation, the proposed model gives rise to efficient spreading and can explain the experimentally observed long-range alignment of motility forces in highly disordered patterns, as well as the buildup of tensile stress throughout the tissue. Our model also qualitatively reproduces the dependence of swirl size and swirl velocity on cell density reported in experiments and exhibits an undulation instability at the edge of the spreading tissue commonly observed in vivo. Finally, we study the dependence of colony spreading speed on important physical and biological parameters and derive simple scaling relations that show that coordination of motility forces leads to an improvement of the wound healing process for realistic tissue parameters. PMID:23345440

Design, synthesis and biological evaluation of novel 4-aminoquinazolines as dual target inhibitors of EGFR-PI3Kα.

PubMed

Ding, Huai-Wei; Deng, Cheng-Long; Li, Dan-Dan; Liu, Dan-Dan; Chai, Shao-Meng; Wang, Wei; Zhang, Yan; Chen, Kai; Li, Xin; Wang, Jian; Song, Shao-Jiang; Song, Hong-Rui

2018-02-25

The overexpression of EGFR correlates with rapidly progressive disease, resistance to chemotherapy and poor prognosis. In certain human cancers, PI3K works synergistically with EGFR to promote proliferation, survival, invasion and metastasis. Development of dual-target drugs against EGFR and PI3K has therapeutic advantage and was an attractive approach against tumors. In this work, based on the molecular docking and previous studies, a series of 4-aminoquinazolines derivatives containing 6-sulfonamide substituted pyridyl group were rationally designed and identified as potent EGFR and PI3K dual inhibitors. The cytotoxicity experiment results showed that this series of compounds could effectively inhibit cell growth. The kinase assay demonstrated that 6c and 6i had high inhibition for EGFR and selectivity for PI3Kα distinguished from other isoforms. Further experiments showed that 6c could induce cell cycle arrest in G1 phase and apoptosis in BT549 cells. The western blot assay indicated that 6c inhibited the proliferation of BT549 cell through EGFR and PI3Kα/Akt signaling pathway. Our study suggested that compound 6c was a potential dual inhibitors of EGFR and PI3Kα. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Microgravity

NASA Image and Video Library

2001-01-24

Interior of a Spacehab module showing the type of rack mounting that will be used, and crew working space that will be available, on the STS-107 Research 1 mission in 2002. Experiments plarned for the mission include soil mechanics, combustion physics, and cell science.

Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Xin-xing; Wang, Jian, E-mail: dr_wangjian@yahoo.com.cn; Wang, Hao-lu

2012-03-23

Highlights: Black-Right-Pointing-Pointer We sorted SP cells from a human gallbladder carcinoma cell lines, SGC-996. Black-Right-Pointing-Pointer SP cells displayed higher proliferation and stronger clonal-generating capability. Black-Right-Pointing-Pointer SP cells showed more migratory and invasive abilities. Black-Right-Pointing-Pointer SP cells were more resistant and tumorigenic than non-SP counterparts. Black-Right-Pointing-Pointer ABCG2 might be a candidate as a marker for SP cells. -- Abstract: The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substratemore » Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer.« less

Repression of mesodermal fate by foxa, a key endoderm regulator of the sea urchin embryo.

PubMed

Oliveri, Paola; Walton, Katherine D; Davidson, Eric H; McClay, David R

2006-11-01

The foxa gene is an integral component of the endoderm specification subcircuit of the endomesoderm gene regulatory network in the Strongylocentrotus purpuratus embryo. Its transcripts become confined to veg2, then veg1 endodermal territories, and, following gastrulation, throughout the gut. It is also expressed in the stomodeal ectoderm. gatae and otx genes provide input into the pregastrular regulatory system of foxa, and Foxa represses its own transcription, resulting in an oscillatory temporal expression profile. Here, we report three separate essential functions of the foxa gene: it represses mesodermal fate in the veg2 endomesoderm; it is required in postgastrular development for the expression of gut-specific genes; and it is necessary for stomodaeum formation. If its expression is reduced by a morpholino, more endomesoderm cells become pigment and other mesenchymal cell types, less gut is specified, and the larva has no mouth. Experiments in which blastomere transplantation is combined with foxa MASO treatment demonstrate that, in the normal endoderm, a crucial role of Foxa is to repress gcm expression in response to a Notch signal, and hence to repress mesodermal fate. Chimeric recombination experiments in which veg2, veg1 or ectoderm cells contained foxa MASO show which region of foxa expression controls each of the three functions. These experiments show that the foxa gene is a component of three distinct embryonic gene regulatory networks.

Novel Anthra[1,2-c][1,2,5]Thiadiazole-6,11-Diones as Promising Anticancer Lead Compounds: Biological Evaluation, Characterization & Molecular Targets Determination.

PubMed

Ali, Ahmed Atef Ahmed; Lee, Yu-Ru; Chen, Tsung-Chih; Chen, Chun-Liang; Lee, Chia-Chung; Shiau, Chia-Yang; Chiang, Chiao-Hsi; Huang, Hsu-Shan

2016-01-01

The novel compounds NSC745885 and NSC757963 developed at our laboratory were tested against a panel of 60 cancer cell lines at the National Cancer Institute, USA, and a panel of 39 cancer cell lines at the Japanese Foundation of Cancer Research. Both compounds demonstrated selective unique multi-log differential patterns of activity, with GI50 values in the sub-micro molar range against cancer cells rather than normal cardiac cells. NSC757963 showed high selectivity towards the leukemia subpanel. Activities of both compounds strongly correlated to expression of NFKB1 and CSNK2B genes, implying that they may inhibit the NF-κB pathway. Immunocytochemical microscopy of OVCAR-3 cells showed clear cytosolic accumulation of the NF-κB p65 subunit following treatment. Western blotting showed dose dependent inhibition of the nuclear expression of the NF-κB p65 subunit with subsequent accumulation in the cytosol following treatment. Docking experiments showed binding of both compounds to the NF-κB activator IKKβ subunit preventing its translocation to the nucleus. Collectively, these results confirm the ability of our compounds to inhibit the constitutively active NF-κB pathway of OVCAR-3 cells. Furthermore, COMPARE analysis indicated that the activity of NSC757963 is similar to the antituberculosis agent rifamycin SV, this was confirmed by testing the antimycobacterial activity of NSC757963 against Mycobacterium tuberculosis, results revealed potent activity suitable for use in clinical practice. Molecular properties and Lipinski's parameters predicted acceptable bioavailability properties with no indication of mutagenicity, tumorigenicity, irritability and reproductive effects. Oral absorption experiments using the human Caco-2 model showed high intestinal absorption of NSC745885 by passive transport mechanism with no intestinal efflux or active transport mechanisms. The unique molecular characterization as well as the illustrated anticancer spectra of activity and bioavailability properties warrant further development of our compounds and present a foundation brick in the pre-clinical investigations to implement such compounds in clinical practice.

CO2 laser annealing of 50-microns-thick silicon solar cells

NASA Technical Reports Server (NTRS)

Walker, F. E.

1979-01-01

A test program is conducted to determine thin solar cell annealing effects using a laser energy source. A CO2 continuous-wave laser was used in annealing experiments on 50 micrometers-thick silicon solar cells after proton irradiation. Test cells were irradiated to a fluence of 1.0 x 10 to the 12th power protons/sq cm with 1.9 MeV protons. After irradiation, those cells receiving full proton dosage were degraded by an average of 30% in output power. In annealing tests laser beam exposure times on the solar cell varied from 2 seconds to 16 seconds reaching cell temperatures of from 400 C to 500 C. Under those conditions annealing test results showed recovery in cell output power of from 33% to 90%.

Child Maltreatment Is Associated with a Reduction of the Oxytocin Receptor in Peripheral Blood Mononuclear Cells

PubMed Central

Krause, Sabrina; Boeck, Christina; Gumpp, Anja M.; Rottler, Edit; Schury, Katharina; Karabatsiakis, Alexander; Buchheim, Anna; Gündel, Harald; Kolassa, Iris-Tatjana; Waller, Christiane

2018-01-01

Background: Child maltreatment (CM) and attachment experiences are closely linked to alterations in the human oxytocin (OXT) system. However, human data about oxytocin receptor (OXTR) protein levels are lacking. Therefore, we investigated oxytocin receptor (OXTR) protein levels in circulating immune cells and related them to circulating levels of OXT in peripheral blood. We hypothesized reduced OXTR protein levels, associated with both, experiences of CM and an insecure attachment representation. Methods: OXTR protein expressions were analyzed by western blot analyses in peripheral blood mononuclear cells (PBMC) and plasma OXT levels were determined by radioimmunoassay (RIA) in 49 mothers. We used the Childhood Trauma Questionnaire (CTQ) to assess adverse childhood experiences. Attachment representations (secure vs. insecure) were classified using the Adult Attachment Projective Picture System (AAP) and levels of anxiety and depression were assessed with the German version of the Hospital Depression and Anxiety scale (HADS-D). Results: CM-affected women showed significantly lower OXTR protein expression with significantly negative correlations between the OXTR protein expression and the CTQ sum score, whereas plasma OXT levels showed no significant differences in association with CM. Lower OXTR protein expression in PBMC were particularly pronounced in the group of insecurely attached mothers compared to the securely attached group. Anxiety levels were significantly higher in CM-affected women. Conclusion: This study demonstrated a significant association between CM and an alteration of OXTR protein expression in human blood cells as a sign for chronic, long-lasting alterations in this attachment-related neurobiological system. PMID:29535656

Alignment of cell division axes in directed epithelial cell migration

NASA Astrophysics Data System (ADS)

Marel, Anna-Kristina; Podewitz, Nils; Zorn, Matthias; Oskar Rädler, Joachim; Elgeti, Jens

2014-11-01

Cell division is an essential dynamic event in tissue remodeling during wound healing, cancer and embryogenesis. In collective migration, tensile stresses affect cell shape and polarity, hence, the orientation of the cell division axis is expected to depend on cellular flow patterns. Here, we study the degree of orientation of cell division axes in migrating and resting epithelial cell sheets. We use microstructured channels to create a defined scenario of directed cell invasion and compare this situation to resting but proliferating cell monolayers. In experiments, we find a strong alignment of the axis due to directed flow while resting sheets show very weak global order, but local flow gradients still correlate strongly with the cell division axis. We compare experimental results with a previously published mesoscopic particle based simulation model. Most of the observed effects are reproduced by the simulations.

Subcollicular projections to the auditory thalamus and collateral projections to the inferior colliculus.

PubMed

Schofield, Brett R; Mellott, Jeffrey G; Motts, Susan D

2014-01-01

Experiments in several species have identified direct projections to the medial geniculate nucleus (MG) from cells in subcollicular auditory nuclei. Moreover, many cochlear nucleus cells that project to the MG send collateral projections to the inferior colliculus (IC) (Schofield et al., 2014). We conducted three experiments to characterize projections to the MG from the superior olivary and the lateral lemniscal regions in guinea pigs. For experiment 1, we made large injections of retrograde tracer into the MG. Labeled cells were most numerous in the superior paraolivary nucleus, ventral nucleus of the trapezoid body, lateral superior olivary nucleus, ventral nucleus of the lateral lemniscus, ventrolateral tegmental nucleus, paralemniscal region and sagulum. Additional sources include other periolivary nuclei and the medial superior olivary nucleus. The projections are bilateral with an ipsilateral dominance (66%). For experiment 2, we injected tracer into individual MG subdivisions. The results show that the subcollicular projections terminate primarily in the medial MG, with the dorsal MG a secondary target. The variety of projecting nuclei suggest a range of functions, including monaural and binaural aspects of hearing. These direct projections could provide the thalamus with some of the earliest (i.e., fastest) information regarding acoustic stimuli. For experiment 3, we made large injections of different retrograde tracers into one MG and the homolateral IC to identify cells that project to both targets. Such cells were numerous and distributed across many of the nuclei listed above, mostly ipsilateral to the injections. The prominence of the collateral projections suggests that the same information is delivered to both the IC and the MG, or perhaps that a common signal is being delivered as a preparatory indicator or temporal reference point. The results are discussed from functional and evolutionary perspectives.

Subcollicular projections to the auditory thalamus and collateral projections to the inferior colliculus

PubMed Central

Schofield, Brett R.; Mellott, Jeffrey G.; Motts, Susan D.

2014-01-01

Experiments in several species have identified direct projections to the medial geniculate nucleus (MG) from cells in subcollicular auditory nuclei. Moreover, many cochlear nucleus cells that project to the MG send collateral projections to the inferior colliculus (IC) (Schofield et al., 2014). We conducted three experiments to characterize projections to the MG from the superior olivary and the lateral lemniscal regions in guinea pigs. For experiment 1, we made large injections of retrograde tracer into the MG. Labeled cells were most numerous in the superior paraolivary nucleus, ventral nucleus of the trapezoid body, lateral superior olivary nucleus, ventral nucleus of the lateral lemniscus, ventrolateral tegmental nucleus, paralemniscal region and sagulum. Additional sources include other periolivary nuclei and the medial superior olivary nucleus. The projections are bilateral with an ipsilateral dominance (66%). For experiment 2, we injected tracer into individual MG subdivisions. The results show that the subcollicular projections terminate primarily in the medial MG, with the dorsal MG a secondary target. The variety of projecting nuclei suggest a range of functions, including monaural and binaural aspects of hearing. These direct projections could provide the thalamus with some of the earliest (i.e., fastest) information regarding acoustic stimuli. For experiment 3, we made large injections of different retrograde tracers into one MG and the homolateral IC to identify cells that project to both targets. Such cells were numerous and distributed across many of the nuclei listed above, mostly ipsilateral to the injections. The prominence of the collateral projections suggests that the same information is delivered to both the IC and the MG, or perhaps that a common signal is being delivered as a preparatory indicator or temporal reference point. The results are discussed from functional and evolutionary perspectives. PMID:25100950

Experience-Dependent Plasticity in Accessory Olfactory Bulb Interneurons following Male-Male Social Interaction.

PubMed

Cansler, Hillary L; Maksimova, Marina A; Meeks, Julian P

2017-07-26

Chemosensory information processing in the mouse accessory olfactory system guides the expression of social behavior. After salient chemosensory encounters, the accessory olfactory bulb (AOB) experiences changes in the balance of excitation and inhibition at reciprocal synapses between mitral cells (MCs) and local interneurons. The mechanisms underlying these changes remain controversial. Moreover, it remains unclear whether MC-interneuron plasticity is unique to specific behaviors, such as mating, or whether it is a more general feature of the AOB circuit. Here, we describe targeted electrophysiological studies of AOB inhibitory internal granule cells (IGCs), many of which upregulate the immediate-early gene Arc after male-male social experience. Following the resident-intruder paradigm, Arc -expressing IGCs in acute AOB slices from resident males displayed stronger excitation than nonexpressing neighbors when sensory inputs were stimulated. The increased excitability of Arc -expressing IGCs was not correlated with changes in the strength or number of excitatory synapses with MCs but was instead associated with increased intrinsic excitability and decreased HCN channel-mediated I H currents. Consistent with increased inhibition by IGCs, MCs responded to sensory input stimulation with decreased depolarization and spiking following resident-intruder encounters. These results reveal that nonmating behaviors drive AOB inhibitory plasticity and indicate that increased MC inhibition involves intrinsic excitability changes in Arc -expressing interneurons. SIGNIFICANCE STATEMENT The accessory olfactory bulb (AOB) is a site of experience-dependent plasticity between excitatory mitral cells (MCs) and inhibitory internal granule cells (IGCs), but the physiological mechanisms and behavioral conditions driving this plasticity remain unclear. Here, we report studies of AOB neuronal plasticity following male-male social chemosensory encounters. We show that the plasticity-associated immediate-early gene Arc is selectively expressed in IGCs from resident males following the resident-intruder assay. After behavior, Arc -expressing IGCs are more strongly excited by sensory input stimulation and MC activation is suppressed. Arc -expressing IGCs do not show increased excitatory synaptic drive but instead show increased intrinsic excitability. These data indicate that MC-IGC plasticity is induced after male-male social chemosensory encounters, resulting in enhanced MC suppression by Arc -expressing IGCs. Copyright © 2017 the authors 0270-6474/17/377240-13$15.00/0.

Experience-Dependent Plasticity in Accessory Olfactory Bulb Interneurons following Male–Male Social Interaction

PubMed Central

Maksimova, Marina A.

2017-01-01

Chemosensory information processing in the mouse accessory olfactory system guides the expression of social behavior. After salient chemosensory encounters, the accessory olfactory bulb (AOB) experiences changes in the balance of excitation and inhibition at reciprocal synapses between mitral cells (MCs) and local interneurons. The mechanisms underlying these changes remain controversial. Moreover, it remains unclear whether MC–interneuron plasticity is unique to specific behaviors, such as mating, or whether it is a more general feature of the AOB circuit. Here, we describe targeted electrophysiological studies of AOB inhibitory internal granule cells (IGCs), many of which upregulate the immediate-early gene Arc after male–male social experience. Following the resident–intruder paradigm, Arc-expressing IGCs in acute AOB slices from resident males displayed stronger excitation than nonexpressing neighbors when sensory inputs were stimulated. The increased excitability of Arc-expressing IGCs was not correlated with changes in the strength or number of excitatory synapses with MCs but was instead associated with increased intrinsic excitability and decreased HCN channel-mediated IH currents. Consistent with increased inhibition by IGCs, MCs responded to sensory input stimulation with decreased depolarization and spiking following resident–intruder encounters. These results reveal that nonmating behaviors drive AOB inhibitory plasticity and indicate that increased MC inhibition involves intrinsic excitability changes in Arc-expressing interneurons. SIGNIFICANCE STATEMENT The accessory olfactory bulb (AOB) is a site of experience-dependent plasticity between excitatory mitral cells (MCs) and inhibitory internal granule cells (IGCs), but the physiological mechanisms and behavioral conditions driving this plasticity remain unclear. Here, we report studies of AOB neuronal plasticity following male–male social chemosensory encounters. We show that the plasticity-associated immediate-early gene Arc is selectively expressed in IGCs from resident males following the resident–intruder assay. After behavior, Arc-expressing IGCs are more strongly excited by sensory input stimulation and MC activation is suppressed. Arc-expressing IGCs do not show increased excitatory synaptic drive but instead show increased intrinsic excitability. These data indicate that MC–IGC plasticity is induced after male–male social chemosensory encounters, resulting in enhanced MC suppression by Arc-expressing IGCs. PMID:28659282

Application of laser tweezers Raman spectroscopy techniques to the monitoring of single cell response to stimuli

NASA Astrophysics Data System (ADS)

Chan, James W.; Liu, Rui; Matthews, Dennis L.

2012-06-01

Laser tweezers Raman spectroscopy (LTRS) combines optical trapping with micro-Raman spectroscopy to enable label-free biochemical analysis of individual cells and small biological particles in suspension. The integration of the two technologies greatly simplifies the sample preparation and handling of suspension cells for spectroscopic analysis in physiologically meaningful conditions. In our group, LTRS has been used to study the effects of external perturbations, both chemical and mechanical, on the biochemistry of the cell. Single cell dynamics can be studied by performing longitudinal studies to continuously monitor the response of the cell as it interacts with its environment. The ability to carry out these measurements in-vitro makes LTRS an attractive tool for many biomedical applications. Here, we discuss the use of LTRS to study the response of cancer cells to chemotherapeutics and bacteria cells to antibiotics and show that the life cycle and apoptosis of the cells can be detected. These results show the promise of LTRS for drug discovery/screening, antibiotic susceptibility testing, and chemotherapy response monitoring applications. In separate experiments, we study the response of red blood cells to the mechanical forces imposed on the cell by the optical tweezers. A laser power dependent deoxygenation of the red blood cell in the single beam trap is reported. Normal, sickle cell, and fetal red blood cells have a different behavior that enables the discrimination of the cell types based on this mechanochemical response. These results show the potential utility of LTRS for diagnosing and studying red blood cell diseases.

Experiment K-7-22: Growth Hormone Regulation Synthesis and Secretion in Microgravity. Part 1; Growth Hormone Regulation Synthesis and Secretion in Microgravity

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Grindeland, R.; Vale, W.; Sawchenko, P.; Ilyina-Kakueva, E. I.

1994-01-01

Changes in the musculoskeletal, immune, vascular, and endocrine system of the rat occur as a result of short-term spaceflight. Since pituitary gland growth hormone (GH) plays a role in the control of these systems, and since the results of an earlier spaceflight mission (Spacelab 3, 1985) showed that GH cell function was compromised in a number of post-flight tests, we repeated and extended the 1985 experiment in two subsequent spaceflights: the 12.5 day mission of Cosmos 1887 (in 1987) and the 14 day mission of Cosmos 2044 (in 1989). The results of these later two flight experiments are the subject of this report. They document repeatable and significant changes in the GH cell system of the spaceflown rat in several post-flight tests.

Fibrin gel as a scaffold for skin substitute – production and clinical experience.

PubMed

Kljenak, Antun; Tominac Trcin, Mirna; Bujić, Marina; Dolenec, Tamara; Jevak, Martina; Mršić, Gordan; Zmiš, Gordana; Barčot, Zoran; Muljačić, Ante; Popović, Maja

2016-06-01

The purpose of this study was to create a fibrin-based human skin substitute in vitro with epidermal and dermal component and to assess its healing potential in deep partial and full thickness burns. Fibrin scaffolds were prepared from commercial fibrin glue kits. Human fibroblasts were cultured in fibrin gel. Human keratinocytes were seeded on the top of the gel. Viability of cells was determined fluorimetrically. Scanning electron microscope and immunocytochemistry analysis of cultured cells were performed. After hydrosurgical preparation of deep burn necrotic tissue, wound bed was prepared for skin substitutes. Progress of healing was documented using visual estimation and photos. Scanning electron microscope images showed good cell attachment and colony spreading of keratinocytes and fibroblasts on fibrin scaff old. Immunofluorescent staining of cell cultures on fibrin scaffold showed expression of vimentin, a marker of fibroblast cells, cytokeratin 19, a marker of epithelial stem cells, as well as involucrin, a marker of differentiated keratinocytes. Clinical results clearly showed that appearance of the skin did not differ significantly from the areas of transplanted skin using split-thickness skin graft techniques. In conclusion, using these fibrin-cultured autografts on massive full-thickness burn resulted in good healing.

Silk fibroin nanoparticles prepared by electrospray as controlled release carriers of cisplatin.

PubMed

Qu, Jing; Liu, Yu; Yu, Yanni; Li, Jing; Luo, Jingwan; Li, Mingzhong

2014-11-01

To maintain the anti-tumor activity of cis-dichlorodiamminoplatinum (CDDP) while avoiding its cytotoxicity and negative influence on normal tissue, CDDP-loaded silk fibroin nanoparticles approximately 59 nm in diameter were successfully prepared by electrospray without using organic solvent. CDDP was incorporated into nanoparticles through metal-polymer coordination bond exchange. In vitro release tests showed that the cisplatin in the nanoparticles could be slowly and sustainably released for more than 15 days. In vitro anti-cancer experiments and intracellular Pt content testing indicated that CDDP-loaded silk fibroin nanoparticles were easily internalized by A549 lung cancer cells, transferring CDDP into cancer cells and then triggering their apoptosis. In contrast, the particles were not easily internalized by L929 mouse fibroblast cells and hence showed weaker cell growth inhibition. The CDDP-loaded silk fibroin nanoparticles showed sustained and efficient killing of tumor cells but weaker inhibition of normal cells. In general, this study provides not only a novel method for preparing CDDP-loaded silk fibroin nanoparticles but also a new carrier system for clinical therapeutic drugs against lung cancers and other tumors. Copyright © 2014 Elsevier B.V. All rights reserved.

Lack of WDR36 leads to preimplantation embryonic lethality in mice and delays the formation of small subunit ribosomal RNA in human cells in vitro.

PubMed

Gallenberger, Martin; Meinel, Dominik M; Kroeber, Markus; Wegner, Michael; Milkereit, Philipp; Bösl, Michael R; Tamm, Ernst R

2011-02-01

Mutations in WD repeat domain 36 gene (WDR36) play a causative role in some forms of primary open-angle glaucoma, a leading cause of blindness worldwide. WDR36 is characterized by the presence of multiple WD40 repeats and shows homology to Utp21, an essential protein component of the yeast small subunit (SSU) processome required for maturation of 18S rRNA. To clarify the functional role of WDR36 in the mammalian organism, we generated and investigated mutant mice with a targeted deletion of Wdr36. In parallel experiments, we used RNA interference to deplete WDR36 mRNA in mouse embryos and cultured human trabecular meshwork (HTM-N) cells. Deletion of Wdr36 in the mouse caused preimplantation embryonic lethality, and essentially similar effects were observed when WDR36 mRNA was depleted in mouse embryos by RNA interference. Depletion of WDR36 mRNA in HTM-N cells caused apoptotic cell death and upregulation of mRNA for BAX, TP53 and CDKN1A. By immunocytochemistry, staining for WDR36 was observed in the nucleolus of cells, which co-localized with that of nucleolar proteins such as nucleophosmin and PWP2. In addition, recombinant and epitope-tagged WDR36 localized to the nucleolus of HTM-N cells. By northern blot analysis, a substantial decrease in 21S rRNA, the precursor of 18S rRNA, was observed following knockdown of WDR36. In addition, metabolic-labeling experiments consistently showed a delay of 18S rRNA maturation in WDR36-depleted cells. Our results provide evidence that WDR36 is an essential protein in mammalian cells which is involved in the nucleolar processing of SSU 18S rRNA.

Reduction of peritoneal carcinomatosis by intraperitoneal administration of phospholipids in rats

PubMed Central

Otto, Jens; Jansen, Petra Lynen; Lucas, Stefan; Schumpelick, Volker; Jansen, Marc

2007-01-01

Background Intraperitoneal tumor cell attachment after resection of gastrointestinal cancer may lead to a developing of peritoneal carcinosis. Intraabdominal application of phospholipids shows a significant decrease of adhesion formation even in case of rising tumor cell concentration. Methods In experiment A 2*106 colonic tumor cells (DHD/K12/Trb) were injected intraperitonely in female BD-IX-rats. A total of 30 rats were divided into three groups with treatments of phospholipids at 6% or 9% and the control group. In experiment B a total of 100 rats were divided into ten groups with treatments of phospholipids at 9% and the control group. A rising concentration of tumor cells (10,000, 50,000, 100,000, 250,000 and 500,000) were injected intraperitonely in female BD-IX-rats of the different groups. After 30 days, the extent of peritoneal carcinosis was determined by measuring the tumor volume, the area of attachment and the Peritoneal Cancer Index (PCI). Results In experiment A, we found a significant reduction (control group: tumor volume: 12.0 ± 4.9 ml; area of tumor adhesion: 2434.4 ± 766 mm2; PCI 28.5 ± 10.0) of peritoneal dissemination according to all evaluation methods after treatment with phospholipids 6% (tumor volume: 5.2 ± 2.2 ml; area of tumor adhesion: 1106.8 ± 689 mm2; PCI 19.0 ± 5.0) and phospholipids 9% (tumor volume: 4.0 ± 3.5 ml; area of tumor adhesion: 362.7 ± 339 mm2; PCI 13.8 ± 5.1). In experiment B we found a significant reduction of tumor volume in all different groups of rising tumor cell concentration compared to the control. As detected by the area of attachment we found a significant reduction in the subgroups 1*104, 25*104 and 50*104. The reduction in the other subgroups shows no significance. The PCI could be reduced significantly in all subgroups apart from 5*104. Conclusion In this animal study intraperitoneal application of phospholipids resulted in reduction of the extent of peritoneal carcinomatosis after intraperitoneal administration of free tumor cells. This effect was exceptionally noticed when the amount of intraperitoneal tumor cells was limited. Consequently, intraperitoneal administration of phospholipids might be effective in reducing peritoneal carcinomatosis after surgery of gastrointestinal tumors in humans. PMID:17584925

Ell3 stimulates proliferation, drug resistance, and cancer stem cell properties of breast cancer cells via a MEK/ERK-dependent signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahn, Hee-Jin; Kim, Gwangil; Park, Kyung-Soon, E-mail: kspark@cha.ac.kr

2013-08-09

Highlights: •Ell3 enhances proliferation and drug resistance of breast cancer cell lines. •Ell3 is related to the cancer stem cell characteristics of breast cancer cell lines. •Ell3 enhances oncogenicity of breast cancer through the ERK1/2 signaling pathway. -- Abstract: Ell3 is a RNA polymerase II transcription elongation factor that is enriched in testis. The C-terminal domain of Ell3 shows strong similarities to that of Ell (eleven−nineteen lysine-rich leukemia gene), which acts as a negative regulator of p53 and regulates cell proliferation and survival. Recent studies in our laboratory showed that Ell3 induces the differentiation of mouse embryonic stem cells bymore » protecting differentiating cells from apoptosis via the promotion of p53 degradation. In this study, we evaluated the function of Ell3 in breast cancer cell lines. MCF-7 cell lines overexpressing Ell3 were used to examine cell proliferation and cancer stem cell properties. Ectopic expression of Ell3 in breast cancer cell lines induces proliferation and 5-FU resistance. In addition, Ell3 expression increases the cancer stem cell population, which is characterized by CD44 (+) or ALDH1 (+) cells. Mammosphere-forming potential and migration ability were also increased upon Ell3 expression in breast cancer cell lines. Through biochemical and molecular biological analyses, we showed that Ell3 regulates proliferation, cancer stem cell properties and drug resistance in breast cancer cell lines partly through the MEK−extracellular signal-regulated kinase signaling pathway. Murine xenograft experiments showed that Ell3 expression promotes tumorigenesis in vivo. These results suggest that Ell3 may play a critical role in promoting oncogenesis in breast cancer by regulating cell proliferation and cancer stem cell properties via the ERK1/2 signaling pathway.« less

Cell-to-Cell Heterogeneity in Cortical Tension Specifies Curvature of Contact Surfaces in Caenorhabditis elegans Embryos

PubMed Central

Fujita, Masashi; Onami, Shuichi

2012-01-01

In the two-cell stage embryos of Caenorhabditis elegans, the contact surface of the two blastomeres forms a curve that bulges from the AB blastomere to the P1 blastomere. This curve is a consequence of the high intracellular hydrostatic pressure of AB compared with that of P1. However, the higher pressure in AB is intriguing because AB has a larger volume than P1. In soap bubbles, which are a widely used model of cell shape, a larger bubble has lower pressure than a smaller bubble. Here, we reveal that the higher pressure in AB is mediated by its higher cortical tension. The cell fusion experiments confirmed that the curvature of the contact surface is related to the pressure difference between the cells. Chemical and genetic interferences showed that the pressure difference is mediated by actomyosin. Fluorescence imaging indicated that non-muscle myosin is enriched in the AB cortex. The cell killing experiments provided evidence that AB but not P1 is responsible for the pressure difference. Computer simulation clarified that the cell-to-cell heterogeneity of cortical tensions is indispensable for explaining the pressure difference. This study demonstrates that heterogeneity in surface tension results in significant deviations of cell behavior compared to simple soap bubble models, and thus must be taken into consideration in understanding cell shape within embryos. PMID:22253922

JPRS Report, Science & Technology, USSR: Life Sciences.

DTIC Science & Technology

1987-08-04

alpha 2 preparation, produced by E. coli cells, is described and discussed. Cytokinin activity was determined by inducement of betacyanin synthesis in...the dark by cytokinin in the presence of thyrosine, used as a substrate. Experiments with A. caudatus sprouts showed that IFN alpha 2 produced...of IFN- alpha 2 appeared at concentration of 0.1 unit/ml but increase of concentration reduced cytokinin activity. In control experiments, induction

[Efficacy of using zinc oxide nanoparticles in nutrition. Experiments on the laboratory animal].

PubMed

Raspopov, R V; Trushina, E N; Mustafina, O K; Tananova, O N; Gmoshinskiĭ, I V; Khotimchenko, S A

2011-01-01

In experiments on rats there was researched bioavailability of zinc oxide (ZnO) nanoparticles. There were determined the content of Zn in blood serum and tibia, intestinal uptake of macromolecules of egg albumin, some hematological, biochemical and immune indices, liver cells apoptosis. The results obtained show that the uptake of nanoparticles of ZnO enables restoration of this microelement status damaged by zinc deficit diet.

All-fiber gas sensor with intracavity photothermal spectroscopy.

PubMed

Zhao, Yan; Jin, Wei; Lin, Yuechuan; Yang, Fan; Ho, Hoi Lut

2018-04-01

We present an all-fiber intracavity photothermal (IC-PT) spectroscopic gas sensor with a hollow-core photonic bandgap fiber (HC-PBF) gas cell. The gas cell is placed inside a fiber-ring laser cavity to achieve higher laser light intensity in the hollow core and hence higher PT modulation signal. An experiment with a 0.62-m-long HC-PBF gas cell demonstrated a noise equivalent concentration of 176 ppb acetylene. Theoretical modeling shows that the IC-PT sensor has the potential of achieving sub-ppb (parts-per-billion) acetylene detection sensitivity.

Proton dynamics of phosphoric acid in HT-PEFCs: Towards "operando" experiments

NASA Astrophysics Data System (ADS)

Khaneft, Marina; Shuai, Liu; Lin, Yu; Janßen, Holger; Lüke, Wiebke; Zorn, Reiner; Ivanova, Oxana; Radulescu, Aurel; Holderer, Olaf; Lehnert, Werner

2018-05-01

High Temperature Polymer Electrolyte Fuel Cells (HT-PEFCs) have been studied with quasielastic neutron scattering, which gives access to the proton diffusion in the fuel cell on local length- and timescales. So far, the different components such as the proton conducting membrane and the electrode layers have been studied separately. Here we show that also operating fuel cells can be investigated and the proton diffusion can be measured under real working conditions. The proton diffusion during power production is compared to that "at rest" but at elevated temperatures.

Biomimicry 1: PC.

PubMed

Cumberland, D C; Gunn, J; Malik, N; Holt, C M

1998-01-01

The surface properties of stents can be modified by coating them, for example with a polymer. Phosphorylcoline (PC) is the major component of the outer layer of the cell membrane. The haemo- and biocompatibility of a PC-containing polymer is thus based on biomimicry, and has been confirmed by several experiments showing much reduced thrombogenicity of PC-coated surfaces, and porcine coronary artery implants showing no sign of adverse effect. Clinical experience with the PC-coated BiodivYsio appears favourable. The PC coating can be tailored for take up and controlled elution of various drugs for stent-based local delivery, a property which is being actively explored.

Mechanism for Collective Cell Alignment in Myxococcus xanthus Bacteria

PubMed Central

Balagam, Rajesh; Igoshin, Oleg A.

2015-01-01

Myxococcus xanthus cells self-organize into aligned groups, clusters, at various stages of their lifecycle. Formation of these clusters is crucial for the complex dynamic multi-cellular behavior of these bacteria. However, the mechanism underlying the cell alignment and clustering is not fully understood. Motivated by studies of clustering in self-propelled rods, we hypothesized that M. xanthus cells can align and form clusters through pure mechanical interactions among cells and between cells and substrate. We test this hypothesis using an agent-based simulation framework in which each agent is based on the biophysical model of an individual M. xanthus cell. We show that model agents, under realistic cell flexibility values, can align and form cell clusters but only when periodic reversals of cell directions are suppressed. However, by extending our model to introduce the observed ability of cells to deposit and follow slime trails, we show that effective trail-following leads to clusters in reversing cells. Furthermore, we conclude that mechanical cell alignment combined with slime-trail-following is sufficient to explain the distinct clustering behaviors observed for wild-type and non-reversing M. xanthus mutants in recent experiments. Our results are robust to variation in model parameters, match the experimentally observed trends and can be applied to understand surface motility patterns of other bacterial species. PMID:26308508

Analysis of cell flux in the parallel plate flow chamber: implications for cell capture studies.

PubMed Central

Munn, L L; Melder, R J; Jain, R K

1994-01-01

The parallel plate flow chamber provides a controlled environment for determinations of the shear stress at which cells in suspension can bind to endothelial cell monolayers. By decreasing the flow rate of cell-containing media over the monolayer and assessing the number of cells bound at each wall shear stress, the relationship between shear force and binding efficiency can be determined. The rate of binding should depend on the delivery of cells to the surface as well as the intrinsic cell-surface interactions; thus, only if the cell flux to the surface is known can the resulting binding curves be interpreted correctly. We present the development and validation of a mathematical model based on the sedimentation rate and velocity profile in the chamber for the delivery of cells from a flowing suspension to the chamber surface. Our results show that the flux depends on the bulk cell concentration, the distance from the entrance point, and the flow rate of the cell-containing medium. The model was then used in a normalization procedure for experiments in which T cells attach to TNF-alpha-stimulated HUVEC monolayers, showing that a threshold for adhesion occurs at a shear stress of about 3 dyn/cm2. Images FIGURE 1 FIGURE 2 PMID:7948702

P2X7 receptor expression levels determine lethal effects of a purine based danger signal in T lymphocytes.

PubMed

Aswad, Fred; Dennert, Gunther

2006-09-01

Contact of T lymphocytes with nicotinamide adenine dinucleotide (NAD) or ATP causes cell death that requires expression of purinergic receptor P2X(7) (P2X(7)R). T cell subsets differ in their responses to NAD and ATP, which awaits a mechanistic explanation. Here, we show that sensitivity to ATP correlates with P2X(7)R expression levels in CD4 cells, CD8 cells and CD4(+)CD25(+) cells from both C57BL/6 and BALB/c mice. But P2X(7)R ligands do not only induce cell death but also shedding of CD62L. It is shown here that in CD62L(high) T cells, CD62L shedding correlates with low expression of P2X(7)Rs and lower cell death, whereas in CD62L(low) cells P2X(7)R expression and death are higher. The possibility is therefore investigated that P2X(7)Rs induce T cell activation. Experiments show that spontaneous T cell proliferation is somewhat higher in cells expressing P2X(7)Rs, but this effect we suggest is caused by P2X(7)R expression on accessory cells.

Expression of checkpoint molecules on myeloid-derived suppressor cells.

PubMed

Ballbach, Marlene; Dannert, Angelika; Singh, Anurag; Siegmund, Darina M; Handgretinger, Rupert; Piali, Luca; Rieber, Nikolaus; Hartl, Dominik

2017-12-01

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population expanded in cancer, infection and autoimmunity capable of suppressing T-cell functions. Checkpoint inhibitors have emerged as a key therapeutic strategy in immune-oncology. While checkpoint molecules were initially associated with T cell functions, recent evidence suggests a broader expression and function in innate myeloid cells. Previous studies provided first evidence for a potential role for checkpoints on MDSCs, yet the human relevance remained poorly understood. Therefore, we investigated the expression and functional relevance of checkpoint molecules in human MDSC-T-cell interactions. Our studies demonstrate that programmed death-ligand 1 (PD-L1) is expressed on granulocytic MDSCs upon co-culture with T cells. Transwell experiments showed that cell-to-cell contact was required for MDSC-T-cell interactions and antibody blocking studies showed that targeting PD-L1 partially impaired MDSC-mediated T-cell suppression. Collectively, these studies suggest a role for PD-L1 in human MDSC function and thereby expand the functionality of this checkpoint beyond T cells, which could pave the way for further understanding and therapeutic targeting of PD-1/PD-L1 in innate immune-mediated diseases. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Biological characteristics of human-urine-derived stem cells: potential for cell-based therapy in neurology.

PubMed

Guan, Jun-Jie; Niu, Xin; Gong, Fei-Xiang; Hu, Bin; Guo, Shang-Chun; Lou, Yuan-Lei; Zhang, Chang-Qing; Deng, Zhi-Feng; Wang, Yang

2014-07-01

Stem cells in human urine have gained attention in recent years; however, urine-derived stem cells (USCs) are far from being well elucidated. In this study, we compared the biological characteristics of USCs with adipose-derived stem cells (ASCs) and investigated whether USCs could serve as a potential cell source for neural tissue engineering. USCs were isolated from voided urine with a modified culture medium. Through a series of experiments, we examined the growth rate, surface antigens, and differentiation potential of USCs, and compared them with ASCs. USCs showed robust proliferation ability. After serial propagation, USCs retained normal karyotypes. Cell surface antigen expression of USCs was similar to ASCs. With lineage-specific induction factors, USCs could differentiate toward the osteogenic, chondrogenic, adipogenic, and neurogenic lineages. To assess the ability of USCs to survive, differentiate, and migrate, they were seeded onto hydrogel scaffold and transplanted into rat brain. The results showed that USCs were able to survive in the lesion site, migrate to other areas, and express proteins that were associated with neural phenotypes. The results of our study demonstrate that USCs possess similar biological characteristics with ASCs and have multilineage differentiation potential. Moreover USCs can differentiate to neuron-like cells in rat brain. The present study shows that USCs are a promising cell source for tissue engineering and regenerative medicine.

The Effect of Antitumor Glycosides on Glioma Cells and Tissues as Studied by Proton HR-MAS NMR Spectroscopy

PubMed Central

García-Álvarez, Isabel; Garrido, Leoncio; Romero-Ramírez, Lorenzo; Nieto-Sampedro, Manuel; Fernández-Mayoralas, Alfonso; Campos-Olivas, Ramón

2013-01-01

The effect of the treatment with glycolipid derivatives on the metabolic profile of intact glioma cells and tumor tissues, investigated using proton high resolution magic angle spinning (1H HR-MAS) nuclear magnetic resonance (NMR) spectroscopy, is reported here. Two compounds were used, a glycoside and its thioglycoside analogue, both showing anti-proliferative activity on glioma C6 cell cultures; however, only the thioglycoside exhibited antitumor activity in vivo. At the drug concentrations showing anti-proliferative activity in cell culture (20 and 40 µM), significant increases in choline containing metabolites were observed in the 1H NMR spectra of the same intact cells. In vivo experiments in nude mice bearing tumors derived from implanted C6 glioma cells, showed that reduction of tumor volume was associated with significant changes in the metabolic profile of the same intact tumor tissues; and were similar to those observed in cell culture. Specifically, the activity of the compounds is mainly associated with an increase in choline and phosphocholine, in both the cell cultures and tumoral tissues. Taurine, a metabolite that has been considered a biomarker of apoptosis, correlated with the reduction of tumor volume. Thus, the results indicate that the mode of action of the glycoside involves, at least in part, alteration of phospholipid metabolism, resulting in cell death. PMID:24194925

Meta-Analysis of Tumor Stem-Like Breast Cancer Cells Using Gene Set and Network Analysis

PubMed Central

Lee, Won Jun; Kim, Sang Cheol; Yoon, Jung-Ho; Yoon, Sang Jun; Lim, Johan; Kim, You-Sun; Kwon, Sung Won; Park, Jeong Hill

2016-01-01

Generally, cancer stem cells have epithelial-to-mesenchymal-transition characteristics and other aggressive properties that cause metastasis. However, there have been no confident markers for the identification of cancer stem cells and comparative methods examining adherent and sphere cells are widely used to investigate mechanism underlying cancer stem cells, because sphere cells have been known to maintain cancer stem cell characteristics. In this study, we conducted a meta-analysis that combined gene expression profiles from several studies that utilized tumorsphere technology to investigate tumor stem-like breast cancer cells. We used our own gene expression profiles along with the three different gene expression profiles from the Gene Expression Omnibus, which we combined using the ComBat method, and obtained significant gene sets using the gene set analysis of our datasets and the combined dataset. This experiment focused on four gene sets such as cytokine-cytokine receptor interaction that demonstrated significance in both datasets. Our observations demonstrated that among the genes of four significant gene sets, six genes were consistently up-regulated and satisfied the p-value of < 0.05, and our network analysis showed high connectivity in five genes. From these results, we established CXCR4, CXCL1 and HMGCS1, the intersecting genes of the datasets with high connectivity and p-value of < 0.05, as significant genes in the identification of cancer stem cells. Additional experiment using quantitative reverse transcription-polymerase chain reaction showed significant up-regulation in MCF-7 derived sphere cells and confirmed the importance of these three genes. Taken together, using meta-analysis that combines gene set and network analysis, we suggested CXCR4, CXCL1 and HMGCS1 as candidates involved in tumor stem-like breast cancer cells. Distinct from other meta-analysis, by using gene set analysis, we selected possible markers which can explain the biological mechanisms and suggested network analysis as an additional criterion for selecting candidates. PMID:26870956

Microgravity

NASA Image and Video Library

1998-01-05

The Interferometer Protein Crystal Growth (IPCG) experiment was designed to measure details of how protein molecules move through a fluid. It was flown on the STS-86 mission for use aboard Russian Space Station Mir in 1998. It studied aspects of how crystals grow - and what conditions lead to the best crystals, details that remain a mystery. IPCG produces interference patterns by spilitting then recombining laser light. This let scientists see how fluid densities - and molecular diffusion - change around a crystal as it grows in microgravity. The heart of the IPCG apparatus is the interferometer cell comprising the optical bench, microscope, other optics, and video camera. IPCG experiment cells are made of optical glass and silvered on one side to serve as a mirror in the interferometer system that visuzlizes crystals and conditions around them as they grow inside the cell. This diagram shows the growth cells. The principal investigator was Dr. Alexander McPherson of University of California, Irvine. Co-investigators are William Witherow and Dr. Marc Pusey of NASA's Marshall Space Flight Center (MSFC).

Microgravity

NASA Image and Video Library

1998-01-05

The Interferometer Protein Crystal Growth (IPCG) experiment was designed to measure details of how protein molecules move through a fluid. It was flown on the STS-86 mission for use aboard Russian Space Station Mir in 1998. It studied aspects of how crystals grow - and what conditions lead to the best crystals, details that remain a mystery. IPCG produces interference patterns by spilitting then recombining laser light. This let scientists see how fluid densities - and molecular diffusion - change around a crystal as it grows in microgravity. The heart of the IPCG apparatus is the interferometer cell comprising the optical bench, microscope, other optics, and video camera. IPCG experiment cells are made of optical glass and silvered on one side to serve as a mirror in the interferometer system that visuzlizes crystals and conditions around them as they grow inside the cell. This view shows a large growth cell. The principal investigator was Dr. Alexander McPherson of University of California, Irvine. Co-investigators are William Witherow and Dr. Marc Pusey of NASA's Marshall Space Flight Center (MSFC).

High-sensitivity NMR beyond 200,000 atmospheres of pressure

NASA Astrophysics Data System (ADS)

Meier, T.; Reichardt, S.; Haase, J.

2015-08-01

Pressure-induced changes in the chemical or electronic structure of solids require pressures well into the Giga-Pascal (GPa) range due to the strong bonding. Anvil cell designs can reach such pressures, but their small and mostly inaccessible sample chamber has severely hampered NMR experiments in the past. With a new cell design that has a radio frequency (RF) micro-coil in the high pressure chamber, NMR experiments beyond 20 Giga-Pascal are reported for the first time. 1 H NMR of water shows sensitivity and resolution obtained with the cells, and 63 Cu NMR on a cuprate superconductor (YBa2Cu3O7-δ) demonstrates that single-crystals can be investigated, as well. 115 In NMR of the ternary chalcogenide AgInTe2 discovers an insulator-metal transition with shift and relaxation measurements. The pressure cells can be mounted easily on standard NMR probes that fit commercial wide-bore magnets with regular cryostats for field- and temperature-dependent measurements ready for many applications in physics and chemistry.

Modeling collective cell migration in geometric confinement

NASA Astrophysics Data System (ADS)

Tarle, Victoria; Gauquelin, Estelle; Vedula, S. R. K.; D'Alessandro, Joseph; Lim, C. T.; Ladoux, Benoit; Gov, Nir S.

2017-06-01

Monolayer expansion has generated great interest as a model system to study collective cell migration. During such an expansion the culture front often develops ‘fingers’, which we have recently modeled using a proposed feedback between the curvature of the monolayer’s leading edge and the outward motility of the edge cells. We show that this model is able to explain the puzzling observed increase of collective cellular migration speed of a monolayer expanding into thin stripes, as well as describe the behavior within different confining geometries that were recently observed in experiments. These comparisons give support to the model and emphasize the role played by the edge cells and the edge shape during collective cell motion.

Modeling collective cell migration in geometric confinement.

PubMed

Tarle, Victoria; Gauquelin, Estelle; Vedula, S R K; D'Alessandro, Joseph; Lim, C T; Ladoux, Benoit; Gov, Nir S

2017-05-03

Monolayer expansion has generated great interest as a model system to study collective cell migration. During such an expansion the culture front often develops 'fingers', which we have recently modeled using a proposed feedback between the curvature of the monolayer's leading edge and the outward motility of the edge cells. We show that this model is able to explain the puzzling observed increase of collective cellular migration speed of a monolayer expanding into thin stripes, as well as describe the behavior within different confining geometries that were recently observed in experiments. These comparisons give support to the model and emphasize the role played by the edge cells and the edge shape during collective cell motion.

Visualization and Measurement of Flow in a Model Rotating-Wall Bioreactor

NASA Astrophysics Data System (ADS)

Brown, Jason B.; Neitzel, G. Paul

1997-11-01

Fluid shear has been observed to have an effect on the in vitro growth of mammalian cells and is expected to play a role in the in vitro development of aggregates of cells into tissue. The interactions between culture media and cell constructs within a circular Couette flow bioreactor with independently rotating cylinders are investigated in model studies using flow visualization. Particle-Image Velocimetry (PIV) is used to quantify the velocity field in a plane perpendicular to the vessel axis which contains a cell construct model. This velocity field is then used to compute the instantaneous shear field. Experiments show the path of the model cell construct is dependent on the rotation rates of the cylinders.

Inhibitory effects of α-pinene on hepatoma carcinoma cell proliferation.

PubMed

Chen, Wei-Qiang; Xu, Bin; Mao, Jian-Wen; Wei, Feng-Xiang; Li, Ming; Liu, Tao; Jin, Xiao-Bao; Zhang, Li-Rong

2014-01-01

Pine needle oil from crude extract of pine needles has anti-tumor effects, but the effective component is not known. In the present study, compounds from a steam distillation extract of pine needles were isolated and characterized. Alpha-pinene was identified as an active anti-proliferative compound on hepatoma carcinoma BEL-7402 cells using the MTT assay. Further experiments showed that α-pinene inhibited BEL-7402 cells by arresting cell growth in the G2/M phase of the cell cycle, downregulating Cdc25C mRNA and protein expression, and reducing cycle dependence on kinase 1(CDK1) activity. Taken together, these findings indicate that α-pinene may be useful as a potential anti-tumor drug.

Red blood cell sedimentation of Apheresis Granulocytes.

PubMed

Lodermeier, Michelle A; Byrne, Karen M; Flegel, Willy A

2017-10-01

Sedimentation of Apheresis Granulocyte components removes red blood cells. It is used to increase the blood donor pool when blood group-compatible donors cannot be recruited for a patient because of a major ABO incompatibility or incompatible red blood cell antibodies in the recipient. Because granulocytes have little ABO and few other red blood cell antigens on their membrane, such incompatibility lies mostly with the contaminating red blood cells. Video Clip S1 shows the process of red blood cell sedimentation of an Apheresis Granulocyte component. This video was filmed with a single smart phone attached to a commercial tripod and was edited on a tablet computer with free software by an amateur videographer without prior video experience. © 2017 AABB.

Vascular smooth muscle cells exhibit a progressive loss of rigidity with serial culture passaging.

PubMed

Dinardo, Carla Luana; Venturini, Gabriela; Omae, Samantha Vieira; Zhou, Enhua H; da Motta-Leal-Filho, Joaquim Maurício; Dariolli, Rafael; Krieger, José Eduardo; Alencar, Adriano Mesquita; Costa Pereira, Alexandre

2012-01-01

One drawback of in vitro cell culturing is the dedifferentiation process that cells experience. Smooth muscle cells (SMC) also change molecularly and morphologically with long term culture. The main objective of this study was to evaluate if culture passages interfere in vascular SMC mechanical behavior. SMC were obtained from five different porcine arterial beds. Optical magnetic twisting cytometry (OMTC) was used to characterize mechanically vascular SMC from different cultures in distinct passages and confocal microscopy/western blotting, to evaluate cytoskeleton and extracellular matrix proteins. We found that vascular SMC rigidity or viscoelastic complex modulus (G) decreases with progression of passages. A statistically significant negative correlation between G and passage was found in four of our five cultures studied. Phalloidin-stained SMC from higher passages exhibited lower mean signal intensity per cell (confocal microscopy) and quantitative western blotting analysis showed a decrease in collagen I content throughout passages. We concluded that vascular SMC progressively lose their stiffness with serial culture passaging. Thus, limiting the number of passages is essential for any experiment measuring viscoelastic properties of SMC in culture.

Therapeutic gene editing in CD34+ hematopoietic progenitors from Fanconi anemia patients.

PubMed

Diez, Begoña; Genovese, Pietro; Roman-Rodriguez, Francisco J; Alvarez, Lara; Schiroli, Giulia; Ugalde, Laura; Rodriguez-Perales, Sandra; Sevilla, Julian; Diaz de Heredia, Cristina; Holmes, Michael C; Lombardo, Angelo; Naldini, Luigi; Bueren, Juan Antonio; Rio, Paula

2017-11-01

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the AAVS1 "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34 + cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34 + cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Correlating measured transient temperature rises with damage rate processes in cultured cells

NASA Astrophysics Data System (ADS)

Denton, Michael L.; Tijerina, Amanda J.; Gonzalez, Cherry C.; Gamboa, B. Giovana; Noojin, Gary D.; Ahmed, Elharith M.; Rickman, John M.; Dyer, Phillip H.; Rockwell, Benjamin A.

2017-02-01

Thermal damage rate processes in biological tissues are usually characterized by a kinetics approach. This stems from experimental data that show how the transformation of a specified biological property of cells or biomolecule (plating efficiency for viability, change in birefringence, tensile strength, etc.) is dependent upon both time and temperature. Here, two disparate approaches were used to study thermal damage rate processes in cultured retinal pigment epithelial cells. Laser exposure (photothermal) parameters included 2-μm laser exposure of non-pigmented cells and 532-nm exposures of cells possessing a variety of melanosome particle densities. Photothermal experiments used a mid-IR camera to record temperature histories with spatial resolution of about 8 μm, while fluorescence microscopy of the cell monolayers identified threshold damage at the boundary between live and dead cells. Photothermal exposure durations ranged from 0.05-20 s, and the effects of varying ambient temperature were investigated. Temperature during heat transfer using a water-jacketed cuvette was recorded with a fast microthermister, while damage and viability of the suspended cells were determined as percentages. Exposure durations for the heat transfer experiments ranged from 50- 60 s. Empirically-determined kinetic parameters for the two heating methods were compared with each other, and with values found in the literature.

Adenylyl cyclase A mRNA localized at the back of cells is actively translated in live chemotaxing Dictyostelium.

PubMed

Wang, Weiye; Chen, Song; Das, Satarupa; Losert, Wolfgang; Parent, Carole A

2018-05-04

Dictyostelium discoideum cells transport adenylyl cyclase A (ACA)-containing vesicles to the back of polarized cells to relay exogenous cAMP signals during chemotaxis. Fluorescence in situ hybridization (FISH) experiments showed that ACA mRNA is also asymmetrically distributed at the back of polarized cells. By using the MS2 bacteriophage system, we now visualize the distribution of ACA mRNA in live chemotaxing cells. We found that the ACA mRNA localization is not dependent on the translation of the protein product and requires multiple cis-acting elements within the ACA-coding sequence. We show that ACA mRNA is associated with actively translating ribosomes and is transported along microtubules towards the back of cells. By monitoring the recovery of ACA-YFP after photobleaching, we observed that local translation of ACA-YFP occurs at the back of cells. These data represent a novel functional role for localized translation in the relay of chemotactic signals during chemotaxis. © 2018. Published by The Company of Biologists Ltd.

In vitro and in vivo evaluation of a new nanocomposite, containing high density polyethylene, tricalcium phosphate, hydroxyapatite, and magnesium oxide nanoparticles.

PubMed

Pourdanesh, Fereydoun; Jebali, Ali; Hekmatimoghaddam, Seyedhossein; Allaveisie, Azra

2014-07-01

In this study, a new nanocomposite, which contained high density polyethylene (HDPE), tricalcium phosphate (Ca3(PO4)2) nanoparticles (TCP NPs), hydroxyapatite nanoparticles (HA NPs), and magnesium oxide nanoparticles (MgO NPs) was prepared. As in vitro experiment, human osteoblasts (HOB) cells were exposed to pristine HDPE and its nanocomposite for a period of 1, 4, and 7 days at 37 °C, and then different assays were carried out, including osteoblast cell proliferation, Trypan blue staining, cell viability, alkaline phosphatase (ALP), and cell adhesion. Antibacterial property of pristine HDPE and its nanocomposite was evaluated, and also their mechanical properties were measured after 2 and 4 months. As in vivo experiment, pristine HDPE and its nanocomposite were separately implanted on calvarium bone of rabbits, and tissue inflammation and osteogenesis were investigated after 2, 4, and 6 months. In case of HOB cells treated with HDPE or nanocomposite, as incubation time was increased, cell proliferation, live/dead ratio, and cell viability were decreased. But, the ALP activity and cell adhesion of HOB cells which treated with nanocomposite were raised after increase of incubation time. This study demonstrated that although the mechanical properties of nanocomposite were similar to HDPE sheet, but their antibacterial property was not similar. The in vivo experiment showed that both pristine HDPE and its nanocomposite had same inflammation responses. Interestingly, osteogenesis was observed after 2 months at bone/nanocomposite interface, and was highly increased after 4 and 6 months. It must be noted that such pattern was not seen at bone/HDPE interface. Copyright © 2014 Elsevier B.V. All rights reserved.

Extremely Low-Frequency Electromagnetic Fields Cause G1 Phase Arrest through the Activation of the ATM-Chk2-p21 Pathway

PubMed Central

Huang, Chao-Ying; Chang, Cheng-Wei; Chen, Chaang-Ray; Chuang, Chun-Yu; Chiang, Chi-Shiun; Shu, Wun-Yi; Fan, Tai-Ching; Hsu, Ian C.

2014-01-01

In daily life, humans are exposed to the extremely low-frequency electromagnetic fields (ELF-EMFs) generated by electric appliances, and public concern is increasing regarding the biological effects of such exposure. Numerous studies have yielded inconsistent results regarding the biological effects of ELF-EMF exposure. Here we show that ELF-EMFs activate the ATM-Chk2-p21 pathway in HaCaT cells, inhibiting cell proliferation. To present well-founded results, we comprehensively evaluated the biological effects of ELF-EMFs at the transcriptional, protein, and cellular levels. Human HaCaT cells from an immortalized epidermal keratinocyte cell line were exposed to a 1.5 mT, 60 Hz ELF-EMF for 144 h. The ELF-EMF could cause G1 arrest and decrease colony formation. Protein expression experiments revealed that ELF-EMFs induced the activation of the ATM/Chk2 signaling cascades. In addition, the p21 protein, a regulator of cell cycle progression at G1 and G2/M, exhibited a higher level of expression in exposed HaCaT cells compared with the expression of sham-exposed cells. The ELF-EMF-induced G1 arrest was diminished when the CHK2 gene expression (which encodes checkpoint kinase 2; Chk2) was suppressed by specific small interfering RNA (siRNA). These findings indicate that ELF-EMFs activate the ATM-Chk2-p21 pathway in HaCaT cells, resulting in cell cycle arrest at the G1 phase. Based on the precise control of the ELF-EMF exposure and rigorous sham-exposure experiments, all transcriptional, protein, and cellular level experiments consistently supported the conclusion. This is the first study to confirm that a specific pathway is triggered by ELF-EMF exposure. PMID:25111195

A particle-based model to simulate the micromechanics of single-plant parenchyma cells and aggregates

NASA Astrophysics Data System (ADS)

Van Liedekerke, P.; Ghysels, P.; Tijskens, E.; Samaey, G.; Smeedts, B.; Roose, D.; Ramon, H.

2010-06-01

This paper is concerned with addressing how plant tissue mechanics is related to the micromechanics of cells. To this end, we propose a mesh-free particle method to simulate the mechanics of both individual plant cells (parenchyma) and cell aggregates in response to external stresses. The model considers two important features in the plant cell: (1) the cell protoplasm, the interior liquid phase inducing hydrodynamic phenomena, and (2) the cell wall material, a viscoelastic solid material that contains the protoplasm. In this particle framework, the cell fluid is modeled by smoothed particle hydrodynamics (SPH), a mesh-free method typically used to address problems with gas and fluid dynamics. In the solid phase (cell wall) on the other hand, the particles are connected by pairwise interactions holding them together and preventing the fluid to penetrate the cell wall. The cell wall hydraulic conductivity (permeability) is built in as well through the SPH formulation. Although this model is also meant to be able to deal with dynamic and even violent situations (leading to cell wall rupture or cell-cell debonding), we have concentrated on quasi-static conditions. The results of single-cell compression simulations show that the conclusions found by analytical models and experiments can be reproduced at least qualitatively. Relaxation tests revealed that plant cells have short relaxation times (1 µs-10 µs) compared to mammalian cells. Simulations performed on cell aggregates indicated an influence of the cellular organization to the tissue response, as was also observed in experiments done on tissues with a similar structure.

The in vivo antitumor effects of type I-interferon against hepatocellular carcinoma: the suppression of tumor cell growth and angiogenesis.

PubMed

Enomoto, Hirayuki; Tao, Lihua; Eguchi, Ryoji; Sato, Ayuko; Honda, Masao; Kaneko, Shuichi; Iwata, Yoshinori; Nishikawa, Hiroki; Imanishi, Hiroyasu; Iijima, Hiroko; Tsujimura, Tohru; Nishiguchi, Shuhei

2017-09-22

Type I-interferon (IFN) is considered to exert antitumor effects through the inhibition of cancer cell proliferation and angiogenesis. Based on the species-specific biological activity of IFN, we evaluated each antitumor mechanism separately. We further examined the antitumor effects of type I-IFN combined with sorafenib. Human IFN (hIFN) significantly inhibited the proliferation of human hepatocellular carcinoma (HCC) Hep3B cells and the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Although mouse IFN (mIFN) did not inhibit the proliferation of Hep3B cells in vitro, mIFN, as well as hIFN, showed significant antitumor effects in mouse Hep3B cell-xenograft model. Furthermore, mIFN treatment amplified the antitumor effects of sorafenib in vivo with the suppression of angiogenesis. The DNA chip analysis showed that the mIFN treatment promoted the antitumor signal pathways of sorafenib, including anti-angiogenic effects. Unlike the effects observed in in vitro experiments, mIFN showed an antitumor effect in the mouse Hep3B cell-xenograft model, suggesting a role of the anti-angiogenic activity in the in vivo tumoricidal effects of type I-IFN. In addition, our findings suggested the clinical utility of combination therapy with type І-IFN and sorafenib for HCC.

Acetylsalicylic acid regulates MMP-2 activity and inhibits colorectal invasion of murine B16F0 melanoma cells in C57BL/6J mice: effects of prostaglandin F(2)alpha.

PubMed

Tsai, Chin-Shaw Stella; Luo, Shue-Fen; Ning, Chung-Chu; Lin, Chien-Liang; Jiang, Ming-Chung; Liao, Ching-Fong

2009-08-01

Epidemiological studies indicate that acetylsalicylic acid may reduce the risk of mortality due to colon cancers. Metastasis is the major cause of cancer death. Matrix metalloproteinases (MMPs) play important roles in tumor invasion regulation, and prostaglandin F(2)alpha (PGF(2)alpha) is a key stimulator of MMP production. Thus, we investigated whether acetylsalicylic acid regulated MMP activity and the invasion of cancer cells and whether PGF(2)alpha attenuated acetylsalicylic acid-inhibited invasion of cancer cells. Gelatin-based zymography assays showed that acetylsalicylic acid inhibited the MMP-2 activity of B16F0 melanoma cells. Matrigel-based chemoinvasion assays showed that acetylsalicylic acid inhibited the invasion of B16F0 cells. Acetylsalicylic acid can inhibit PGF(2)alpha synthesis and PGF(2)alpha is a key stimulator of MMP-2 production. Our data showed that PGF(2)alpha treatment attenuated the acetylsalicylic acid-inhibited invasion of B16F0 cells. In animal experiments, acetylsalicylic acid reduced colorectal metastasis of B16F0 cells in C57BL/6J mice by 44%. Our results suggest that PGF(2)alpha is a therapeutic target for metastasis inhibition and acetylsalicylic acid may possess anti-metastasis ability.

Development of a statistical model for cervical cancer cell death with irreversible electroporation in vitro.

PubMed

Yang, Yongji; Moser, Michael A J; Zhang, Edwin; Zhang, Wenjun; Zhang, Bing

2018-01-01

The aim of this study was to develop a statistical model for cell death by irreversible electroporation (IRE) and to show that the statistic model is more accurate than the electric field threshold model in the literature using cervical cancer cells in vitro. HeLa cell line was cultured and treated with different IRE protocols in order to obtain data for modeling the statistical relationship between the cell death and pulse-setting parameters. In total, 340 in vitro experiments were performed with a commercial IRE pulse system, including a pulse generator and an electric cuvette. Trypan blue staining technique was used to evaluate cell death after 4 hours of incubation following IRE treatment. Peleg-Fermi model was used in the study to build the statistical relationship using the cell viability data obtained from the in vitro experiments. A finite element model of IRE for the electric field distribution was also built. Comparison of ablation zones between the statistical model and electric threshold model (drawn from the finite element model) was used to show the accuracy of the proposed statistical model in the description of the ablation zone and its applicability in different pulse-setting parameters. The statistical models describing the relationships between HeLa cell death and pulse length and the number of pulses, respectively, were built. The values of the curve fitting parameters were obtained using the Peleg-Fermi model for the treatment of cervical cancer with IRE. The difference in the ablation zone between the statistical model and the electric threshold model was also illustrated to show the accuracy of the proposed statistical model in the representation of ablation zone in IRE. This study concluded that: (1) the proposed statistical model accurately described the ablation zone of IRE with cervical cancer cells, and was more accurate compared with the electric field model; (2) the proposed statistical model was able to estimate the value of electric field threshold for the computer simulation of IRE in the treatment of cervical cancer; and (3) the proposed statistical model was able to express the change in ablation zone with the change in pulse-setting parameters.

Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

PubMed

Leyva-Leyva, Margarita; López-Díaz, Annia; Barrera, Lourdes; Camacho-Morales, Alberto; Hernandez-Aguilar, Felipe; Carrillo-Casas, Erika M; Arriaga-Pizano, Lourdes; Calderón-Pérez, Jaime; García-Álvarez, Jorge; Orozco-Hoyuela, Gabriel; Piña-Barba, Cristina; Rojas-Martínez, Augusto; Romero-Díaz, Víktor; Lara-Arias, Jorge; Rivera-Bolaños, Nancy; López-Camarillo, César; Moncada-Saucedo, Nidia; Galván-De los Santos, Alejandra; Meza-Urzúa, Fátima; Villarreal-Gómez, Luis; Fuentes-Mera, Lizeth

2015-11-01

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated β1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in orthopedic bioengineering.

Absence of single critical dose for the amorphization of quartz under ion irradiation

NASA Astrophysics Data System (ADS)

Zhang, S.; Pakarinen, O. H.; Backholm, M.; Djurabekova, F.; Nordlund, K.; Keinonen, J.; Wang, T. S.

2018-01-01

In this work, we first simulated the amorphization of crystalline quartz under 50 keV 23 Na ion irradiation with classical molecular dynamics (MD). We then used binary collision approximation algorithms to simulate the Rutherford backscattering spectrometry in channeling conditions (RBS-C) from these irradiated MD cells, and compared the RBS-C spectra with experiments. The simulated RBS-C results show an agreement with experiments in the evolution of amorphization as a function of dose, showing what appears to be (by this measure) full amorphization at about 2.2 eVṡatom-1 . We also applied other analysis methods, such as angular structure factor, Wigner-Seitz, coordination analysis and topological analysis, to analyze the structural evolution of the irradiated MD cells. The results show that the atomic-level structure of the sample keeps evolving after the RBS signal has saturated, until the dose of about 5 eVṡatom-1 . The continued evolution of the SiO2 structure makes the definition of what is, on the atomic level, an amorphized quartz ambiguous.

Absence of single critical dose for the amorphization of quartz under ion irradiation.

PubMed

Zhang, S; Pakarinen, O H; Backholm, M; Djurabekova, F; Nordlund, K; Keinonen, J; Wang, T S

2018-01-10

In this work, we first simulated the amorphization of crystalline quartz under 50 keV [Formula: see text]Na ion irradiation with classical molecular dynamics (MD). We then used binary collision approximation algorithms to simulate the Rutherford backscattering spectrometry in channeling conditions (RBS-C) from these irradiated MD cells, and compared the RBS-C spectra with experiments. The simulated RBS-C results show an agreement with experiments in the evolution of amorphization as a function of dose, showing what appears to be (by this measure) full amorphization at about 2.2 eV⋅[Formula: see text]. We also applied other analysis methods, such as angular structure factor, Wigner-Seitz, coordination analysis and topological analysis, to analyze the structural evolution of the irradiated MD cells. The results show that the atomic-level structure of the sample keeps evolving after the RBS signal has saturated, until the dose of about 5 eV⋅[Formula: see text]. The continued evolution of the [Formula: see text] structure makes the definition of what is, on the atomic level, an amorphized quartz ambiguous.

Induction of a bystander mutagenic effect of alpha particles in mammalian cells

NASA Technical Reports Server (NTRS)

Zhou, H.; Randers-Pehrson, G.; Waldren, C. A.; Vannais, D.; Hall, E. J.; Hei, T. K.; Chatterjee, A. (Principal Investigator)

2000-01-01

Ever since the discovery of X-rays was made by Rontgen more than a hundred years ago, it has always been accepted that the deleterious effects of ionizing radiation such as mutation and carcinogenesis are attributable mainly to direct damage to DNA. Although evidence based on microdosimetric estimation in support of a bystander effect appears to be consistent, direct proof of such extranuclear/extracellular effects are limited. Using a precision charged particle microbeam, we show here that irradiation of 20% of randomly selected A(L) cells with 20 alpha particles each results in a mutant fraction that is 3-fold higher than expected, assuming no bystander modulation effect. Furthermore, analysis by multiplex PCR shows that the types of mutants induced are significantly different from those of spontaneous origin. Pretreatment of cells with the radical scavenger DMSO had no effect on the mutagenic incidence. In contrast, cells pretreated with a 40 microM dose of lindane, which inhibits cell-cell communication, significantly decreased the mutant yield. The doses of DMSO and lindane used in these experiments are nontoxic and nonmutagenic. We further examined the mutagenic yield when 5-10% of randomly selected cells were irradiated with 20 alpha particles each. Results showed, likewise, a higher mutant yield than expected assuming no bystander effects. Our studies provide clear evidence that irradiated cells can induce a bystander mutagenic response in neighboring cells not directly traversed by alpha particles and that cell-cell communication process play a critical role in mediating the bystander phenomenon.

Probing the Role of Nascent Helicity in p27 Function as a Cell Cycle Regulator

PubMed Central

Otieno, Steve; Kriwacki, Richard

2012-01-01

p27 regulates the activity of Cdk complexes which are the principal governors of phase transitions during cell division. Members of the p27 family of proteins, which also includes p21 and p57, are called the Cip/Kip cyclin-dependent kinase regulators (CKRs). Interestingly, the Cip/Kip CKRs play critical roles in cell cycle regulation by being intrinsically unstructured, a characteristic contrary to the classical structure-function paradigm. They exhibit nascent helicity which has been localized to a segment referred to as sub-domain LH. The nascent helicity of this sub-domain is conserved and we hypothesize that it is an important determinant of their functional properties. To test this hypothesis, we successfully designed and prepared p27 variants in which domain LH was either more or less helical with respect to the wild-type protein. Thermal denaturation experiments showed that the ternary complexes of the p27 variants bound to Cdk2/Cyclin A were less stable compared to the wild-type complex. Isothermal titration calorimetry experiments showed a decrease in the enthalpy of binding for all the mutants with respect to p27. The free energies of binding varied within a much narrower range. In vitro Cdk2 inhibition assays showed that the p27 variants exhibited disparate inhibitory potencies. Furthermore, when over-expressed in NIH 3T3 mouse fibroblast cells, the less helical p27 variants were less effective in causing cell cycle arrest relative to the wild-type p27. Our results indicate that the nascent helicity of sub-domain LH plays a key role mediating the biological function of p27. PMID:23071750

An age-structured model of hiv infection that allows for variations in the production rate of viral particles and the death rate of productively infected cells.

PubMed

Nelson, Patrick W; Gilchrist, Michael A; Coombs, Daniel; Hyman, James M; Perelson, Alan S

2004-09-01

Mathematical models of HIV-1 infection can help interpret drug treatment experiments and improve our understanding of the interplay between HIV-1 and the immune system. We develop and analyze an age- structured model of HIV-1 infection that allows for variations in the death rate of productively infected T cells and the production rate of viral particles as a function of the length of time a T cell has been infected. We show that this model is a generalization of the standard differential equation and of delay models previously used to describe HIV-1 infection, and provides a means for exploring fundamental issues of viral production and death. We show that the model has uninfected and infected steady states, linked by a transcritical bifurcation. We perform a local stability analysis of the nontrivial equilibrium solution and provide a general stability condition for models with age structure. We then use numerical methods to study solutions of our model focusing on the analysis of primary HIV infection. We show that the time to reach peak viral levels in the blood depends not only on initial conditions but also on the way in which viral production ramps up. If viral production ramps up slowly, we find that the time to peak viral load is delayed compared to results obtained using the standard (constant viral production) model of HIV infection. We find that data on viral load changing over time is insufficient to identify the functions specifying the dependence of the viral production rate or infected cell death rate on infected cell age. These functions must be determined through new quantitative experiments.

Development of a high-sensitivity and portable cell using Helmholtz resonance for noninvasive blood glucose-level measurement based on photoacoustic spectroscopy.

PubMed

Tachibana, K; Okada, K; Kobayashi, R; Ishihara, Y

2016-08-01

We describe the possibility of high-sensitivity noninvasive blood glucose measurement based on photoacoustic spectroscopy (PAS). The demand for noninvasive blood glucose-level measurement has increased due to the explosive increase in diabetic patients. We have developed a noninvasive blood glucose-level measurement based on PAS. The conventional method uses a straight-type resonant cell. However, the cell volume is large, which results in a low detection sensitivity and difficult portability. In this paper, a small-sized Helmholtz-type resonant cell is proposed to improve detection sensitivity and portability by reducing the cell dead volume. First, the acoustic property of the small-sized Helmholtz-type resonant cell was evaluated by performing an experiment using a silicone rubber. As a result, the detection sensitivity of the small-sized Helmholtz-type resonant cell was approximately two times larger than that of the conventional straight-type resonant cell. In addition, the inside volume was approximately 30 times smaller. Second, the detection limits of glucose concentration were estimated by performing an experiment using glucose solutions. The experimental results showed that a glucose concentration of approximately 1% was detected by the small-sized Helmholtz-type resonant cell. Although these results on the sensitivity of blood glucose-level measurement are currently insufficient, they suggest that miniaturization of a resonance cell is effective in the application of noninvasive blood glucose-level measurement.

Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.

PubMed

Lopata, Anna; Hughes, Ruth; Tiede, Christian; Heissler, Sarah M; Sellers, James R; Knight, Peter J; Tomlinson, Darren; Peckham, Michelle

2018-04-26

Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.

Three-dimensional growth of human endothelial cells in an automated cell culture experiment container during the SpaceX CRS-8 ISS space mission - The SPHEROIDS project.

PubMed

Pietsch, Jessica; Gass, Samuel; Nebuloni, Stefano; Echegoyen, David; Riwaldt, Stefan; Baake, Christin; Bauer, Johann; Corydon, Thomas J; Egli, Marcel; Infanger, Manfred; Grimm, Daniela

2017-04-01

Human endothelial cells (ECs) were sent to the International Space Station (ISS) to determine the impact of microgravity on the formation of three-dimensional structures. For this project, an automatic experiment unit (EU) was designed allowing cell culture in space. In order to enable a safe cell culture, cell nourishment and fixation after a pre-programmed timeframe, the materials used for construction of the EUs were tested in regard to their biocompatibility. These tests revealed a high biocompatibility for all parts of the EUs, which were in contact with the cells or the medium used. Most importantly, we found polyether ether ketones for surrounding the incubation chamber, which kept cellular viability above 80% and allowed the cells to adhere as long as they were exposed to normal gravity. After assembling the EU the ECs were cultured therein, where they showed good cell viability at least for 14 days. In addition, the functionality of the automatic medium exchange, and fixation procedures were confirmed. Two days before launch, the ECs were cultured in the EUs, which were afterwards mounted on the SpaceX CRS-8 rocket. 5 and 12 days after launch the cells were fixed. Subsequent analyses revealed a scaffold-free formation of spheroids in space. Copyright © 2017 Elsevier Ltd. All rights reserved.

Persistence of Only a Minute Viable Population in Chlorotic Microcystis aeruginosa PCC 7806 Cultures Obtained by Nutrient Limitation.

PubMed

Meireles, Diogo de Abreu; Schripsema, Jan; Arnholdt, Andrea Cristina Vetö; Dagnino, Denise

2015-01-01

Cultures from the cyanobacterial strain Microcystis aeruginosa PCC 7806 submitted to nutrient limitation become chlorotic. When returned to nutrient rich conditions these cultures regain their green colour. The aim of this study was to verify whether the cells in these cultures could be considered resting stages allowing the survival of periods of nutrient starvation as has been reported for Synechococcus PCC 7942. The experiments with Microcystis were carried out in parallel with Synechococcus cultures to rule out the possibility that any results obtained with Microcystis were due to our particular experimental conditions. The results of the experiments with Synechococcus PCC 7942 cultures were comparable to the reported in the literature. For Microcystis PCC 7806 a different response was observed. Analysis of chlorotic Microcystis cultures by flow cytometry showed that the phenotype of the cells in the population was not homogenous: the amount of nucleic acids was about the same in all cells but only around one percent of the population emitted red autofluorescence indicating the presence of chlorophyll. Monitoring of the reversion of chlorosis by flow cytometry showed that the re-greening was most likely the result of the division of the small population of red autofluorescent cells originally present in the chlorotic cultures. This assumption was confirmed by analysing the integrity of the DNA and the membrane permeability of the cells of chlorotic cultures. Most of the DNA of these cultures was degraded and only the autofluorescent population of the chlorotic cultures showed membrane integrity. Thus, contrary to what has been reported for other cyanobacterial genera, most of the cells in chlorotic Microcystis cultures are not resting stages but dead. It is interesting to note that the red autofluorescent cells of green and chlorotic cultures obtained in double strength ASM-1 medium differ with respect to metabolism: levels of emission of red autofluorescence are higher in the cells of green cultures and the ability to convert fluorescein diacetate of these cells are heterogeneous when compared to the autofluorescent cells of chlorotic cultures. Thus, the small population of the red autofluorescent cells of chlorotic cultures are in a differentiated metabolic state that allow them to persist in conditions in which most of the population loses viability; persistent cells can be detected in chlorotic cultures maintained for more than a year.

Persistence of Only a Minute Viable Population in Chlorotic Microcystis aeruginosa PCC 7806 Cultures Obtained by Nutrient Limitation

PubMed Central

de Abreu Meireles, Diogo; Schripsema, Jan; Vetö Arnholdt, Andrea Cristina; Dagnino, Denise

2015-01-01

Cultures from the cyanobacterial strain Microcystis aeruginosa PCC 7806 submitted to nutrient limitation become chlorotic. When returned to nutrient rich conditions these cultures regain their green colour. The aim of this study was to verify whether the cells in these cultures could be considered resting stages allowing the survival of periods of nutrient starvation as has been reported for Synechococcus PCC 7942. The experiments with Microcystis were carried out in parallel with Synechococcus cultures to rule out the possibility that any results obtained with Microcystis were due to our particular experimental conditions. The results of the experiments with Synechococcus PCC 7942 cultures were comparable to the reported in the literature. For Microcystis PCC 7806 a different response was observed. Analysis of chlorotic Microcystis cultures by flow cytometry showed that the phenotype of the cells in the population was not homogenous: the amount of nucleic acids was about the same in all cells but only around one percent of the population emitted red autofluorescence indicating the presence of chlorophyll. Monitoring of the reversion of chlorosis by flow cytometry showed that the re-greening was most likely the result of the division of the small population of red autofluorescent cells originally present in the chlorotic cultures. This assumption was confirmed by analysing the integrity of the DNA and the membrane permeability of the cells of chlorotic cultures. Most of the DNA of these cultures was degraded and only the autofluorescent population of the chlorotic cultures showed membrane integrity. Thus, contrary to what has been reported for other cyanobacterial genera, most of the cells in chlorotic Microcystis cultures are not resting stages but dead. It is interesting to note that the red autofluorescent cells of green and chlorotic cultures obtained in double strength ASM-1 medium differ with respect to metabolism: levels of emission of red autofluorescence are higher in the cells of green cultures and the ability to convert fluorescein diacetate of these cells are heterogeneous when compared to the autofluorescent cells of chlorotic cultures. Thus, the small population of the red autofluorescent cells of chlorotic cultures are in a differentiated metabolic state that allow them to persist in conditions in which most of the population loses viability; persistent cells can be detected in chlorotic cultures maintained for more than a year. PMID:26181753

Design of quantum dot-conjugated lipids for long-term, high-speed tracking experiments on cell surfaces.

PubMed

Murcia, Michael J; Minner, Daniel E; Mustata, Gina-Mirela; Ritchie, Kenneth; Naumann, Christoph A

2008-11-12

The current study reports the facile design of quantum dot (QD)-conjugated lipids and their application to high-speed tracking experiments on cell surfaces. CdSe/ZnS core/shell QDs with two types of hydrophilic coatings, 2-(2-aminoethoxy)ethanol (AEE) and a 60:40 molar mixture of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine and 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol-2000], are conjugated to sulfhydryl lipids via maleimide reactive groups on the QD surface. Prior to lipid conjugation, the colloidal stability of both types of coated QDs in aqueous solution is confirmed using fluorescence correlation spectroscopy. A sensitive assay based on single lipid tracking experiments on a planar solid-supported phospholipid bilayer is presented that establishes conditions of monovalent conjugation of QDs to lipids. The QD-lipids are then employed as single-molecule tracking probes in plasma membranes of several cell types. Initial tracking experiments at a frame rate of 30 frames/s corroborate that QD-lipids diffuse like dye-labeled lipids in the plasma membrane of COS-7, HEK-293, 3T3, and NRK cells, thus confirming monovalent labeling. Finally, QD-lipids are applied for the first time to high-speed single-molecule imaging by tracking their lateral mobility in the plasma membrane of NRK fibroblasts with up to 1000 frames/s. Our high-speed tracking data, which are in excellent agreement with previous tracking experiments that used larger (40 nm) Au labels, not only push the time resolution in long-time, continuous fluorescence-based single-molecule tracking but also show that highly photostable, photoluminescent nanoprobes of 10 nm size can be employed (AEE-coated QDs). These probes are also attractive because, unlike Au nanoparticles, they facilitate complex multicolor experiments.

Ultrastructural and functional characterization of circulating hemocytes from Galleria mellonella larva: Cell types and their role in the innate immunity.

PubMed

Wu, Gongqing; Liu, Yi; Ding, Ying; Yi, Yunhong

2016-08-01

Galleria mellonella larvae have been widely used as a model to study the virulence of various human pathogens. Hemocytes play important roles in the innate immune response of G. mellonella. In this study, the hemocytes of G. mellonella larvae were analyzed by transmission electron microscope, light microscope, and cytochemistry. The cytological and morphological analyses revealed four types of hemocytes; Plasmatocytes, granular cells, spherule cells and oenocytoids. Differential hemocyte counts showed that under our conditions plasmatocytes and granular cells were the most abundant circulating cell types in the hemolymph. We also investigated the role of different types of hemocytes in the cellular and humoral immune defenses. The in-vivo experiment showed that plasmatocytes, granular cells and oenocytoids phagocytized FITC-labelled Escherichia coli bacteria in larvae of G. mellonella, whereas the granular cells exhibited the strongest phagocytic ability against these microbial cells. After incubation with L-DOPA, plasmatocytes, granular cells and oenocytoids are stained brown, indicating the presence of phenoloxidase activity. These results shed new light on our understanding of the immune function of G. mellonella hemocytes. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2016-06-01

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

Study on activity measurement of Nostoc flagelliforme cells based on color identification

NASA Astrophysics Data System (ADS)

Wang, Yizhong; Su, Jianyu; Liu, Tiegen; Kong, Fanzhi; Jia, Shiru

2008-12-01

In order to measure the activities of Nostoc flagelliforme cells, a new method based on color identification was proposed in this paper. N. flagelliforme cells were colored with fluoreseein diaeetate. Then, an image of colored N. flagelliforme cells was taken, and changed from RGB model to HIS model. Its histogram of hue H was calculated, which was used as the input of a designed BP network. The output of the BP network was the description of measured activity of N. flagelliforme cells. After training, the activity of N. flagelliforme cells was identified by the BP network according to the histogram of H of their colored image. Experiments were conducted with satisfied results to show the feasibility and usefulness of activity measurement of N. flagelliforme cells based on color identification.

Cell diameter measurements obtained with a handheld cell counter could be used as a surrogate marker of G2/M arrest and apoptosis in colon cancer cell lines exposed to SN-38

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tahara, Makiko; Department of Gastrointestinal Surgery, Jichi Medical University, Shimotsuke, Tochigi; Inoue, Takeshi

2013-05-17

Highlights: •Chemo-sensitivity to SN-38 was assayed by the automated cell counter. •Colon cancer cell line, HCT116 cells were more sensitive to SN-38 than HT29 cells. •Increase of cell size reflects G2/M arrest. •Appearance of small particles indicates cell apoptosis. -- Abstract: In vitro assessment of chemosensitivity are important for experiments evaluating cancer therapies. The Scepter 2.0 cell counter, an automated handheld device based on the Coulter principle of impedance-based particle detection, enables the accurate discrimination of cell populations according to cell size and volume. In this study, the effects of SN-38, the active metabolite of irinotecan, on the colon cancermore » cell lines HCT116 and HT29 were evaluated using this device. The cell count data obtained with the Scepter counter were compared with those obtained with the {sup 3}H-thymidine uptake assay, which has been used to measure cell proliferation in many previous studies. In addition, we examined whether the changes in the size distributions of these cells reflected alterations in the frequency of cell cycle arrest and/or apoptosis induced by SN-38 treatment. In our experiments using the Scepter 2.0 cell counter, the cell counts were demonstrated to be accurate and reproducible measure and alterations of cell diameter reflected G2/M cell cycle arrest and apoptosis. Our data show that easy-to-use cell counting tools can be utilized to evaluate the cell-killing effects of novel treatments on cancer cells in vitro.« less

Cutaway line drawing of STS-34 middeck experiment Polymer Morphology (PM)

NASA Technical Reports Server (NTRS)

1989-01-01

Cutaway line drawing shows components of STS-34 middeck experiment Polymer Morphology (PM). Generic Electronics Module (GEM) components include the control housing, circulating fans, hard disk, tape drives, computer boards, and heat exchanger. PM, a 3M-developed organic materials processing experiment, is designed to explore the effects of microgravity on polymeric materials as they are processed in space. The samples of polymeric materials being studied in the PM experiment are thin films (25 microns or less) approximately 25mm in diameter. The samples are mounted between two infrared transparent windows in a specially designed infrared cell that provides the capability of thermally processing the samples to 200 degrees Celsius with a high degree of thermal control. The samples are mounted on a carousel that allows them to be positioned, one at a time, in the infrared beam where spectra may be acquired. The GEM provides all carousel and sample cell control (SCC). The first flight of P

Measuring ignitability for in situ burning of oil spills weathered under Arctic conditions: from laboratory studies to large-scale field experiments.

PubMed

Fritt-Rasmussen, Janne; Brandvik, Per Johan

2011-08-01

This paper compares the ignitability of Troll B crude oil weathered under simulated Arctic conditions (0%, 50% and 90% ice cover). The experiments were performed in different scales at SINTEF's laboratories in Trondheim, field research station on Svalbard and in broken ice (70-90% ice cover) in the Barents Sea. Samples from the weathering experiments were tested for ignitability using the same laboratory burning cell. The measured ignitability from the experiments in these different scales showed a good agreement for samples with similar weathering. The ice conditions clearly affected the weathering process, and 70% ice or more reduces the weathering and allows a longer time window for in situ burning. The results from the Barents Sea revealed that weathering and ignitability can vary within an oil slick. This field use of the burning cell demonstrated that it can be used as an operational tool to monitor the ignitability of oil spills. Copyright © 2011 Elsevier Ltd. All rights reserved.

The NASA light-emitting diode medical program-progress in space flight and terrestrial applications

NASA Astrophysics Data System (ADS)

Whelan, Harry T.; Houle, John M.; Whelan, Noel T.; Donohoe, Deborah L.; Cwiklinski, Joan; Schmidt, Meic H.; Gould, Lisa; Larson, David L.; Meyer, Glenn A.; Cevenini, Vita; Stinson, Helen

2000-01-01

This work is supported and managed through the NASA Marshall Space Flight Center-SBIR Program. Studies on cells exposed to microgravity and hypergravity indicate that human cells need gravity to stimulate cell growth. As the gravitational force increases or decreases, the cell function responds in a linear fashion. This poses significant health risks for astronauts in long termspace flight. LED-technology developed for NASA plant growth experiments in space shows promise for delivering light deep into tissues of the body to promote wound healing and human tissue growth. This LED-technology is also biologically optimal for photodynamic therapy of cancer. .

Hermetic encapsulation technique for solar arrays

NASA Technical Reports Server (NTRS)

Deminet, C.; Horne, W. E.

1980-01-01

A concept is presented for encapsulating solar cells between two layers of glass either individually, in panels, or in a continuous process. The concept yields an integral unit that is hermetically sealed and that is tolerant to high temperature thermal cycling and to particulate radiation. Data are presented on both high temperature solar cells and special glasses that soften at low temperatures for use with the concept. The results of encapsulating experiments are presented which show the successful application of the concept to the special high temperature cells. The mechanical feasibility of encapsulating 2 mil cells between two layers of 2 mil glass is also demonstrated.

Cavity enhanced atomic magnetometry

PubMed Central

Crepaz, Herbert; Ley, Li Yuan; Dumke, Rainer

2015-01-01

Atom sensing based on Faraday rotation is an indispensable method for precision measurements, universally suitable for both hot and cold atomic systems. Here we demonstrate an all-optical magnetometer where the optical cell for Faraday rotation spectroscopy is augmented with a low finesse cavity. Unlike in previous experiments, where specifically designed multipass cells had been employed, our scheme allows to use conventional, spherical vapour cells. Spherical shaped cells have the advantage that they can be effectively coated inside with a spin relaxation suppressing layer providing long spin coherence times without addition of a buffer gas. Cavity enhancement shows in an increase in optical polarization rotation and sensitivity compared to single-pass configurations. PMID:26481853

Dynamics of cell area and force during spreading.

PubMed

Brill-Karniely, Yifat; Nisenholz, Noam; Rajendran, Kavitha; Dang, Quynh; Krishnan, Ramaswamy; Zemel, Assaf

2014-12-16

Experiments on human pulmonary artery endothelial cells are presented to show that cell area and the force exerted on a substrate increase simultaneously, but with different rates during spreading; rapid-force increase systematically occurred several minutes past initial spreading. We examine this theoretically and present three complementary mechanisms that may accompany the development of lamellar stress during spreading and underlie the observed behavior. These include: 1), the dynamics of cytoskeleton assembly at the cell basis; 2), the strengthening of acto-myosin forces in response to the generated lamellar stresses; and 3), the passive strain-stiffening of the cytoskeleton. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A thermodynamical model for stress-fiber organization in contractile cells.

PubMed

Foucard, Louis; Vernerey, Franck J

2012-01-02

Cell mechanical adaptivity to external stimuli is vital to many of its biological functions. A critical question is therefore to understand the formation and organization of the stress fibers from which emerge the cell's mechanical properties. By accounting for the mechanical aspects and the viscoelastic behavior of stress fibers, we here propose a thermodynamic model to predict the formation and orientation of stress fibers in contractile cells subjected to constant or cyclic stretch and different substrate stiffness. Our results demonstrate that the stress fibers viscoelastic behavior plays a crucial role in their formation and organization and shows good consistency with various experiments.

Stabilisation of cables of fibronectin with micromolar concentrations of copper: in vitro cell substrate properties.

PubMed

Ahmed, Zubair; Briden, Anita; Hall, Susan; Brown, Robert A

2004-02-01

We have previously described the production of large cables of fibronectin, a large extracellular matrix cell adhesion glycoprotein, which has a potential application in tissue engineering. Here we have stabilised these cables for longer survival and looked at their ultrastructural cell-substrate behaviour in vitro. Dissolution experiments showed that low concentrations of copper not only caused significant material stabilisation but left pores which could promote cell ingrowth, as we have previously reported with Fn-mats. Indeed, the greatest amount of cell ingrowth was observed for copper treated cables. Immunostaining showed S-100(+) multi-layers of cells around the edge of cables while ultrastructural analysis confirmed the presence of a mixture of fibroblasts and bipolar cells associated with fragments of basal lamina, which is a Schwann cell phenotype. Interestingly, the outermost layers of cells consisted of S-100(-) cells, presumed fibroblasts, apparently 'capping' the Schwann cells. Toxicity tests revealed that Schwann cells were only able to grow at the lowest concentration of copper used (1microM) while fibroblasts grew at all concentrations tested. These results could be used to design biomaterials with optimum properties for promoting cellular ingrowth and survival in tissue engineered grafts which may be used to improve peripheral nerve repair.

Keratinocyte Motility Is Affected by UVA Radiation-A Comparison between Normal and Dysplastic Cells.

PubMed

Niculiţe, Cristina M; Nechifor, Marina T; Urs, Andreea O; Olariu, Laura; Ceafalan, Laura C; Leabu, Mircea

2018-06-07

UVA radiation induces multiple and complex changes in the skin, affecting epidermal cell behavior. This study reports the effects of UVA exposure on normal (HaCaT) and dysplastic (DOK) keratinocytes. The adherence, spreading and proliferation were investigated by time-lapse measurement of cell layer impedance on different matrix proteins. Prior to UVA exposure, the time required for adherence and spreading did not differ significantly for HaCaT and DOK cells, while spreading areas were larger for HaCaT cells. Under UVA exposure, HaCaT and DOK cells behavior differed in terms of movement and proliferation. The cells' ability to cover the denuded surface and individual cell trajectories were recorded by time-lapse videomicroscopy, during wound healing experiments. Dysplastic keratinocytes showed more sensitivity to UVA, exhibiting transient deficiencies in directionality of movement and a delay in re-coating the denuded area. The actin cytoskeleton displayed a cortical organization immediately after irradiation, in both cell lines, similar to mock-irradiated cells. Post-irradiation, DOK cells displayed a better organization of stress fibers, persistent filopodia, and new, stronger focal contacts. In conclusion, after UVA exposure HaCaT and DOK cells showed a different behavior in terms of adherence, spreading, motility, proliferation, and actin cytoskeleton dynamics, with the dyplastic keratinocytes being more sensitive.

Neutral endopeptidase promotes phorbol ester-induced apoptosis in prostate cancer cells by inhibiting neuropeptide-induced protein kinase C delta degradation.

PubMed

Sumitomo, M; Shen, R; Goldberg, J S; Dai, J; Navarro, D; Nanus, D M

2000-12-01

Phorbol esters induce apoptosis in androgen-sensitive LNCaP cells, which express neutral endopeptidase (NEP), but not in androgen-independent prostate cancer (PC) cells, which lack NEP expression. We investigated the role of NEP in PC cell susceptibility to 12-O-tetradecanoylphorbol-13-acetate (TPA). Western analysis showed that expression of NEP and protein kinase Cdelta (PKCdelta) correlated with PC cell sensitivity to TPA-induced growth arrest and apoptosis in LNCaP cells and in TSU-Prl cells expressing an inducible wild-type NEP protein. Inhibition of NEP enzyme activity using the specific NEP inhibitor CGS24592, or inhibition of PKCdelta using Rottlerin at concentrations that inhibit PKCdelta but not PKCalpha, significantly inhibited TPA-induced growth inhibition and cell death. Furthermore, pulse-chase experiments showed PKCdelta is stabilized in LNCaP cells and in TSU-Pr1 cells overexpressing wild-type NEP compared with PC cells lacking NEP expression. This results from NEP inactivation of its neuropeptide substrates (bombesin and endothelin-1), which in the absence of NEP stimulate cSrc kinase activity and induce rapid degradation of PKCdelta protein. These results indicate that expression of enzymatically active NEP by PC cells is necessary for TPA-induced apoptosis, and that NEP inhibits neuropeptide-induced, cSrc-mediated PKCdelta degradation.

Morphogenesis of a higher plant from cultured cells and embryos in space

NASA Technical Reports Server (NTRS)

Krikorian, A. D.; Dutcher, F. R.; Quinn, C. E.; Steward, F. C.

1982-01-01

Reference is made to the Cosmos 782 experiment, which showed that cultured totipotent cells of carrot can give rise to embryos with well-developed roots but minimally developed shoots at near-zero g. The problem of whether the development of leafy shoots is sensitive to near-zero g conditions is considered. A test system that would allow this problem to be resolved in a future space flight is described.

Myosin II dynamics are regulated by tension in intercalating cells.

PubMed

Fernandez-Gonzalez, Rodrigo; Simoes, Sérgio de Matos; Röper, Jens-Christian; Eaton, Suzanne; Zallen, Jennifer A

2009-11-01

Axis elongation in Drosophila occurs through polarized cell rearrangements driven by actomyosin contractility. Myosin II promotes neighbor exchange through the contraction of single cell boundaries, while the contraction of myosin II structures spanning multiple pairs of cells leads to rosette formation. Here we show that multicellular actomyosin cables form at a higher frequency than expected by chance, indicating that cable assembly is an active process. Multicellular cables are sites of increased mechanical tension as measured by laser ablation. Fluorescence recovery after photobleaching experiments show that myosin II is stabilized at the cortex in regions of increased tension. Myosin II is recruited in response to an ectopic force and relieving tension leads to a rapid loss of myosin, indicating that tension is necessary and sufficient for cortical myosin localization. These results demonstrate that myosin II dynamics are regulated by tension in a positive feedback loop that leads to multicellular actomyosin cable formation and efficient tissue elongation.

Characterization of a BODIPY Dye as an Active Species for Redox Flow Batteries.

PubMed

Kosswattaarachchi, Anjula M; Friedman, Alan E; Cook, Timothy R

2016-12-08

An all-organic redox flow battery (RFB) employing a fluorescent boron-dipyrromethene (BODIPY) dye (PM567) was investigated. In a RFB, the stability of the electrolyte in all charged states is critically linked to coulombic efficiency. To evaluate stability, bulk electrolysis and cyclic voltammetry (CV) experiments were performed. Oxidized and reduced, PM567 does not remain intact; however, the products of bulk electrolysis evolve over time to show stable redox behavior, making the dye a precursor for the active species of an RFB. A theoretical cell potential of 2.32 V was predicted from CV experiments with a working discharge voltage of approximately 1.6 V in a static test cell. Mass spectrometry was used to identify the products of bulk electrolysis. Related experiments were carried out using ferrocene and cobaltocenium hexafluorophosphate as redox-stable benchmarks to further explain the stability results. The coulombic efficiency of a model cell using PM567 as a precursor for charge carriers stabilized around 73 %. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Flight Test of a Technology Transparent Light Concentration Panel on SMEX/WIRE

NASA Technical Reports Server (NTRS)

Stern, Theodore G.; Lyons, John

2000-01-01

A flight experiment has demonstrated a modular solar concentrator that can be used as a direct substitute replacement for planar photovoltaic panels in spacecraft solar arrays. The Light Concentrating Panel (LCP) uses an orthogrid arrangement of composite mirror strips to form an array of rectangular mirror troughs that reflect light onto standard, high-efficiency solar cells at a concentration ratio of approximately 3:1. The panel area, mass, thickness, and pointing tolerance has been shown to be similar to a planar array using the same cells. Concentration reduces the panel's cell area by 2/3, which significantly reduces the cost of the panel. An opportunity for a flight experiment module arose on NASA's Small Explorer / Wide-Field Infrared Explorer (SMEX/WIRE) spacecraft, which uses modular solar panel modules integrated into a solar panel frame structure. The design and analysis that supported implementation of the LCP as a flight experiment module is described. Easy integration into the existing SMEX-LITE wing demonstrated the benefits of technology transparency. Flight data shows the stability of the LCP module after nearly one year in Low Earth Orbit.

Humanized CD7 nanobody-based immunotoxins exhibit promising anti-T-cell acute lymphoblastic leukemia potential

PubMed Central

Yu, Yuan; Li, Jialu; Zhu, Xuejun; Tang, Xiaowen; Bao, Yangyi; Sun, Xiang; Huang, Yuhui; Tian, Fang; Liu, Xiaomei; Yang, Lin

2017-01-01

Background Nanobodies, named as VHHs (variable domain of heavy chain of HCAb [heavy-chain antibodies]), are derived from heavy-chain-only antibodies that circulate in sera of camelids. Their exceptional physicochemical properties, possibility of humanization, and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components, including immunotoxins. In our previous efforts, we have successfully generated the monovalent and bivalent CD7 nanobody-based immunotoxins, which can effectively trigger the apoptosis of CD7-positive malignant cells. To pursue the possibility of translating those immunotoxins into clinics, we humanized the nanobody sequences (designated as dhuVHH6) as well as further truncated the Pseudomonas exotoxin A (PE)-derived PE38 toxin to produce a more protease-resistant form, which is named as PE-LR, by deleting majority of PE domain II. Methods and results Three new types of immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. These recombinant immunotoxins were expressed in Escherichia coli and showed that nanobody immunotoxins have the benefits of easy soluble expression in a prokaryotic expression system. Flow cytometry results revealed that all immunotoxins still maintained the ability to bind specifically to CD7-positive T lymphocyte strains without binding to CD7-negative control cells. Laser scanning confocal microscopy revealed that these proteins can be endocytosed into the cytoplasm after binding with CD7-positive cells and that this phenomenon was not observed in CD7-negative cells. WST-8 experiments showed that all immunotoxins retained the highly effective and specific growth inhibition activity in CD7-positive cell lines and primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Further in vivo animal model experiments showed that humanized dhuVHH6-PE38 immunotoxin can tolerate higher doses and extend the survival of NOD-Prkdcem26Il2rgem26Nju (NCG) mice transplanted with CEM cells without any obvious decrease in body weight. Further studies on NCG mice model with patient-derived T-ALL cells, dhuVHH6-PE38 treatment, significantly prolonged mice survival with ~40% survival improvement. However, it was also noticed that although dhuVHH6-PE-LR showed strong antitumor effect in vitro, its in vivo antitumor efficacy was disappointing. Conclusion We have successfully constructed a targeted CD7 molecule-modified nanobody (CD7 molecule-improved nanobody) immunotoxin dhuVHH6-PE38 and demonstrated its potential for treating CD7-positive malignant tumors, especially T-cell acute lymphoblastic leukemia. PMID:28331319

Cytotoxic effects in 3T3-L1 mouse and WI-38 human fibroblasts following 72 hour and 7 day exposures to commercial silica nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stępnik, Maciej, E-mail: mstep@imp.lodz.pl; Arkusz, Joanna; Smok-Pieniążek, Anna

The potential toxic effects in murine (3T3-L1) and human (WI-38) fibroblast cell lines of commercially available silica nanoparticles (NPs), Ludox CL (nominal size 21 nm) and CL-X (nominal size of 30 nm) were investigated with particular attention to the effect over long exposure times (the tests were run after 72 h exposure up to 7 days). These two formulations differed in physico-chemical properties and showed different stabilities in the cell culture medium used for the experiments. Ludox CL silica NPs were found to be cytotoxic only at the higher concentrations to the WI-38 cells (WST-1 and LDH assays) but notmore » to the 3T3-L1 cells, whereas the Ludox CL-X silica NPs, which were less stable over the 72 h exposure, were cytotoxic to both cell lines in both assays. In the clonogenic assay both silica NPs induced a concentration dependent decrease in the surviving fraction of 3T3-L1 cells, with the Ludox CL-X silica NPs being more cytotoxic. Cell cycle analysis showed a trend indicating alterations in both cell lines at different phases with both silica NPs tested. Buthionine sulfoximine (γ-glutamylcysteine synthetase inhibitor) combined with Ludox CL-X was found to induce a strong decrease in 3T3-L1 cell viability which was not observed for the WI-38 cell line. This study clearly indicates that longer exposure studies may give important insights on the impact of nanomaterials on cells. However, and especially when investigating nanoparticle effects after such long exposure, it is fundamental to include a detailed physico-chemical characterization of the nanoparticles and their dispersions over the time scale of the experiment, in order to be able to interpret eventual impacts on cells. -- Highlights: ► Ludox CL silica NPs are cytotoxic to WI-38 fibroblasts but not to 3T3-L1 fibroblasts. ► Ludox CL-X silica NPs are cytotoxic to both cell lines. ► In clonogenic assay both silica NPs induce cytotoxicity, higher for CL-X silica. ► Cell cycle analysis shows alterations in both cell lines with both silica NP tested. ► Buthionine sulfoximine enhances cytotoxicity of Ludox CL-X in 3T3-L1 cells.« less